biomolecules

Review
Erectile Function and Sexual Behavior: A Review of the Role of
Nitric Oxide in the Central Nervous System
Maria Rosaria Melis * and Antonio Argiolas

Department of Biomedical Sciences, Section of Neurosciences and Clinical Pharmacology, University of Cagliari,
09124 Cagliari, Italy; argiolas@unica.it
* Correspondence: mrmelis@unica.it

Abstract: Nitric oxide (NO), the neuromodulator/neurotransmitter formed from L-arginine by neu-
ronal, endothelial and inducible NO synthases, is involved in numerous functions across the body,
from the control of arterial blood pressure to penile erection, and at central level from energy home-
ostasis regulation to memory, learning and sexual behavior. The aim of this work is to review earlier
studies showing that NO plays a role in erectile function and sexual behavior in the hypothalamus
and its paraventricular nucleus and the medial preoptic area, and integrate these findings with
those of recent studies on this matter. This revisitation shows that NO influences erectile function
and sexual behavior in males and females by acting not only in the paraventricular nucleus and
medial preoptic area but also in extrahypothalamic brain areas, often with different mechanisms.
Most importantly, since these areas are strictly interconnected with the paraventricular nucleus and
medial preoptic area, send to and receive neural projections from the spinal cord, in which sexual
communication between brain and genital apparatus takes place, this review reveals that central NO



participates in concert with neurotransmitters/neuropeptides to a neural circuit controlling both the


consummatory (penile erection, copulation, lordosis) and appetitive components (sexual motivation,
Citation: Melis, M.R.; Argiolas, A.
arousal, reward) of sexual behavior.
Erectile Function and Sexual
Behavior: A Review of the Role of
Keywords: nitric oxide; nitric oxide donors; nitric oxide targets; penile erection; sexual behavior; rat
Nitric Oxide in the Central Nervous
System. Biomolecules 2021, 11, 1866.
https://doi.org/10.3390/
biom11121866
1. Introduction
Academic Editor: Jesus Tejero Nitric oxide (NO), the gaseous highly diffusible compound produced from the amino
acid L-arginine by NO synthases, a family of Ca2+ -calmodulin-dependent iron contain-
Received: 6 November 2021 ing enzymes (e.g., neuronal, endothelial or inducible NO synthase) ([1–4] and references
Accepted: 8 December 2021 therein), has been added to the list of neurotransmitters and/or neuromodulators at the
Published: 11 December 2021 level of the peripheral and central nervous system in the 1990s [5–12]. Since then, NO has
been involved in numerous functions at peripheral and central level. Among these, one of
Publisher’s Note: MDPI stays neutral the best known is certainly its key role in relaxing cavernous corpora smooth muscles at
with regard to jurisdictional claims in the penile level (a key passage for the induction of penile erection) by activating guany-
published maps and institutional affil- late cyclase, the enzyme that converts guanosine triphosphate (GTP) in cyclic guanosine
iations. monophosphate (cGMP), the second messenger that—together with the other second mes-
senger cyclic adenosine monophosphate (cAMP) formed from adenosine triphosphate
(ATP) by the enzyme adenylate cyclase—is well known for its key role in practically every
cellular function in all living animals. NO in cavernous corpora is produced mainly by
Copyright: © 2021 by the authors. the endothelial NO synthase isoform localized in endothelial cells that overlay smooth
Licensee MDPI, Basel, Switzerland. muscle cells upon activation by an increased Ca2+ influx secondary to the activity of cholin-
This article is an open access article ergic and non-cholinergic neurons that impinge on them. However, at penile level also
distributed under the terms and the neuronal NO synthase isoform is present, mainly in non-adrenergic–non-cholinergic
conditions of the Creative Commons neurons, which participate in the relaxation of cavernous smooth muscles together with
Attribution (CC BY) license (https://
the cholinergic ones (reviews of the mechanism controlling cavernous corpora smooth
creativecommons.org/licenses/by/
muscles relaxation are found in [9,13–17]). The potency of NO in activating guanylate
4.0/).

Biomolecules 2021, 11, 1866. https://doi.org/10.3390/biom11121866 https://www.mdpi.com/journal/biomolecules

Biomolecules 2021, 11, 1866 2 of 40

cyclase and increasing cGMP concentration that mediates the relaxation of cavernous
smooth muscles led researchers to consider NO as the key physiological neurotransmitter
of penile erection at local level [10,11,18]. Together with the selective presence in cavernous
corpora smooth muscles of phosphodiesterase type V enzyme, which selectively inactivates
cGMP to GMP leading to the end of the cavernous corpora relaxation and then of penile
erection, this has opened the way to the discovery of selective, orally active inhibitors of
this phosphodiesterase enzyme isoform clinically used today for the treatment of erectile
dysfunction (reviews on phosphodiesterase type V and its functions are found in [19,20]).
Studies with selective endothelial or neuronal NO synthase knockout mice revealed that
both NO synthase isoforms play a role in the relaxation of the cavernous smooth muscles,
with the neuronal isoform playing a main role in the initiation and the endothelial one in
the maintenance of penile erection [21]. Most importantly, these studies revealed also that
neuronal NO synthase knockout mice mate normally and have a NO-dependent erectile
response [21]. This is due to endothelial NO synthase and alternative spliced neuronal
NO synthase variants surviving gene deletion, synthetizing NO and maintaining erectile
function even in the absence of neuronal NO synthase [22,23].
NO synthase isoforms are also present in the central nervous system, where a NO-
guanylate cyclase operating system has been also identified in several brain areas. Among
these, particular attention has been dedicated to the hippocampus and the cerebellum,
which play a key role in learning and memory formation and in which mechanisms
strongly related to these central functions, such as long-term potentiation and long-term
depression, are well known to take place [6,7,12,24,25]. In these areas, neuronal NO
synthase that produces NO is localized in the cell bodies of neurons on which impinge
excitatory (usually glutamatergic) synapses. The activation of these neurons through
excitatory amino acid receptors of the AMPA [(±)-a-amino-3-hydroxy-5-methyl-isoxaxole-
4-propionic acid] first and then of NMDA (N-methyl-D-aspartic acid) subtypes (NMDA
receptors, which are coupled to voltage-dependent Ca2+ channels), leads to an increased
Ca2+ influx and subsequent NO synthase activation in these neuronal cell bodies. Newly
produced NO travels as a retrograde messenger from the cell bodies in which it has been
produced to the excitatory glutamatergic synapses, where guanylate cyclase is localized,
activating its production of cGMP which activates the mechanisms that produce long term
potentiation in the hippocampus or long-term depression in the cerebellum or vice versa,
depending on the neuronal system involved [6,7,12,24]. This initial picture is changing
continuously, with the discovery of new modes of operation of NO ranging from local
signaling to volume-type transmission across various brain regions and nuclei ([26] and
references therein).
Another area of the brain rich in NO synthase is the hypothalamus and in particular
the paraventricular nucleus (PVN), which appears to be one of the brain nuclei richest
in NO synthase [27]. Detailed immunocytochemical studies have shown that the NO
synthase in the PVN is localized in the cell bodies and dendrites of neurons, which contain
oxytocin, vasopressin and somatostatin [9,28–30] often found close to neurons containing
corticotrophin-releasing hormone (CRH), leading researchers to suggest that NO in the
PVN might be involved in the release of these two hormones in the neurohypophysis
and of CRH in the median eminence ([31] and references therein). NO synthase was also
detected and found involved in the activation of luteinizing hormone-releasing hormone
(LH-RH) neurons in the medial preoptic area ([31] and references therein). The localization
of a Ca2+ -dependent NO synthase in PVN oxytocinergic neurons [8] in close proximity
with dopamine [32,33], serotonin (5-HT) [34] and excitatory amino acid projections [35]
and the above-mentioned possible involvement of NO in the release of CRH that controls
corticotrophin (ACTH) release, and of LH-RH that control the release of both follicle
stimulating hormone (FSH) and luteinizing hormone (LH) from the adenohypophysis, led
the authors of this review and other research groups to investigate on a possible role of NO
in the central control of erectile function and sexual behavior first in the PVN [36–43] and in
the medial preoptic area [44,45] and later in other brain areas. It is now clear that NO plays a
Biomolecules 2021, 11, 1866 3 of 40

role in the control of erectile function and sexual behavior not only at the hypothalamic and
medial preoptic area level, but also in other extrahypothalamic brain areas. Among these,
the most studied are the hippocampal formation [46], the ventral tegmental area [47,48],
the ventral subiculum of the hippocampus, the posteromedial cortical nucleus of the
amygdala [49–51], the ventral medulla and the spinal cord [52]. In many of these areas
NO exerts its sexual effects by acting in concert with other neurotransmitters and/or
neuropeptides (see Section 6 and [53–57]). Against the role of central NO in erectile function
and sexual behavior supported by the above studies, mating and penile erection are normal
in neuronal NO synthase knockout mice [21], in spite of an excess of inappropriate sexual
behavior and a marked increase in aggressive behavior [58]. In fact, this finding suggests
that this NO synthase isoform is not necessary for sexual behavior, apparently ruling out
the involvement of central NO in erectile function and sexual behavior. It is likely that this
discrepancy is only apparent, first because the other NO synthase isoforms are also present
in the central nervous system and can produce NO to replace that missing for the absence of
neuronal NO synthase as found at penile level (see above), and second, because this finding
may simply indicate only that NO is one of the many agents controlling sexual behavior.
Thus, it is likely that the failure of the absence of neuronal NO synthase to abolish/reduce
mating and penile erection reflects the redundancy of systems involved in reproductive
physiology, which has an evolutionary origin, as it is fundamental to guarantee the passage
of genes to the next generations and thus the species survival, rather than showing no
role of NO in erectile function and sexual behavior [57]. However, the hypothesis that
NO-synthase knockout mice copulate normally may be due to the fact that erectile function
and copulatory behavior in male mice are organized differently from those of male rats
cannot be completely ruled out. In fact, (i) while male rats need a few minutes to reach the
first ejaculation, which may be followed by many others, male mice need much more time
to reach only one ejaculation rarely followed by others [59–61] and (ii) differences in the
spinal control of penile reflexes have been found to occur between male mice and rats [62].
This review has been prepared by considering the results of numerous published high-
quality studies, including those made in the authors’ laboratories, on the role of central NO
in erectile function and sexual behavior in laboratory animals (mainly rats), from penile
erection in males to copulation in males and females, and sexual intercourse in humans
when available, in order to provide an updated picture of the most recent discoveries and
to identify new possible advances that may be useful for realizing new strategies based on
the modulation of central NO activity for the therapy of erectile dysfunctions and other
sexual disorders.

2. Central NO and Erectile Function

Penile erection is considered the main component of the male sexual response, as it
allows sexual intercourse to occur with the female counterpart. It is produced by a complex
neural central and peripheral interaction that induces muscle and vascular changes at
the level of the erectile tissues of the male genital apparatus (cavernous corpora, corpus
spongiosum, bulbocavernosus and ischiocavernosus muscles and other perineal muscles,
which are differently organized and controlled by spinal cord projections in males and
females). This is further complicated by humoral and endocrine influences, exerted mainly
by testosterone and its metabolites estradiol and 5α-dihydrotestosterone, which take place
at central and peripheral levels. Although the aim of this review is to focus on the sexual
effects of central NO, it is important to recall that testosterone and its metabolites are key
regulators of male erectile function and sexual behavior since, through the modulation of
genomic processes, they exert upstream effects on all signaling pathways activated by all
the neurotransmitters and neuropeptides cited in this review, including NO synthase itself
at both central and peripheral sites [16,59,60].
Penile erection may occur not only during sexual activity but also in other contexts,
such as after simple manipulation of the genitalia, or during sleep or erotic fantasies
in humans, or in male rats put in the presence of an inaccessible receptive female (non-
 ing pathways activated by all the neurotransmitters and neuropeptides cited in this re-
view, including NO synthase itself at both central and peripheral sites [16,59,60].
Penile erection may occur not only during sexual activity but also in other contexts,
such as after simple manipulation of the genitalia, or during sleep or erotic fantasies in
Biomolecules 2021, 11, 1866 humans, or in male rats put in the presence of an inaccessible receptive female 4 of 40

(non-contact erections), or after treatment with several classes of drugs (i.e., dopamine
agonists, serotonin agonists, NO donors, phosphodiesterase inhibitors, soluble guanylate
cyclase
contactactivators,
erections),RhoA-Rho kinase inhibitors,
or after treatment with several etc.) and of
classes neuropeptides [i.e., adrenocor-
drugs (i.e., dopamine agonists,
ticotropin (ACTH)-melanocyte stimulating hormone (α-MSH)-related
serotonin agonists, NO donors, phosphodiesterase inhibitors, soluble guanylate peptides, oxytocin,
cyclase
hexarelin
activators, analogues,
RhoA-RhoVGF-related peptides
kinase inhibitors, etc.) (produced
and neuropeptides by proteolytic cleavage of the
[i.e., adrenocorticotropin
protein VGF) and others],
(ACTH)-melanocyte stimulatingacting in the(α-MSH)-related
hormone central nervous systemoxytocin,
peptides, or peripherally
hexarelin
[16,40,54–57,63–75].
analogues, VGF-related Depending
peptideson(produced
the context by in which penile
proteolytic erection
cleavage of theoccurs,
proteinitVGF)
is gen-
and
erally accepted that different central and peripheral neural and/or
others], acting in the central nervous system or peripherally [16,40,54–57,63–75]. Depend- humoral endocrine
mechanisms may participate
ing on the context in the erection
in which penile regulation of this
occurs, it issexual response,
generally accepted often
thatindifferent
a very
complex manner [67] (see Figure 1 for a schematic representation
central and peripheral neural and/or humoral endocrine mechanisms may participate in of central and periph-
eral
theneural
regulationpathways
of thiscontrolling
sexual response,erectile function
often in a veryandcomplex
sexual behavior
manner [67] in mammals).
(see FigureThe 1 for
involvement
a schematic of central NO in
representation ofthe control
central andofperipheral
penile erectionneuralwas first shown
pathways in the erectile
controlling 1990s
byfunction
studiesand thatsexual
revealed the inhibitory
behavior in mammals). effectThe of NO synthase of
involvement inhibitors
central NO given notcontrol
in the only
systemically (i.e., by
of penile erection wasacting mainlyin
first shown atthe
the1990s
penile bylevel)
studies butthat
alsorevealed
into thethe lateral ventricles
inhibitory effect
(i.c.v.) (i.e., by acting at the central level) on this sexual response
of NO synthase inhibitors given not only systemically (i.e., by acting mainly at the penile induced by several
drugs
level)andbut peptides
also into (often together
the lateral with yawning)
ventricles (i.c.v.) (i.e.,inbymale ratsat[37,40,76].
acting the central These
level)studies
on this
were
sexualfollowed
response by induced
other studies showing
by several thatand
drugs these compounds
peptides (oftenwere also able
together withtoyawning)
reduce
in expression
the male rats [37,40,76].
of penile These
erectionstudies were place
that takes followed by other studies
in physiological showing
contexts, for that these
example
compounds
when sexuallywere potentalsomale
ablerats
to reduce
are putthe expression
into the presence of penile
of anerection
inaccessiblethat takes place
sexually re-in
physiological
ceptive female contexts, for example
rats (non-contact when sexually
erections) or whenpotent
copulation male rats
was are put into
allowed thewhen
(e.g., presence
in
of an inaccessible sexually receptive female rats (non-contact
copula penile erections take place) [77], and even when penile erections are induced by erections) or when copulation
was allowed
direct stimulation (e.g.,ofwhen in copula
the penis (e.g., penile erections take
reflex erections) [42].place) [77], and even when penile
erections are induced by direct stimulation of the penis (e.g., reflex erections) [42].

Figure 1. A schematic representation of the neural pathways controlling the male and female genital
Figure 1. A schematic representation of the neural pathways controlling the male and female gen-
apparatus.
ital When
apparatus. When sexual (visual,
sexual auditory,
(visual, olfactory,
auditory, tactile
olfactory, and even
tactile imaginative
and even in humans)
imaginative stimuli
in humans)
reach the
stimuli brain
reach the high
braincenters, this activates
high centers, neural pathways,
this activates to date to
neural pathways, still unknown,
date leading leading
still unknown, to penile
toerection in malesin
penile erection and clitoris
males anderection
clitoris and vaginal
erection andlubrication in females,inallowing
vaginal lubrication females,sexual intercourse.
allowing sexual
These pathways
intercourse. Thesetravel from the
pathways brain,
travel frommainly from the
the brain, hypothalamus
mainly and its nuclei (paraventricular
from the hypothalamus and its nuclei
(paraventricular nucleus,nucleus),
nucleus, ventromedial ventromedial nucleus),
and medial and medial
preoptic preoptic
area, through thearea, through
medulla the medulla
oblongata and the
oblongata andtothe
spinal cord, thespinal cord,
genital to the genital
apparatus. apparatus.
The latter The latter
is innervated byispudendal
innervated by pudendal
nerves originatingnerves
from
originating from the sacral S2–S4 spinal tract and containing the primary afferent
the sacral S2–S4 spinal tract and containing the primary afferent sensory from and motor pathways tosensory from and
the penis in the male and to the clitoris in the female, and by cavernous nerves containing the primary
efferent sympathetic and parasympathetic pathways originating in the pelvic plexuses. These receive
neural inputs from (i) pelvic nerves originating in the sacral S2–S4 spinal tract, (ii) hypogastric nerves
originating in the thoracic-lumbar (T4-L2) spinal tract and (iii) post-ganglia fibers originating from
the paravertebral sympathetic ganglia of the thoracic–lumbar tract of the spinal cord (T11-L2). Details
are found in [15–17,40,53,54,56,57,59,60,63–65,67,68,75].
Biomolecules 2021, 11, 1866 5 of 40

2.1. Central NO Facilitates Erectile Function in Male Rats: Effect of NO Synthase Inhibitors
As recalled above, in the 1990s the involvement of central NO in the control of penile
erection was first shown by studies that revealed an inhibitory effect of NO synthase
inhibitors given endovenously or i.c.v. on these responses induced by drugs (i.e., apomor-
phine, a mixed D1/D2 dopamine receptor agonist given systemically or into the PVN and
the excitatory amino acid NMDA, agonist of the NMDA receptor, but not AMPA, agonist
of the AMPA receptor or trans (±)-1-amino-1,3-cyclopentane-dicarboxilic acid (ACPD),
agonist of the metabotropic receptor [78], given into the PVN or 5-HT receptor agonists
of the 5-HT1C subtype [i.e., 1-(3-chlorophenyl)-piperazine (m-CPP) and N-(3-trifluoro-
methylphenyl)-piperazine (TFMPP)] given systemically, and peptides (i.e., oxytocin given
i.c.v. or into the PVN and ACTH 1-24 given i.c.v.) in male rats (Table 1) [36,37,41,77,79–84].
In these earlier studies, two well-characterized competitive inhibitors, namely L-arginine
analogs, such as NG -nitro-L-arginine methyl ester (L-NAME), NG -monomethyl-L-arginine
(L-NMMA) and its inactive isomer NG -monomethyl-D-arginine (D-NMMA) [1] were used.
The potency of these compounds in preventing penile erection induced by the above
compounds in male rats was correlated with their potency in inhibiting NO synthase,
being L-NAME 4–5 times more potent than L-NMMA and D-NMMA ineffective as ex-
pected [36,41,77,79–85]. That the L-NAME prevention of apomorphine-, oxytocin-, NMDA-,
5-HT1C receptor agonists and ACTH-induced penile erection is mediated by the compet-
itive inhibition of NO synthase and not by other effects of this compound was further
supported by the ability of i.c.v. L-arginine, the physiological substrate of NO synthase,
to prevent the inhibitory effect of L-NAME, in spite of its scarce ability to induce, per se,
this behavioral response [86] as discussed below (Sections 2.2.1 and 2.2.2). Recent studies
have shown that L-NAME and S-methyl-thio-citrulline, a selective neuronal NO synthase
inhibitor [87] given i.c.v. are also able to prevent penile erection induced not only by apo-
morphine, oxytocin, and NMDA, but also by other compounds such as hexarelin analogue
peptides [88–91], VGF-derived peptides [92,93] and even when this sexual response was
induced by SR 141716A (Rimonabant, [N-(piperidin-1-yl)-5-(4-chlorophenyl)-4-methyl-1H-
pyrazole- 3-carboxyamide]), a selective antagonist of the cannabinoid receptor of the CB1
subtype [94], injected into the PVN of male rats [95] (Table 1).

Table 1. Drug- and peptide-induced penile erection in male rats: effect of NO synthase inhibitors given systemically,
intracerebroventricularly or in the PVN.

Effect on Penile Erection

Penile Erection L-NAME D-NMMA
L-NMMA
Induced by
Given Given Given
i.p. i.c.v PVN i.p. i.c.v. PVN i.p. i.c.v. PVN
Dopamine agonists ↓ ↓ ↓ ↓ ↓ ↓ == == ==
Oxytocin ↓ ↓ ↓ ↓ ↓ ↓ == == ==
NMDA ↓ ↓ ↓ ↓ ↓ ↓ == == ==
5-HT1C agonists ↓ ↓ == ↓ ↓ == == == ==
ACTH 1-24 ↓ ↓ == ↓ ↓ == == == ==
Hexarelin peptides n.a. n.a. ↓ n.a. n.a. ↓ n.a. n.a. ==
VGF peptides n.a. n.a. ↓ n.a. n.a. ↓ n.a. n.a. ==
SR 141716A n.a. n.a. ↓ n.a. n.a. ↓ n.a. n.a. ==

↓ = prevention; == = no effect; n.a. = not available; i.p. = intraperitoneally; i.c.v. = intracerebroventricularly; PVN = into the PVN. Dopamine
agonists = dopamine receptor agonists of the D2 receptor family (D2 and D4 receptor subtype); L-NAME = NG -nitro-L-arginine methyl
ester; L-NMMA = NG -monomethyl-L-arginine; D-NMMA = NG -monomethyl-D-arginine. Dopamine agonists were given systemically or
into the PVN, oxytocin i.c.v. or in the PVN, N-methyl-D-aspartic acid in the PVN, 5-HT1C agonists systemically or into the PVN, ACTH 1-24
i.c.v., hexarelin analogues, VGF-derived peptides and SR 141716A into the PVN. Details are found in [36–38,41,43,79–84,86,90,91,93,96–101].
Biomolecules 2021, 11, 1866 6 of 40

2.2. Central NO, PVN and Erectile Function

The major problem with NO synthase inhibitors given systemically is that it is impos-
sible to separate the local (at penile level) and systemic effects (i.e., marked hypotension)
from the central effect of these compounds. For this reason, the above studies were soon
followed by other studies in which NO synthase inhibitors were injected i.c.v. or into
specific brain areas of male rats. These studies revealed that the PVN was one of the
brain areas where NO synthase inhibitors act to prevent apomorphine-, oxytocin- and
NMDA-induced penile erection and where an increase in the NO content, such as that
obtained by injecting the so-called NO donors (drugs that release or produce NO), causes
penile erection as will be discussed below [37,86] (see Sections 2.2.1 and 2.2.2).

2.2.1. NO Synthase Inhibitors Injected into the PVN Abolish/Reduce Drug- and
Peptide-Induced Penile Erection in Male Rats
As recalled above NO synthase inhibitors were found able to prevent apomorphine-,
oxytocin-, and NMDA-, but not 5HT1C or ACTH 1-24-induced penile erection when injected
into the PVN of male rats. Indeed L-NAME was found able to prevent this sexual response
induced by apomorphine, NMDA and oxytocin not only when injected i.c.v. but also when
injected in this hypothalamic nucleus but not in surrounding structures [79–81,83,85,86]
(Table 1). These findings are in line with the fact that apomorphine, oxytocin and NMDA
and other compounds such as hexarelin analogues, VGF related peptides and even SR
141716A, a compound that block cannabinoid receptors of the CB1 subtype, induce penile
erection by activating oxytocinergic neurons whose cell bodies are located in the PVN
and surrounding area, which project to extra-hypothalamic brain areas and to the spinal
cord as discussed below (Figure 2) (Section 2.2.4) [53–57,73]. Conversely, the failure of
L-NAME injected into the PVN of male rats to prevent penile erection induced by ACTH
1-24 and 5-HT1C receptor agonists, in spite of its ability to prevent such responses when
injected i.c.v., suggest that both these compounds induce penile erection by acting on NO
synthase at brain sites different from the PVN with a mechanism that does not involve
central oxytocinergic neurotransmission. In line with this possibility, in male rats (i) ACTH
1-24-induced penile erection is not abolished by lesions of the PVN [117], which destroy
central oxytocinergic neurons, nor by the blockade of central oxytocinergic receptors with
d(CH2 )5 Tyr(Me)-Orn8 -vasotocin, a potent oxytocin receptor antagonist [118], injected into
the PVN [119] or by ω-conotoxin, a potent and selective inhibitor of N-type Ca2+ channels
present mainly in the nervous tissues [120,121] injected in the PVN [102], (ii) ACTH 1-24
induces penile erection when injected in areas surrounding the hypothalamic portion of
the third ventricle but not in the PVN [122], and finally (iii) the ACTH symptomatology
(which include also stretching and yawning) differs from that induced by dopamine
agonists, NMDA and oxytocin, because it begins 25–30 min after the treatment and lasts
for several hours [123]. Similarly to that reported above for ACTH 1-24, penile erection
induced by 5-HT1C receptor agonists given i.c.v., is not mediated by the activation of central
oxytocinergic neurons. Accordingly, the erectile response induced by i.c.v. 5-HT1C receptor
agonists is not prevented by the blockade of oxytocinergic receptors, and (ii) 5-HT1C
receptor agonists do not induce penile erection when injected in the PVN of male rats [82].
Perhaps more importantly, L-NAME injected into the PVN is also unable to prevent 5-HT1C
receptor agonist-induced penile erection [83]. Together the above results suggest that NO
synthase inhibitors given i.c.v. prevent penile erection induced by these compounds by
acting at sites located downstream to oxytocinergic neurons in a yet undiscovered brain
area. In this regard, it is pertinent to recall that a serotoninergic pathway originating in
the nucleus paragigantocellularis of the ventral medulla sending projections to the spinal
cord in the region of the spinal nucleus of the bulbocavernosus (spinal lumbosacral tract
L2-S1) and which inhibits penile erection, has been identified in male rats [124–126]. More
intriguingly, NO synthase is found localized in neurons of the ventral medulla [127] as well
as in the spinal cord [52], raising the possibility that NO acts in these regions to facilitate
the pro-erectile effect of these compounds as discussed below (see Section 3).
 Biomolecules 2021, 11, 1866 7 of 40
Biomolecules 2021, 11, x 7 of 42

Figure
Figure TheThe
2. 2. mainmain mechanism
mechanism by which
by which dopamine
dopamine (DA) (DA) agonists
agonists of theof D2
thefamily
D2 family (D2 D
(D2 and and D
4 re- 4
receptor
ceptor subtype),
subtype), oxytocin,
oxytocin, NMDA,NMDA, hexarelin
hexarelin peptides
peptides andandVGF VGF peptides
peptides facilitate
facilitate penile
penile erection
erection
andand copulatory
copulatory activity
activity in in male
male rats
rats is is
thethe activation
activation of of oxytocinergic
oxytocinergic neurons
neurons originating
originating in in
thethe
PVNPVN and and projecting
projecting to to
thethe spinal
spinal cord
cord and
and totoextrahypothalamic
extrahypothalamic brain
brain areas.Apparently
areas. Apparently allall
thethe
above
above compounds,
compounds, byby acting
actingonontheir
theirown
ownreceptors
receptorslocalized
localizedin in the
the cell
cell bodies of these
these oxytocin-
oxyto-
cinergic neurons, increase
ergic neurons, increase Ca 2+ ions influx in the cell bodies of these neurons, in turn causing the
Ca2+ ions influx in the cell bodies of these neurons, in turn causing the
activation
activation of of
NONO synthase
synthase (NOS),
(NOS),thethe
Ca2+Ca calmodulin-dependent
2+ calmodulin-dependent enzyme
enzymepresent in oxytocinergic
present in oxytociner-
cell bodies that converts the amino acid L-arginine (L-Arg) to nitric oxide (NO). NO in turn activates
gic cell bodies that converts the amino acid L-arginine (L-Arg) to nitric oxide (NO). NO in turn
oxytocinergic neurons to release oxytocin in the spinal cord and in extrahypothalamic brain areas
activates oxytocinergic neurons to release oxytocin in the spinal cord and in extrahypothalamic
inducing penile erection and facilitating sexual behavior by a mechanism not involving the
brain areas
guanylate inducing penile
cyclase-cyclic erectionmonophosphate
guanosine and facilitating sexual
(GC-cGMP)behavior by a mechanism
pathway, not involving
as discussed below
the guanylate cyclase-cyclic guanosine monophosphate (GC-cGMP) pathway,
(Section 2.4.2). PVN oxytocinergic neurons facilitate penile erection and sexual activity not only as discussed below
(Section
when 2.4.2).by
activated PVNtheoxytocinergic
above compounds, neuronsbut facilitate
also bypenile
drugs erection and sexual
that increase PVNactivity not only when
NO concentration
(NOactivated
donors),byby the
theabove compounds,
blockade of PVN CB1 but also by drugs
receptors (whichthatare
increase PVN NO
not located concentrationcell
on oxytocinergic (NO
bodies but increase
donors), the activity
by the blockade of the
of PVN CB1latter ones by
receptors increasing
(which are notglutamatergic neurotransmission
located on oxytocinergic in
cell bodies
thebut
PVN) (see Section
increase 2.2.4/Figure
the activity 3), and
of the latter onesbybyphysiological sexual stimulineurotransmission
increasing glutamatergic (e.g., pheromones and in the
others).
PVN) (see Conversely, when these3), oxytocinergic
Section 2.2.4/Figure and by physiologicalneurons are stimuli
sexual inhibited,
(e.g.,for instance and
pheromones by gam-
others).
ma-amino-butyric acid GABA, opioid peptides/opiate drugs or by drugs that inhibit NOS activity,
Conversely, when these oxytocinergic neurons are inhibited, for instance by gamma-amino-butyric
the spontaneous (i.e., physiologically activated) or drug/neuropeptide-stimulated erectile function
acid GABA, opioid peptides/opiate drugs or by drugs that inhibit NOS activity, the spontaneous
and sexual activity is reduced. Details are found in [16,17,40,53–57,73,77,88–93,100–116].
(i.e., physiologically activated) or drug/neuropeptide-stimulated erectile function and sexual activity
is reduced. Details are found in [16,17,40,53–57,73,77,88–93,100–116].
Biomolecules 2021,2021,
Biomolecules 11, x11, 1866 8 of 42
8 of 40

Figure 3. A synthetic representation of two possible mechanisms by means of which endocannabi-

Figure 3. A synthetic representation of two possible mechanisms by means of which endocanna-
noids
binoids maymay inhibit
inhibit penile
penile erection
erection at the
at the PVN PVN level
level in male
in male rats.rats. Briefly,
Briefly, endocannabinoids
endocannabinoids inhibit
inhibit
the release of glutamic acid (GLU) from glutamatergic synapses impinging on the cell bodies of of
the release of glutamic acid (GLU) from glutamatergic synapses impinging on the cell bodies
oxytocinergic
oxytocinergic neurons
neurons (OXY) (OXY) by acting
by acting mainlymainly on CB1
on CB1 receptors
receptors located
located on these
on these glutamatergic
glutamatergic
synapses; alternatively,
synapses; theythey
alternatively, can can
act on
actCB1
on CB1receptors localized
receptors on inhibitory
localized GABAergic
on inhibitory GABAergicsynapses
synapses
that that
impinge on these
impinge glutamatergic
on these glutamatergic synapses
synapses andand
whose
whoseactivation
activation inhibits glutamic
inhibits glutamicacid
acidrelease.
release. In
In both
bothcases
casesthis
thiscauses
causesa areduction
reductionininCa
2+ influx through Ca2+ 2+
Ca2+ influx through Ca channels-coupled
channels-coupled NMDA
NMDA receptors
receptors in
in the cell bodies of oxytocinergic neurons
the cell bodies of oxytocinergic neurons mediating
mediatingpenile erection
penile erectionfollowed by aby
followed reduction in NO
a reduction in NO
synthase activity, thereby inhibiting oxytocinergic neurons and penile erection. The above mecha-
synthase activity, thereby inhibiting oxytocinergic neurons and penile erection. The above mecha-
nisms may also be not mutually exclusive and operate in concert to inhibit erectile function. Both
nisms may also be not mutually exclusive and operate in concert to inhibit erectile function. Both
these mechanisms have been identified in other brain areas rich in CB1 receptors. (+) = activation;
(−) =these mechanisms
inhibition. Details have been in
are found identified in other brain areas rich in CB1 receptors. (+) = activation;
[95,100,101,112,113].
(−) = inhibition. Details are found in [95,100,101,112,113].
PVN2.2.2.
of male
NO ratsDonors to prevent
Injectedpenile erection
i.c.v. or Directlyinduced
into theby ACTH
PVN Induce1-24Penile
and 5-HT 1C receptor
Erection in
agonists, in
Male Rats spite of its ability to prevent such responses when injected i.c.v., suggest that
both these compounds induce penile erection by acting on NO synthase at brain sites
The studies reviewed above are in line with the hypothesis that NO is involved in
different from
the regulation the ofPVNpenilewith a mechanism
erection inducedthat does not involve
by dopamine agonists,central
oxytocin,oxytocinergic
NMDA, VGF-
neurotransmission. In line with this possibility, in male rats (i)
related peptides and hexarelin analogue peptides at the PVN level, while its involvement ACTH 1-24-induced pe-
nile in
erection is not abolished by lesions of the PVN [117], which
this sexual response induced by ACTH and 5-HT1C agonists seems to occur at sites destroy central oxyto-
cinergic
distinct neurons,
from thenor PVN byyettheto beblockade
identifiedof (seecentral
above).oxytocinergic
Further supportreceptorsfor the with
above
d(CH 2)5Tyr(Me)-Orn -vasotocin, a potent oxytocin receptor antagonist [118], injected into
8
hypothesis is provided by the results of the studies reviewed in this section, which show
the PVN
that NO [119]donors
or by ω-conotoxin,
injected i.c.v.a or potent and into
directly selective inhibitor
the PVN of N-type
of male rats canCa2+induce
channelsper se
present mainly in the nervous tissues [120,121] injected in
spontaneous penile erections indistinguishable from those induced by the drugs the PVN [102], (ii) ACTH 1-24and
induces
peptidespenile erection when
mentioned above. injected
In thisinregard,
areas surrounding
it is pertinent thetohypothalamic portion ofNO
recall that numerous
the third ventricle but not in the PVN [122], and finally (iii)
donors have been tested and found effective as agents for increasing NO content and the ACTH symptomatology
(which include
causing also stretching
relaxation of cavernous and yawning)
smooth musclesdiffers
in infrom
vitro that inducedmade
experiments by dopamine
with isolated
agonists,
stripsNMDA and oxytocin,
of cavernous corporabecause it begins
tissue and/or 25–30 min
isolated after thecavernous
or cultured treatment corpora
and lastscells
for several
of several hours [123]. Similarly
mammals from rat,torabbit,
that reported
dog andabove cat to for ACTHand
primates 1-24,man
penile erectionNO
[128–133].
induced by 5-HT receptor agonists given i.c.v., is not mediated
donors have also been tested in double-blind crossover trials as a treatment of human
1C by the activation of
central oxytocinergic neurons. Accordingly, the erectile response induced
erectile dysfunction, but with scarce success [133]. Nonetheless, only a few of the available by i.c.v. 5-HT 1C

receptor agonists
NO donors haveis not
beenprevented by the blockade
injected centrally in male rats of oxytocinergic receptors,
to test their ability and (ii)
to induce penile
5-HT 1C receptor
erection (Tableagonists
2) [103].doThese
not induce
includepenile erection when
nitroglycerin, injected in NO
a well-known the PVNdonor ofclinically
male
rats used
[82]. for
Perhaps
its potentmore importantly,
vasodilating L-NAMEand
properties injected
for theinto the PVN
treatment is also pectoris
of angina unable to [134],
prevent
isoamyl5-HT 1C receptor
nitrite, a NO agonist-induced
donor clinically used penile
forerection
the therapy[83].of Together the above
angina [134] and wellresults
known
suggest that
for its NO synthase
aphrodisiac inhibitors
effect given i.c.v.
[135], sodium prevent penile
nitroprusside, erection
another induced by
well-known NO these
donor
clinicallyby
compounds used for at
acting itssites
potent antihypertensive
located downstreameffect [134], hydroxylamine,
to oxytocinergic neurons in awhich is not
yet un-
used clinically
discovered brain area. but Inis converted
this regard,toitNO by various
is pertinent cellular
to recall thatenzymes [24] and pathway
a serotoninergic S-nitroso-N-
Biomolecules 2021, 11, 1866 9 of 40

acetyl-D-penicillamine (SNAP), which is considered a spontaneous and effective releaser

of NO in aqueous solutions [24]. Finally, L-arginine, the physiological substrate of NO
synthase and direct precursor of NO, is usually added to the list of NO donors tested for a
potential pro-erectile effect. As shown in Table 2, L-arginine, but not D-arginine, injected
i.c.v. at doses up to 1000 µg is unable to induce penile erection, but is able to reduce
the inhibitory effect of NO synthase inhibitors on apomorphine-, oxytocin- and NMDA-
induced penile erection in male rats. Despite its ineffectiveness when given i.c.v., L-arginine,
but not D-arginine, is able to induce penile erection when injected into the PVN in more than
70% of the treated rats [86]. These findings confirm that L-arginine-induced penile erection
is mediated by its conversion to NO because of the selective substrate stereospecificity of
NO synthase (e.g., the L-amino acid only is converted to NO and citrulline). In line with
these studies L-arginine (500 nmol/0.5µL) injected into the PVN (i) increases intracavernous
blood pressure in male rats kept under pentobarbital anesthesia [43], (ii) increases reflex
penile erections in awake rats [42], and (iii) both these responses were abolished by the
concomitant administration of L-NAME (500 nmol) [42,43]. At variance from L-arginine,
nitroglycerin is able to induce penile erection either when given i.c.v. (33–99 µg) or into
the PVN (1.6–6.6 µg) in a dose-dependent manner [37,85]. Like nitroglycerin, isoamyl
nitrite is able to induce penile erection immediately after i.c.v. injection at doses between
20 and 100 µg. However, it has not been tested after injection into the PVN because of its
incompatibility and/or insolubility with the majority of available solvents [103]. Unlike
nitroglycerin and isoamyl nitrite, sodium nitroprusside does not induce penile erection
when injected i.c.v. at a dose up to 100 µg, but is able to do so when injected into the PVN
at doses higher than 10 µg. The failure of sodium nitroprusside to induce penile erection in
male rats when given i.c.v. is probably due to the appearance of collateral behavioral effects,
i.e., hyperactivity, hypermotility, sniffing, rearing and convulsions often followed by death
within 2–3 h after treatment [103]. These effects mask the expression of penile erection
and might be due to the release of highly toxic cyanide ions [136]. Accordingly, despite
its ability to induce penile erection when injected into the PVN, sodium nitroprusside
has been reported able to reduce sexual competence of male rats during copulatory tests
with a receptive female rat when given systemically in a dose-dependent manner, with
the highest dose tested (60 µg/kg) found highly toxic and causing high mortality in the
treated animals [137]. Like sodium nitroprusside, when injected i.c.v. at doses between 10
and 100 µg hydroxylamine also induces collateral effects such as convulsions that mask the
appearance of penile erection in male rats. However, the compound is able to induce penile
erection when injected directly into the PVN at doses between 10 and 50 µg, which failed
to induce convulsions or other abnormal behaviors [103,113]. Unexpectedly, S-nitroso-D-
acetyl-penicillamine is found able to induce dose-dependent gross behavioral changes, e.g.,
excitation, hypermotility, sniffing and rearing, which mask the expression of penile erection
when given i.c.v. at doses up to 100 µg in male rats. The compound is also ineffective
when injected into the PVN at the dose of 10 µg. Higher doses have not been tested for
the insolubility of the compound in physiological solvents and even in organic solvents
(DMSO) [103]. Thus, it is unknown if this NO donor induces penile erection or not in male
rats. As recalled above, many other NO donors have been synthesized and a few also tested
in in vivo and in in vitro experiments in penile tissues, such as 3-morpholino-sydnonimine
hydrochloride (SIN-1) or z-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-
1,2-diolate (PAPA/NONOate), but to our knowledge, not tested at the central level for
their effect on penile erection. This is true also for new NO donors under development
for the treatment of erectile dysfunction [138,139]. Intriguingly, some of these are also
light-controllable, e.g., when administered in the cavernous corpora, they can be activated
by light to release NO that becomes immediately available for the activation of guanylate
cyclase, thereby increasing cGMP that induces the cavernous corpora relaxation and penile
erection [140].
Biomolecules 2021, 11, 1866 10 of 40

Table 2. Effect of L- and D-arginine and NO donors given i.c.v. or into the PVN on penile erection in
male rats.

Penile Erection
NO Donor Given i.c.v. Into the PVN
L -arginine == ↑
D -arginine == ==
Nitroglycerin ↑ ↑
Sodium nitroprusside == ↑
Isoamyl nitrite ↑ n.a.
Hydroxylamine ↑ n.a.
S-nitroso-D-acetyl-penicillamine n.a. n.a.
↑ = increase; == = no effect; n.a. = not available. Details are found in [37,85,86,103,141].

2.2.3. Dopamine Agonists, Oxytocin, NMDA, Hexarelin Analogue Peptides, VGF-Related

Peptides Induce Penile Erection by Increasing NO Production in the PVN of Male Rats
The ability of NO synthase inhibitors given into the PVN to prevent apomorphine-,
oxytocin-, NMDA-, hexarelin analogue peptide- and VGF derived peptide-induced pe-
nile erection in male rats is in line with the hypothesis that the stimulation by the above
substances of their own receptors in the PVN is related to a consequent activation of NO
synthase in the PVN. Support for this possibility is obtained by measuring the production
of NO in the PVN in vivo. This is achieved by measuring the concentration of the reaction
products of newly formed NO with O2 and H2 O, NO2 − and NO3 − (nitrites and nitrates),
which represent an indirect but reliable indicator of NO production in vivo [10,104,142], in
the dialysate collected from a vertical microdialysis probe implanted in the PVN of male
rats, after the administration of the above substances. As expected, and as summarized
in Table 3, apomorphine-, oxytocin-, NMDA-, hexarelin analogue peptide-, VGF derived
peptide- and SR 141766A-induced penile erection occur concomitantly to an increase in
NO production in the PVN, as demonstrated by the increased concentration of NO2 − and
NO3 − found in the PVN dialysate [89–91,93,96,97,104,143]. The increased NO production
induced by the above compounds was reduced/abolished by the prior inhibition of neu-
ronal NO synthase by L-NAME or S-methyl-thio-citrulline given in the PVN of male rats
at a dose that also reduces/abolishes the sexual response [89–91,93,96,97,104,143]. The
prevention/reduction of the increase in NO production and of the concomitant erectile
response induced by each one of the above compounds is usually also obtained with the
selective blockade of the receptors on which each compound acts in the PVN to increase
NO production and penile erection; that is, apomorphine responses are selectively abol-
ished by the blockade of PVN dopaminergic receptors of the D2 family (e.g., receptors
of the D2 and D4 subtype), for instance with haloperidol, sulpiride, cis-flupentixol or
L-745,870 (3-[4-(4-chlorophenyl)piperazin-1-ylmethyl]-1H-pyrrolo [2,3-b]pyridine trihy-
drochloride), a selective D4 receptor antagonist [98,99,104,144–146], but not with SCH
23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro- 1H-3-benzazepine
hydro chloride)], which blocks D1 receptors [97,140], the oxytocin effect by the selec-
tive blockade of oxytocinergic receptors with d(CH2 )5 Tyr(Me)-Orn8 -vasotocin, the effect
of NMDA by the selective blockade of NMDA receptors with (+)-MK-801 (dizocilpine,
[(5R,10S)-(+)-5-methyl-10,11-dihydro-5Hdibenzo-[a,d]cyclohepten-5,10-imine hydrogen
maleate]), a potent non-competitive NMDA receptor antagonist [147], but not with CNQX
(6-cyano-7-nitro-quinoxaline-2,3-dione), an AMPA receptor antagonist [96,97]. This mecha-
nism cannot be proved for hexarelin analogue peptide- and VGF-related peptide-induced
penile erection and the concomitant increase in NO production, as no selective antagonist
is available so far of the putative receptors on which these two classes of compounds act,
although some evidence for the existence of a receptor specific for hexarelin analogues that
mediates penile erection and different from those that mediate growth hormone release
Biomolecules 2021, 11, 1866 11 of 40

induced by these peptides has been obtained in male rats [73,90,91,148]. Conversely, the
increase in PVN NO production induced by apomorphine, NMDA, hexarelin analogue
peptides and VGF derived peptides is not reduced/abolished by d(CH2 )5 Tyr(Me)-Orn8 -
vasotocin given i.c.v. despite its ability to reduce/abolish penile erection induced by these
compounds and by oxytocin as well (Figure 2) [88–91,93,96,97,104,123].
Table 3. Dopamine agonists (of the D2 and D4 receptor subtype), oxytocin, NMDA, hexarelin
analogue peptides, VGF-related peptides injected into the PVN of male rats induce penile erection
and increase NO production in the PVN: effect of NO synthase inhibitors (L-NAME or SMTC) and/or
drug/peptide receptor antagonists given in the PVN or i.c.v. when indicated.

PVN Pretreatment PVN Treatment NO Production Penile Erection

Vehicle/Drug/Peptide Drug/Peptide
L-NAME/SMTC DA agonists ↓ ↓
Haloperidol DA agonists ↓ ↓
SCH 23390 DA agonists == ==
Oxy-Ant apomorphine == ==
OXY-Ant i.c.v. apomorphine == ↓
(+)MK-801 apomorphine == ==
L-NAME/SMTC oxytocin ↓ ↓
Haloperidol oxytocin == ==
Oxy-Ant oxytocin ↓ ↓
Oxy-Ant i.c.v. oxytocin ↓ ↓
(+)MK-801 oxytocin == ==
L-NAME/SMTC NMDA ↓ ↓
Haloperidol NMDA == ==
Oxy-Ant NMDA == ==
Oxy-Ant i.c.v. NMDA == ↓
(+)MK-801 NMDA ↓ ↓
L-NAME VGF 588-617 ↓ ↓
Cis-flupenthixol VGF 588-617 n.a. ==
Oxy-Ant VGF 588-617 == ==
Oxy-Ant i.c.v. VGF 588-617 == ↓
(+)MK-801 VGF 588-617 n.a. ==
L-NAME EP 80661 ↓ ↓
Cys-flupenthixol EP 80661 n.a. ==
Oxy-Ant EP 80661 == ==
Oxy-Ant i.c.v. EP 80661 == ↓
(+)MK-801 EP 80661 n.a. ==
↓ = prevention; == = no effect; n.a. = not available; DA agonists = dopamine agonists of the D2 receptor family
(D2 and D4 receptor subtype); haloperidol, cys-flupenthixol and SCH23390 are dopamine receptor antagonists of
the D2, D1/D2 and D1 receptors, respectively; L-NAME = NG -nitro-L-arginine methyl ester; SMTC = S-methyl-
thio-citrulline; NMDA = N-methyl-D-aspartic acid, (+)MK-801 is a NMDA receptor antagonist; Oxy-Ant is
d(CH2 )5 Tyr(Me)-Orn8 vasotocin, an oxytocin receptor antagonist; EP 80661 is an hexarelin peptide analogue, VGF
588-671 is a VGF derived peptide. Details are found in [36,37,79–83,88,93,96–99,102,104,143,149,150].

The studies reviewed above show that the activation of NO synthase in the PVN
induced by the stimulation of dopamine, oxytocin and NMDA receptors, and possibly
other receptors present in the PVN of male rats, leads to penile erection. Perhaps more
importantly, the activation of NO synthase takes place in the PVN not only when penile
Biomolecules 2021, 11, 1866 12 of 40

erection is induced by drugs and/or peptides acting in the PVN, but also when this sexual
response occurs in physiological contexts, for instance when sexually potent male rats are
put in the presence of an inaccessible sexually receptive female rat (e.g., when male rats
show non-contact erections) or during copulation, when in copula penile erections occur
(Table 4) (see also Sections 2.2.3 and 3) [54,77,105]. Indeed, an increase in NO production
(measured by an increase in the concentration of NO2 − and NO3 − ) is found in the PVN
dialysate of sexually potent male rats that show non-contact erections in the presence of
an inaccessible receptive female rat and during copulation [77]. That the NO synthase
activation and concomitant increase in NO production in the PVN are important for the
induction of penile erection in physiological contexts is further supported by the ability
of L-NAME, which inhibits NO synthase, injected into the PVN of sexually potent male
rats to prevent not only the increase in NO production in the PVN dialysate but also
to abolish/reduce non-contact erections and impair copulation with a receptive female
rat [77,148].

Table 4. NO production increases in the PVN of male rats during non-contact erections and copula-
tion: reversal by the NO synthase inhibitor L-NAME, GABA agonists and the opiate morphine but
not by methylene blue, hemoglobin, or d(CH2 )5 Tyr(Me)-Orn8 vasotocin (Oxy-Ant) injected into the
PVN, in spite of the ability of the latter to abolish non-contact erections and impair copulation when
given i.c.v.

PVN Treatment NO Production Non-Contact Erections Copulation

L-NAME ↓ ↓ ↓
Muscimol ↓ ↓ ↓
Baclofen == == ==
Morphine ↓ ↓ ↓
U-69,593 == == ==
Methylene Blue == == ==
Hemoglobin == == ==
Oxy-Ant == == ==
Oxy-Ant i.c.v. == ↓ ↓
↓ = prevention; == = no effect; n.a. = not available; L-NAME = NG -nitro-L-arginine methyl ester; musci-
mol is a GABAA receptor agonist, baclofen a GABAB receptor agonist, morphine a µ-opioid receptor ago-
nist, U-69,593 a k-opioid receptor agonist, methylene blue a guanylate cyclase inhibitor, hemoglobin a NO
scavenger and d(CH2 )5 Tyr(Me)-Orn8 vasotocin (Oxy-Ant) an oxytocin receptor antagonist. Details are found
in [77,104,105,107,114–116].

2.2.4. GABA Agonists, Opioid Peptides/Opiates and Cannabinoids Reduce Spontaneous

or Drug- and Peptide-Induced Penile Erection by Inhibiting NO Synthase in the PVN of
Male Rats
Changes in the activity of NO synthase in the PVN of male rats also occur when the
pro-erectile effect of dopamine agonists, oxytocin, NMDA, hexarelin analogue peptides
and VGF-related peptides, on penile erection—or when penile erection occurs in physio-
logical contexts (the presence of an inaccessible receptive female rat and copulation)—is
reduced or abolished by drugs that act in the PVN on GABAA receptors, opioid receptors
of the µ subtype and cannabinoid receptors of the CB1 subtype. Accordingly, the increase
in NO production that occurs during non-contact erections and copulation or when in-
duced by the above drugs and/or peptides given at doses that induce penile erection is
reduced/abolished by the injection into the PVN of muscimol, a GABAA receptor agonist,
but not by blacofen, a GABAB receptor agonist [105–107], by morphine, a µ-opioid receptor
agonist, but not by the k-opioid receptor agonist U-69,593 [106,108–111], given at doses
that abolish penile erection without inducing other gross behavioral changes (Figure 2,
Tables 4 and 5). As muscimol effects are abolished by bicuculline, a GABAA receptor
antagonist [105–107], and morphine effects are abolished by naloxone, a µ-opioid receptor
Biomolecules 2021, 11, 1866 13 of 40

antagonist [77,109–111], these results show that muscimol reduces the increase in NO syn-
thase activity that leads to penile erection by stimulating GABAA receptors and morphine
by stimulating µ-opioid receptors in the PVN.

Table 5. Dopamine agonists (of the D2 and D4 receptor subtype), oxytocin, NMDA, hexarelin
analogue peptides, VGF-related peptides injected into the PVN induce penile erection and increase
NO production in the PVN of male rats: reversal by GABA agonists and the opiate morphine injected
into the PVN.

PVN Pretreatment PVN Treatment NO Production Penile Erection

Vehicle/Drug/Peptide Drug/Peptide
Muscimol DA agonists ↓ ↓
Baclofen DA agonists == ==
Morphine DA agonists ↓ ↓
U-69,593 DA agonists == ==
Muscimol oxytocin ↓ ↓
Baclofen oxytocin == ==
Morphine oxytocin ↓ ↓
U-69,593 oxytocin == ==
Muscimol NMDA ↓ ↓
Baclofen NMDA == ==
Morphine NMDA ↓ ↓
U-69,593 NMDA n.a. ==
Muscimol VGF 588-617 ↓ ↓
Baclofen VGF 588-617 n.a. ==
Morphine VGF 588-617 n.a. ↓
U-69,593 VGF 588-617 n.a. ==
Muscimol EP 80661 ↓ ↓
Baclofen EP 80661 n.a. ==
Morphine EP 80661 ↓ ↓
U-69,593 EP 80661 n.a. ==
↓ = prevention; == = no effect; n.a. = not available. DA agonists are dopamine receptor agonists of the D2
receptor family (D2 and D4 receptor subtype); NMDA N-methyl-D-aspartic acid; muscimol a GABAA receptor
agonist, baclofen a GABAB receptor agonist, morphine a µ-opioid receptor agonist, U-69,593 a k-opioid recep-
tor agonist. EP 80661 a hexarelin peptide analogue, VGF 588-617 a VGF derived peptide. Details are found
in [106,108–112,116].

Changes in NO synthase activity at the level of the PVN are also involved in the
inhibitory effect of cannabinoid receptors of the CB1 subtype on penile erection. This
became evident when a selective antagonist of the CB1 receptor subtype SR 141716A [94],
was found capable of inducing penile erection when injected into the PVN of male rats [95].
Apparently, the blockade of CB1 receptors in the PVN induces penile erection by activating
glutamatergic neurotransmission in the PVN. Glutamic acid, in turn, increases Ca2+ influx
in the cell bodies of PVN oxytocinergic neurons causing the activation of NO synthase
and the increase in NO production leading to penile erection (Figure 3). Accordingly, SR
141716A-induced penile erection occurs with an increase in extracellular glutamic acid
and NO production in the male rat PVN dialysate [100,101]. Penile erection and the in-
crease in NO production, but not in extracellular glutamic acid induced by SR 141716A,
are antagonized by the blockade of NMDA receptors in the PVN with (+)-MK-801, and
by the inhibition of NO synthase in the PVN by L-NAME [95,100,101]. In contrast, SR
141716A-induced penile erection, increased NO production and glutamic acid release in the
Biomolecules 2021, 11, 1866 14 of 40

male rat PVN dialysate were all antagonized by morphine [112]. As to the activation of glu-
tamatergic neurotransmission that causes penile erection by stimulating PVN oxytocinergic
neurons mediating penile erection secondary to the blockade of CB1 receptors in the PVN,
this may be due to CB1 receptors that directly inhibit glutamatergic synapses impinging on
oxytocinergic neurons mediating penile erection or to CB1 receptors located in inhibitory
GABAergic synapses impinging on glutamatergic synapses and whose blockade decreases
the GABA-mediated inhibition of these synapses [100,101,112,113] (Figure 3). Further
studies are required to discover if one of the two mechanisms is the most relevant or if
the two mechanisms are not mutually exclusive. Irrespective of the exact mechanisms by
which cannabinoids inhibit erectile function, this inhibitory effect is mediated at least in
part by a reduction in NO activity at PVN level [100,101,112,113].

2.3. NO, Erectile Function and Extrahypothalamic Brain Areas

Recent studies have shown that in male rats, NO is involved in the control of erectile
function not only at the level of the PVN but also in other brain areas. Among these,
the best known are the ventral tegmental area, the posteromedial cortical nucleus of the
amygdala, the bed nucleus of the stria terminalis and the spinal cord (Table 6).

Table 6. NO facilitates erectile function and sexual behavior in rats by acting in different brain areas and with different
mechanisms: a brief summary.

Brain Area NO Synthesis Site Mechanism of Action

Cell bodies of oxytocinergic neurons Activation of central oxytocin neurotransmission apparently by
PVN projecting to extrahypohalamic brain acting as intracellular messenger and with a mechanism that
areas and spinal cord does not involve guanylate cyclase yet to be identified.
Activation of dopamine release by insert hypothalamic
Medial preoptic cells yet to be
Medial preoptic area dopaminergic neurons by acting as a retrograde messenger with
identified
a mechanism that involves the activation of guanylate cyclase.
Ventromedial nucleus Activation of the lordosis response by a still unknown
Neurons yet to be identified
of the hypothalamus mechanism.
Activation of mesolimbic dopaminergic neurons projecting to
Cell bodies of
the nucleus accumbens and medial prefrontal cortex by acting
Ventral tegmental area mesolimbic/mesocortical
as intracellular messenger with a mechanism that involves the
dopaminergic neurons
activation of guanylate cyclase.
Activation of glutamatergic neurons projecting directly or
indirectly (through the medial prefrontal cortex) to the ventral
Ventral subiculum of Glutamatergic neurons of the
tegmental area in order to activate mesolimbic dopaminergic
the hippocampus subiculum
neurons by acting as intercellular messenger with a mechanism
involving guanylate cyclase.
Activation of glutamatergic neurons projecting directly or
Posteromedial cortical indirectly (through the medial prefrontal cortex) to the ventral
Glutamatergic neurons of the
nucleus of the tegmental area in order to activate mesolimbic dopaminergic
amygdalar nucleus
amygdala neurons by acting as intercellular messenger, possibly with a
mechanism involving guanylate cyclase.
Activation of glutamatergic neurons projecting to the PVN and
Bed nucleus of the Glutamatergic neurons of the bed other extrahypothalamic brain areas mediating erectile function.
stria terminalis nucleus It is still unknown if NO acts as intercellular or intracellular
messenger and if guanylate cyclase is involved or not.
Ventral medulla
5-HT cell bodies of neurons projecting Activation of 5HT neurons projecting from the ventral medulla
(nucleus
to the spinal cord (L2-S2)? to the spinal tract L2-S2, with a still-unknown mechanism?
paragigantocellularis
Activation of spinal neurons projecting to the pelvic plexuses
Spinal cord Spinal neurons of the L2-S2 spinal and pudendal nerves that reach the penis to control cavernous
(L2-S2) tract corpora relaxation and the muscles at the basis of the penis
involved in penile reflexes and reflex erection.
Biomolecules 2021, 11, 1866 15 of 40

In the ventral tegmental area, NO plays a role in the activation of mesolimbic dopamin-
ergic neurons induced by the injection of oxytocin apparently with a mechanism similar
only in part to that found in the PVN. Accordingly, the available data suggest that oxy-
tocin injected into the caudal part of the ventral tegmental area of male rats stimulates
oxytocinergic receptors located in the cell bodies of mesolimbic dopaminergic neurons.
This causes an increased Ca2+ influx inside the cell bodies of these dopaminergic neurons,
thereby activating neuronal NO synthase. At variance from the PVN, newly formed NO, in
turn, activates guanylate cyclase, which is co-localized with NO synthase in the cell bodies
of dopaminergic neurons. As discussed below, this increases cGMP levels and activates
mesolimbic dopamine neurons to release dopamine in the nucleus accumbens causing
penile erection [47,48] (see also Section 2.4.1, Section 2.4.3, Section 3, and Section 3.1.1).
NO is also involved in the induction of penile erection induced by oxytocin injected
into the ventral subiculum of the hippocampus, the posteromedial cortical nucleus of
the amygdala [49–51] and the bed nucleus of the stria terminalis [151,152] of male rats.
Accordingly, oxytocin injected into the ventral subiculum and the posteromedial cortical
nucleus of the amygdala induces penile erection that occurs with a concomitant activation
of NO synthase and an increase in NO production, as measured by the increased NO2 −
and NO3 − levels in the dialysate obtained from these two brain areas. In the ventral
subiculum, newly produced NO acts as an intercellular messenger and activates by a
mechanism still unknown excitatory (glutamatergic) efferent, yet to be identified, direct
or indirect projections from this area to the ventral tegmental area. Here, the increase
in extracellular glutamic acid activates mesolimbic dopaminergic neurons leading to an
increased release of dopamine in the nucleus accumbens [49–51], as reported above for
oxytocin injected into the ventral tegmental area of male rats [47,48]. The activation of
NO synthase in the ventral subiculum is necessary for the pro-erectile effect of oxytocin,
as this is abolished by NO synthase inhibition. It is likely that a mechanism similar to
that described above for the ventral subiculum operates also in the posteromedial cortical
nucleus of the amygdala. Oxytocin induces penile erection also when injected in the bed
nucleus of the stria terminalis of male rats by increasing extracellular glutamic acid that
acts on NMDA receptors located in glutamatergic neurons containing NO synthase, which
project from the bed nucleus to the PVN and other extrahypothalamic areas controlling
penile erection. As for the ventral subiculum and the posteromedial cortical nucleus, the
activation of NO synthase is required for the pro-erectile effect of oxytocin injected in
this nucleus, since it is abolished by S-methyl-thio-citrulline injected into the bed nucleus
before oxytocin [151,152]. As for the posteromedial cortical nucleus of the amygdala, it is
unknown whether NO in this area acts as intercellular or intracellular messenger and if
guanylate cyclase is the main target responsible for penile erection induced by the injection
of oxytocin.
Finally, NO may also play a role in the control of penile erection at the level of
the ventral medulla and the spinal cord. Accordingly, as recalled above (Section 2.1),
inhibition by NO synthase by L-NAME given i.c.v. but not in the PVN of male rats
prevents penile erection induced by 5-HT1C receptor agonists when injected i.c.v. with
a mechanism that does not involve oxytocin [82,83]. This raises the possibility that NO
synthase inhibitors prevent penile erection induced by these compounds by acting at sites
located downstream to oxytocinergic neurons in a yet undiscovered brain area. In this
regard, it is pertinent to recall that NO synthase is found localized in neurons of the ventral
medulla [103] as well as in the spinal cord [52] of male rats. Since electrolytic lesions of the
nucleus paragigantocellularis of the reticular formation of the ventral medulla facilitate
penile reflexes and copulatory behavior in male rats [153] and drugs that enhance 5-HT
transmission, especially 5-HTA receptor agonists, impair these sexual responses [154–156],
it is tempting to speculate that NO synthase inhibitors prevent 5-HT1C receptor agonist-
induced penile erection by inhibiting NO synthase in the ventral medulla or in the spinal
cord [83].
Biomolecules 2021, 11, 1866 16 of 40

2.4. Facilitation of Erectile Function by Central NO: Mechanism of Action

The studies reviewed above made in male rats show that NO facilitates erectile
function at the central level by means of mechanisms which may be different depending
on the brain area considered. Among these, the most studied is the activation of central
oxytocinergic neurotransmission at the level of the PVN. Here and in other brain areas,
numerous experiments aimed at identifying the molecular mechanism activated by NO
to facilitate penile erection have been performed, but only in the ventral tegmental area
has convincing evidence for a specific mechanism (e.g., activation of guanylate cyclase)
been obtained so far. However, some hypotheses on how NO facilitates erectile function
when injected into the ventral subiculum have been also proposed, as conducted for
the PVN and the ventral tegmental area of male rats where NO acts as an intracellular
messenger to facilitate erectile function while it acts as an intercellular messenger in the
ventral subiculum.

2.4.1. NO Facilitates Erectile Function in Male Rats by Activating Central

Oxytocinergic Neurotransmission
As extensively discussed above, (i) numerous compounds (i.e., dopamine receptor
agonists, oxytocin, NMDA, hexarelin analogue peptides, VGF-derived peptides and even
the CB1 receptor antagonist SR 141716A) induce penile erection and concomitantly in-
crease NO production in the PVN of male rats; (ii) both these behavioral and biochemical
responses are prevented by the inhibition of neuronal NO synthase, which may be direct
with non-selective and selective competitive inhibitors, or indirect through the stimulation
of GABAA , µ-opioid and CB1 cannabinoid receptors in the PVN; (iii) the NO synthase
inhibition-induced prevention of drug-and peptide-induced responses is usually reversed
by the concomitant administration of L-arginine, the physiological substrate of the enzyme
and (iiii) the increase in PVN NO production that occurs during non-contact erection and
copulation is antagonized by the inhibition of PVN NO synthase by L-NAME and by the
blockade of oxytocin receptors with d(CH2 )5 Tyr(Me)-Orn8 -vasotocin given i.c.v. but not
in the PVN (Tables 3 and 4). These findings provide convincing evidence that in male
rats NO plays a key role in the control of erectile function at central level. Such a role is
further strengthened by the ability of NO donors injected in the PVN to induce penile
erection episodes indistinguishable from those induced by the above substances. More
importantly, penile erection induced by oxytocin, dopamine agonists, NMDA, hexarelin
analogue peptides and VGF-derived peptides are prevented not only by NO synthase
inhibition (see above) but also by the blockade of oxytocinergic receptors with the oxytocin
receptor antagonist d(CH2 )5 Tyr(Me)-Orn8 -vasotocin given i.c.v., in line with the hypothesis
that these compounds induce penile erection by activating the central oxytocinergic neuro-
transmission (Figure 2) [53,73]. Since d(CH2 )5 Tyr(Me)-Orn8 -vasotocin given i.c.v. but not in
the PVN also antagonizes the pro-erectile effect of NO donors in male rats (Table 7), these
findings make it reasonable to assume that the sexual response induced by NO donors
injected into the PVN is also mediated by the activation of the same oxytocinergic neurons
projecting to extrahypothalamic brain areas, which are activated by dopamine receptor
agonists, NMDA, hexarelin analogue peptides, VGF-derived peptides and oxytocin itself.
In agreement with this hypothesis, penile erection induced by nitroglycerin, sodium ni-
troprusside and hydroxylamine injected in the PVN or by isoamyl nitrite given i.c.v. are
prevented by d(CH2 )5 Tyr(Me)-Orn8 -vasotocin given i.c.v. but not in the PVN at the dose
of 0.1 µg, 10 min before the NO donors [85,103]. Conversely, nitroglycerin-induced penile
erections are not prevented by haloperidol (0.5 mg/kg i.p. 30 min before the NO donor),
suggesting that the NO donor effect on penile erection is not mediated by dopamine in
the PVN or in other brain areas [84]. It is likely that haloperidol does not prevent the
pro-erectile response induced by the other NO donors found active in inducing penile
erection, but experiments supporting this hypothesis are not available to our knowledge,
since no data supporting this hypothesis have been found in the PubMed and Google
Scholar medlines made by the authors when writing this review.
Biomolecules 2021, 11, 1866 17 of 40

Table 7. Effect of L- and D-arginine and NO donors given i.c.v. or into the PVN or after pretreatment
with the NO synthase inhibitor L-NAME or the oxytocin receptor antagonist d(CH2 )5 Tyr(Me)-Orn8 -
vasotocin (Oxy Ant) given i.c.v or into the PVN on penile erection in male rats.

Penile Erection
i.c.v. Pretreatment PVN Pretreatment NO Donor Given i.c.v PVN
Vehicle Vehicle
Drug Drug
Peptide Peptide
Saline D -arginine ==
Saline D -arginine ==
Saline L -arginine ==
Saline L -arginine ↑
Oxy-Ant L -arginine ↓
Oxy-Ant L -arginine ↑
L-NAME L -arginine ↓
L-NAME L -arginine ↓
Vehicle Nitroglycerin ↑
Vehicle Nitroglycerin ↑
Oxy-Ant Nitroglycerin ↓
Oxy-Ant Nitroglycerin ↑
L-NAME Nitroglycerin ↑
L-NAME Nitroglycerin ↑
vehicle SNP ↑
Oxy-Ant SNP ↓
Oxy-Ant SNP ↑
L-NAME SNP ↑
L-NAME SNP ↑
Vehicle Isoamyl nitrite ↑
Oxy-Ant Isoamyl nitrite ↓
L-NAME Isoamyl nitrite ↑
Vehicle Hydroxylamine ↑
Oxy-Ant Hydroxylamine ↓
L-NAME Hydroxylamine ↑
↓ = prevention; ↑ increase; == = no effect; n.a. = not available. SNP = sodium nitroprusside. Details are found in
[37,85,103,136].

2.4.2. Is Guanylate Cyclase the NO Target in the PVN?

The studies reviewed above support the hypothesis that either endogenous NO
formed by the stimulation of dopamine, NMDA, oxytocin, hexarelin analogue peptide
and VGF-derived peptide receptors in the PVN, or by NO donors injected in the PVN,
activates oxytocinergic transmission to induce penile erection in male rats. Unfortunately,
these studies do not provide any evidence about the mechanism by means of which NO
activates oxytocinergic neurons. However, these studies suggest that guanylate cyclase,
one of the best-known targets of NO in peripheral tissues and in several brain areas as
well [5,11,12,25,157] is not involved in the facilitatory effect of NO on erectile function in
the PVN. Accordingly, the microinjection into the PVN of methylene blue and LY 83583
[6-(phenylamino)-5,6-quinoline-dione], two putative inhibitors of this enzyme [158,159]
Biomolecules 2021, 11, 1866 18 of 40

at doses between 5–20 µg are unable to prevent penile erection induced by apomorphine,
oxytocin, NMDA or NO donors in male rats (Table 8) [79,80,85,103]. This suggests that
NO acts in the PVN on other targets in order to activate oxytocinergic neurons projecting
to extra-hypothalamic brain areas and the spinal cord mediating penile erection. In this
regard it is pertinent to recall that NO might interact with numerous other enzymes that,
like guanylate cyclase, bind metal ions such iron, and that other targets of NO, such as
cellular ADP-ribosyl-transferases, have been identified (reviews on other targets of NO
are found in [4,12,26]). However, this interpretation is complicated in part by the ability of
both methylene blue (100–400 µg) and LY 83583 (100–200 µg) to prevent penile erection
induced by apomorphine, oxytocin, NMDA, NO donors and 5HT1C receptor agonists
when injected i.c.v. in male rats (Table 8) [79,80,85,103]. One possible explanation for
this discrepancy is that cGMP is involved in the expression of penile erection induced
by the above substances in some yet undiscovered brain area distant from the PVN. In
agreement with this possibility, methylene blue given i.c.v. is unable to prevent the increase
in NO production in the PVN induced by apomorphine, oxytocin and NMDA despite its
ability to prevent penile erection (Table 8). That guanylate cyclase might not be the target
of NO in the PVN for the induction of penile erection is also suggested by experiments
showing that (i) 8-Bromo-cGMP, an active and stable (phosphodiesterase-resistant) cGMP
analog that would be expected to mimic the effect of endogenous cGMP, is unable to
induce penile erection when injected into the PVN at doses between 0.1 and 50 µg in
male rats [96,103,104,143,160], and (ii) methylene blue injected into the PVN is unable
to reduce/abolish both non-contact erections in sexually potent male rats put into the
presence of an inaccessible sexually receptive female rats and copulation when in copula
erections take place and the concomitant increase in NO production that occurs in the PVN
in these physiological contexts (Table 4) [76] (see also Sections 2.2.3 and 3). In this regard, it
must be noted that a NO-cGMP signaling pathway might not exist or be active in the PVN of
male rats, although such pathway has been well characterized not only by biochemical but
also by immunocytochemical studies in other brain areas, such as the hippocampus and the
cerebellum [25]. In agreement with this hypothesis, the PVN of male rats contains only very
low amounts of guanylate cyclase immunoreactivity [25] and PVN oxytocinergic neurons,
labeled by NO synthase-directed antibodies, are not labeled by guanylate cyclase-directed
antibodies [29]. Irrespective of the mechanism by which NO acts in the PVN to activate central
oxytocinergic neurotransmission, as will be discussed below, together with other findings
these results also support the hypothesis that NO facilitates penile erection by acting as an
intracellular messenger in the male rat PVN (Figure 4) (see Section 2.4.4).

Table 8. PVN drug-, peptide- and NO donor-induced penile erection and PVN NO production in male rats: effects of guanylate
cyclase (GC) inhibitors (methylene blue or LY 83583) and the NO scavenger hemoglobin given i.c.v. or into the PVN.

Penile Erection NO Production

Drug, Peptide, NO Donor into the PVN GC Inhibitors Hemoglobin GC Inhibitors Hemoglobin
Given Given
i.c.v. PVN i.c.v. PVN i.c.v. PVN i.c.v. PVN
DA agonists ↓ == == == == == ↓ ↓
Oxytocin ↓ == == == == == ↓ ↓
NMDA ↓ == == == == == ↓ ↓
EP 80661 ↓ == == == n.a. n.a. ↓ ↓
VGF 588-617 ↓ == == == n.a. n.a. ↓ ↓
5-HT1C agonists ↓ == == == n.a. n.a. ↓ ↓
NO donors ↓ == == == n.a. n.a. ↓ ↓
↓ = prevention; == = no effect; n.a. = not available. GC inhibitors for methylene blue or LY 83586, DA agonists for dopamine receptor
agonists of the D2 family (D2 and D4 receptor subtype); NMDA for N-methyl-D-aspartic acid; EP 80661 is a hexarelin peptide and VGF
678-617 is a VGF peptide. Details are found in [82,83,85,86,90–93,96,97,103,104,143].
Biomolecules 2021, 11, 1866 19 of 40
Biomolecules 2021, 11, x 20 of 42

Figure 4. NO influences penile erection by acting in several rat brain areas including the PVN, the
Figure 4. NO influences penile erection by acting in several rat brain areas including the PVN, the
ventral tegmentalarea,
ventral tegmental area,thetheventral
ventralsubiculum
subiculum of of the
the hippocampus,
hippocampus, the the posteromedial
posteromedial cortical
cortical nucleus
nucleus
of the amygdala and the bed nucleus of the stria terminalis. In these
of the amygdala and the bed nucleus of the stria terminalis. In these areas NO acts by means of dif- areas NO acts by means of
different mechanisms. In the PVN, NO activates oxytocinergic neurons
ferent mechanisms. In the PVN, NO activates oxytocinergic neurons projecting to the spinal cord that projecting to the spinal cord
mediate penile
that mediate erection
penile and to
erection extrahypothalamic
and to extrahypothalamic areasareas
including the ventral
including tegmental
the ventral areaarea
tegmental and and
the
posteromedial cortical nucleus of the amygdala. Here, NO acts intracellularly
the posteromedial cortical nucleus of the amygdala. Here, NO acts intracellularly in the neurons in in the neurons in which
itwhich
is formed by a yetby
it is formed unknown mechanism
a yet unknown unrelated
mechanism to the guanylate
unrelated cyclase-cGMP
to the guanylate pathway.
cyclase-cGMP Addi-
pathway.
tionally, in the ventral tegmental area (VTA), NO produced by the activation
Additionally, in the ventral tegmental area (VTA), NO produced by the activation of NO synthase of NO synthase local-
ized in the cell bodies of mesolimbic-mesocortical dopaminergic neurons acts intracellularly in the
localized in the cell bodies of mesolimbic-mesocortical dopaminergic neurons acts intracellularly
dopaminergic neurons in which is produced by the activation of oxytocinergic receptors that induce
in the dopaminergic neurons in which is produced by the activation of oxytocinergic receptors
penile erection. However in these neurons, at variance from the PVN, NO activates guanylate cyclase
tothat induce
increase penile
cGMP erection.
levels, whichHowever
lead to theinactivation
these neurons, at variance
of dopaminergic from the
neurons PVN, NO
to release activates
dopamine in
guanylate
the nucleuscyclase
accumbens to increase
and incGMP levels,prefrontal
the medial which lead to the
cortex andactivation
to induceofpeniledopaminergic
erection. neurons
It is likelyto
release
that dopaminepathway
a NO-cGMP in the nucleus
is also accumbens
operative inand the in the medial
ventral prefrontal
subiculum (VS) andcortex
theand to induce penile
posteromedial cor-
tical nucleus
erection. It isoflikely
the amygdala (Am) in pathway
that a NO-cGMP neurons activated by oxytocin
is also operative in the given in these
ventral areas at
subiculum a dose
(VS) and
that induces penile erection. At variance from the ventral tegmental area,
the posteromedial cortical nucleus of the amygdala (Am) in neurons activated by oxytocin given in here NO acts after being
released
these areas fromat athe
doseneurons in which
that induces is formed
penile erection. as At
an variance
intercellular
frommessenger
the ventraltotegmental
activate excitatory
area, here
neurons projecting directly or indirectly to the ventral tegmental area
NO acts after being released from the neurons in which is formed as an intercellular messenger in order to activate mesolimbic to
and mesocortical dopaminergic neurons to release dopamine in the nucleus accumbens and in the
activate excitatory neurons projecting directly or indirectly to the ventral tegmental area in order
medial prefrontal cortex and induce penile erection. In the bed nucleus of the stria terminalis (BNST),
to activate mesolimbic and mesocortical dopaminergic neurons to release dopamine in the nucleus
oxytocin releases glutamic acid that acts on the cell bodies of glutamatergic neurons containing NO
accumbens
synthase andand in the medial
projecting to the prefrontal
PVN, ventral cortex and induce
tegmental area,penile erection. and
hippocampus In the bed nucleus
amygdala. HowofNO the
stria terminalis
formed in the bed (BNST),
nucleusoxytocin
acts toreleases glutamic
facilitate erectile acid that acts
function on unknown.
is still the cell bodies of glutamatergic
Details are found in
neurons containing NO synthase and projecting to the PVN, ventral tegmental area, hippocampus
[47–51,53–57,96–99,143,144,151,152,160].
and amygdala. How NO formed in the bed nucleus acts to facilitate erectile function is still unknown.
2.4.3.
Details Guanylate
are found in Cyclase Is the NO Target in the Ventral Tegmental Area of Male Rats
[47–51,53–57,96–99,143,144,151,152,160].

2.4.3.AtGuanylate
variance from theIsPVN,
Cyclase theTarget
the NO available data
in the suggest
Ventral that NO,Area
Tegmental produced byRats
of Male the ac-
tivation of NO synthase located in the cell bodies of mesolimbic dopaminergic neurons
At variance from the PVN, the available data suggest that NO, produced by the activa-
by the increased Ca2+ influx that takes place after activation of oxytocinergic receptors
tion of NO synthase located in the cell bodies of mesolimbic dopaminergic neurons by the
expressed in 2+these cell bodies by oxytocin, activates guanylate cyclase in the ventral
increased Ca influx that takes place after activation of oxytocinergic receptors expressed
tegmental area of male rats in order to facilitate penile erection induced by oxytocin in-
in these cell bodies by oxytocin, activates guanylate cyclase in the ventral tegmental area
jected into this mesencephalic area [47,48] (Table 9). In agreement with this hypothesis, (i)
of male rats in order to facilitate penile erection induced by oxytocin injected into this
the erectile response induced by oxytocin injected into the male rat caudal ventral teg-
mesencephalic area [47,48] (Table 9). In agreement with this hypothesis, (i) the erectile
mental area occurs concomitantly to an increased NO production and both behavioral
Biomolecules 2021, 11, 1866 20 of 40

response induced by oxytocin injected into the male rat caudal ventral tegmental area
occurs concomitantly to an increased NO production and both behavioral and neuro-
chemical response are antagonized not only by the blockade of oxytocinergic receptors by
d(CH2 )5 Tyr(Me)-Orn8 -vasotocin but also by the neuronal NO synthase inhibitor S-methyl-
thio-citrulline injected into the caudal ventral tegmental area before oxytocin [47,160];
(ii) the erectile response but not the increase in NO production is also antagonized by ODQ
(1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one), a potent inhibitor of guanylate cyclase [25],
injected into the caudal ventral tegmental area before oxytocin [47,160]; (iii) 8-bromo-cGMP
induces penile erection episodes (indistinguishable from those induced by oxytocin) when
injected into the caudal ventral tegmental area of male rats with a U-inverted dose response
curve, being the maximum response found at the dose of 3 µg [47,48]. Further support for
the involvement of the NO-cGMP signaling pathway in the induction of penile erection
by oxytocin in the ventral tegmental area of male rats comes from immunohistochemistry
experiments showing that the ventral tegmental area contains mesolimbic dopaminergic
cell bodies that are labeled for both NO synthase and guanylate cyclase (the two enzymes
are located in the same cell body), and which run close to oxytocinergic fibers originating
from the PVN [47]. These results support the hypothesis that NO synthase and guanylate
cyclase co-exist in the cell bodies of mesolimbic dopaminergic neurons whose activation fa-
cilitates penile erection. In agreement with this hypothesis, experimental evidence showing
that NO synthase and guanylate cyclase may also coexist in specific neuronal populations
is also available, especially in mesencephalic structures, favoring an intracellular autocrine
role of a NO-cGMP pathway in these neurons [161] (Figure 4). This is in spite of the fact
that NO synthase and guanylate cyclase are often found in neurons juxtaposed with NO
synthase in the cell bodies of and guanylate cyclase in nerve endings of excitatory (often
glutamatergic) neurons that impinge on the NO synthase containing cell bodies in many
parts of the CNS [6,12,25]. In these areas NO is commonly accepted to act as a retrograde
messenger molecule in many neural processes mainly related to long-term potentiation
and/or depression [162]. Together with the inability of the NO scavenger hemoglobin
to inhibit penile erection despite its ability to reduce NO2 concentration in the dialysate
obtained from the caudal ventral tegmental area of male rats, these findings confirm that
NO acts mainly as an intracellular messenger in the ventral tegmental area, as it will be
discussed below (see Section 2.4.4). That cGMP may have a role in facilitating erectile
function at level of the ventral tegmental area is also supported by the ability of sildenafil
and vardenafil, two orally active phosphodiesterase type 5 inhibitors clinically used for
the therapy of erectile dysfunction, to increase the number of non-contact erections in rats
screened for showing or not this sexual response compared to control rats when injected
directly into the ventral tegmental area [163]. Accordingly, the two drugs are expected
to increase cGMP levels in the ventral tegmental area (due to the inhibition of cGMP
degradation by phosphodiesterase type V). Interestingly, in this physiological context, the
increase in the number of non-contact erections takes place concomitantly to an increase
in extracellular dopamine in the dialysate obtained from the nucleus accumbens of these
rats [163], as found with oxytocin injected into the VTA [47,48].
A NO-cGMP signaling pathway might play a role in the induction of penile erection
by oxytocin injected into the ventral subiculum or into the posteromedial cortical nucleus
of the amygdala of male rats. In fact, oxytocin injected in these two areas induces penile
erection [49] and increases NO production [50] as found in the ventral tegmental area.
Both these responses are antagonized by the oxytocin receptor antagonist d(CH2 )5 Tyr(Me)-
Orn8 -vasotocin and by the NO synthase inhibitor S-methyl-thio-citrulline [50]. Apparently,
oxytocin injected into the ventral subiculum induces penile erection by acting on its own
receptors localized in yet unidentified neurons whose activity when changed by the stimula-
tion of oxytocin receptors causes an increase in glutamic acid neurotransmission (Figure 4).
Accordingly, an increase in extracellular glutamic acid is found in the dialysate from the
ventral subiculum of male rats 15 min after the oxytocin injection and usually 15 min
before the appearance of penile erection, which instead occurs 25–30 min after oxytocin
Biomolecules 2021, 11, 1866 21 of 40

injection. Glutamic acid in turn causes penile erection, possibly activating excitatory (possi-
bly glutamatergic) efferents projecting from the ventral subiculum to extra-hippocampal
areas such as the medial prefrontal cortex. This leads to the activation of glutamatergic
neurons projecting from the prefrontal cortex to the ventral tegmental area, in turn acti-
vating mesolimbic mesocortical dopaminergic neurons and penile erection ([50,51] and
references therein). Alternatively, glutamic acid in the ventral subiculum might activate
glutamatergic neurons projecting directly to the ventral tegmental area, since in male
rats a direct glutamatergic pathway from the ventral subiculum to the ventral tegmen-
tal area, which activates mesolimbic dopaminergic neurons, cannot be ruled out ([50,51]
and references therein). It is likely that the neurons expressing oxytocin receptors also
contain NO synthase whose activation leads to an increase in NO that, when produced,
activates glutamatergic neurons projecting to the medial prefrontal cortex or the ventral
tegmental area with a still unknown mechanism. Accordingly, since hemoglobin, a NO
scavenger, injected into the ventral subiculum before oxytocin prevents both the increase
in NO production and penile erection in male rats, it is also likely that newly formed NO
travels intercellularly to reach glutamatergic neurons that, once activated, lead to penile
erection mediated by the increased glutamatergic activity in the ventral tegmental area
followed by that of mesolimbic dopaminergic neurons [50,51] (see Section 2.4.4). Further
experiments are required to verify if guanylate cyclase is involved in the facilitatory effect
of oxytocin injected in the ventral subiculum and to ascertain if mechanisms similar to
those summarized above are also operative in the posteromedial cortical nucleus of the
amygdala and in the bed nucleus of the stria terminalis of male rats after the injection of
oxytocin at doses that increase NO production and induce penile erection [49,151,152].

Table 9. Oxytocin-induced penile erection and NO production increase in the ventral tegmental area
and the ventral subiculum of male rats: effect of the NO synthase inhibitor L-NAME, the oxytocin
receptor antagonist d(CH2 )5 Tyr(Me)-Orn8 -vasotocin (Oxy-Ant), the guanylate cyclase inhibitor
ODQ and the NO scavenger hemoglobin given in the ventral tegmental area and in the ventral
subiculum, respectively.

Oxytocin Injected in the

Pretreatment Ventral Tegmental Area Ventral Subiculum
Penile Erection NO Production Penile Erection NO Production
L-NAME ↓ ↓ ↓ ↓
Oxy-Ant ↓ ↓ ↓ ↓
ODQ ↓ == n.a. n.a.
Haemoglobin == ↓ ↓ ↓
↓ = prevention; == = no effect; n.a. = not available. Details are found in [47–51,160].

2.4.4. NO Facilitates Erectile Function in Male Rats by Acting Intracellularly in the PVN
and in the Ventral Tegmental Area and Intercellularly in the Ventral Subiculum
The studies reviewed above performed on male rats also show that NO may act as
an intracellular or intercellular messenger depending on the brain area where it acts to
control erectile function. As to the PVN and the ventral tegmental area, the available data
support a role for NO as an intracellular messenger (Figure 4). Accordingly, hemoglobin, a
potent NO scavenger [164] injected either i.c.v. (100–400 µg) or in the PVN (10–20 µg), is
unable to prevent penile erection induced by oxytocin, apomorphine, NMDA, NO donors
or 5 HT1C agonists injected into the PVN (Table 8) [79–81,103] or by oxytocin injected into
the caudal ventral tegmental area [47]. This occurs in spite of the ability of hemoglobin-
injected i.c.v. or in the PVN to abolish/reduce the increase in NO metabolites in the male
rat PVN extracellular dialysate induced by the above substances [79–81,96,104] or when
this increase occurs in physiological contexts, [e.g., in the presence of a sexually receptive
female rat (non-contact erections) or during copulation] [77] (Table 4), or when induced by
oxytocin injected into the caudal ventral tegmental area as well [47] (Table 9). Although
Biomolecules 2021, 11, 1866 22 of 40

these findings may be considered as evidence against the role of guanylate cyclase in the
control of erectile function in the PVN at variance from the ventral tegmental area, they are
far from being decisive since hemoglobin, due to its high molecular weight, would bind
NO exclusively in the extracellular space, being unable to cross cellular membranes. Hence,
the inability of hemoglobin to prevent penile erection induced by the above substances
including oxytocin when injected into the PVN, or when this sexual response occurs in
physiological contexts, which all increase NO production in the PVN of male rats, may
be explained by assuming that NO is acting intracellularly in those neurons in which it is
formed to induce penile erection rather than after having been released in the extracellular
space to reach other neuronal targets in these two brain areas (Tables 4, 8 and 9). However,
this assumption does not rule out that NO might act also as an intercellular messenger
in the PVN and in the caudal ventral tegmental area and perhaps also in the other brain
areas considered in this review, where the injection of oxytocin induces penile erection by
activating NO synthase and increasing NO production. Indeed, irrespective of the brain
area considered, NO released out from the neurons in which it is formed and escaped to
the scavenging effect of hemoglobin, might well act on and mediate other effects by acting
on neurons close to those from which it has been released.
At variance from the PVN and the ventral tegmental area, in the ventral subiculum of
the hippocampus NO acts as an intercellular messenger to facilitate penile erection induced
by oxytocin injected in this area (Figure 4). Accordingly, in male rats hemoglobin injected
into the ventral subiculum before oxytocin prevents both the increase in NO production
and penile erection (Table 9). This suggests that once produced by the stimulation of
oxytocinergic receptors, NO travels intercellularly to reach glutamatergic neurons that,
once activated, lead to penile erection mediated by the increased glutamatergic activity in
the ventral tegmental area. Unfortunately, in the experiments summarized above [50,51],
the effect of a guanylate cyclase inhibitor injected into the ventral subiculum or of 8-Bromo-
cGMP, which would mimic the effect of endogenous cGMP, was not tested in male rats
to ascertain if NO was acting on guanylate cyclase to activate the glutamatergic neurons,
which are proposed to activate glutamic acid neurotransmission in the ventral tegmental
area for inducing penile erection as discussed above. Further experiments are required to
verify if NO acts as an intracellular or intercellular messenger in the posteromedial cortical
nucleus of the amygdala and in the bed nucleus of the stria terminalis after the injection of
oxytocin at doses that induce penile erection and increase NO production [49,151,152].

3. Central NO and Male Sexual Behavior

It is well known that sexual behavior plays a key role in the reproduction of all living
animals, from insects to mammals, including humans. As already recalled in Section 2,
in male mammals sexual behavior is testosterone dependent and is organized in two
main phases, anticipatory and consummatory, and several well-defined and quantifiable
parameters have been identified in each phase and in both males and females. The studies
on male sexual behavior have usually been and are still performed mainly in rats because
of their availability and the well-characterized sequence of copulatory behavior and its
parameters [59,60,165,166]. However, data on male sexual behavior in other animal species
(mice, rabbits, hamsters, voles, primates, and even birds, as the Japanese quail and the zebra
finch) are also available [61,167–172]. Penile erection, seminal emission and ejaculation are
the main components of the consummatory phase of the male sexual response and are pre-
ceded by an anticipatory phase, which includes motivation towards and searching for an
adequate partner for copulation [59,60,173]. Briefly, when sexual (visual, auditory, olfactory,
tactile and even imaginative in humans) stimuli reach the central nervous system, neural
pathways are activated which convey sexual information from the higher brain centers
through the spinal cord and the autonomous nervous system to the genital apparatus to
induce penile erection, thus allowing copulation that will culminate with ejaculation with
the female [16,18,59–61,75,173]. It is well known that numerous neurotransmitters and neu-
ropeptides are involved at the central and peripheral level in the control of both phases of
Biomolecules 2021, 11, 1866 23 of 40

male sexual behavior. Among neurotransmitters, the best known are dopamine, serotonin,
glutamic acid and gamma-amino-butyric acid (GABA) [40,53,56,63,70,73,114,115]. They
influence sexual behavior by acting in different brain areas, from the hypothalamus and its
nuclei (e.g., PVN and lateral hypothalamus), to the medial preoptic area and other brain
areas, which include the ventral tegmental area, the nucleus accumbens, the prefrontal
cortex, the hippocampus, the amygdala, the bed nucleus of the stria terminalis, the medulla
oblongata and the spinal cord. In these brain areas neurotransmitters often interact in a
concerted manner with each other and/or with neuropeptides, such as oxytocin, adreno-
corticotropin (ACTH), α-melanocyte stimulating hormone (α-MSH) and opioid peptides
in the control of several aspects of sexual behavior, from sexual motivation and arousal
to sexual performance or both [53,55–57,66,73,102,116,149,150,174]. As recalled above, NO
was added to the list of neurotransmitters/neuromodulators that play a role in sexual
behavior in 1990s when it was discovered that this molecule formed from L-arginine by
NO synthase is the physiological neurotransmitter of penile erection at local level (see
Section 1). This is due to the key role of NO in the activation of guanylate cyclase and the
consequent increase in cGMP levels that are responsible for the relaxation of the cavernous
corpora smooth muscles allowing penile erection to occur (see Section 1). This discovery
was soon followed by studies showing that NO plays a role in erectile function (discussed
above) and sexual behavior also at the central level [37,38,41]. The medial preoptic area
and the PVN have been identified as the two main brain sites where NO acts to facilitate
male sexual behavior, although some experimental evidence for the involvement of the
ventral tegmental area is also available.

3.1. Medial Preoptic Area

As found with erectile function, the first evidence for a facilitatory role of NO in sexual
behavior was obtained with the NO synthase inhibitor L-NAME given intraperitoneally,
which was found able to impair copulatory behavior in a dose dependent manner in male
rats, while L-arginine induced only modest effects [38,41,175]. The inhibitory effect of
L-NAME was found much more marked after i.c.v. injection, being the drug found able
to completely prevent ejaculation in sexually naive rats, but not in sexually experienced
rats [41]. The latter study also reported that in the PVN, but not in the amygdala or the bed
nucleus of the stria terminalis, of sexually potent male rats, NO synthase messenger RNA
content was twice that found in sexually impotent rats (e.g., rats that do not copulate when
put together with a sexually receptive female rat). These findings led us to suggest that
NO may facilitate copulatory behavior in male rats by acting not only at penile level but
also at the central level and in particular at the level of the PVN. The medial preoptic area
was soon identified as one of the brain areas where central NO may facilitate copulatory
behavior (Table 7). In this regard it is pertinent to recall that the medial preoptic area plays
a key role in sexual behavior and that several neurotransmitters interact to control penile
reflexes and copulation at this level [59,60]. Among these, the most studied are dopamine,
serotonin and glutamic acid [176,177]. In particular, it is well known that dopamine is
released in this area during copulation and that dopamine receptor agonists infused into
the medial preoptic area facilitate, and antagonists impair, copulatory behavior in male
rats [178,179]. More important for this review, (i) the administration of L-arginine (e.g., the
NO precursor) also increases dopamine release in the medial preoptic area, (ii) the infusion
of L-NAME at the concentration of 400 mM in the dialysis buffer via the microdialysis
probe, but not its inactive isomer D-NAME into the medial preoptic area for 3 h prior to
the introduction of a sexually receptive female rat in the mating arena, antagonized the
extracellular dopamine increase that occurs in the medial preoptic area during copulation
and impairs copulation [180]. This led to suggest that NO in the medial preoptic area
facilitates copulation by increasing dopamine release in male rats (Table 10).
Biomolecules 2021, 11, 1866 24 of 40

Table 10. NO facilitates copulatory behavior by increasing dopamine release in the medial preoptic
area of male rats engaged in copulation with a sexually receptive female: effect of NO synthase
inhibitors (L-NAME and L-NMMA), NO donors (SNP), guanylate cyclase inhibitors (ODQ) and
8-Bromo-cGMP injected into the medial preoptic area on extracellular dopamine and copulation.

MPOA Treatment Dopamine Copulation

L -arginine ↑ ↑
L-NAME ↓ ↓
D-NAME ↑ ↑
L-NAME+L-Arginine ↓ ↓
SNP ↑ ↑
ODQ ↓ ↓
ODQ+SNP ↓ ↓
8-Bromo-cGMP ↑ ↑
L-NMMA+8-Bromo-cGMP ↑ ↑
↑ = increase; ↓ = prevention; n.a. = not available. L-NAME is NG -nitro-L-arginine methyl ester, L-NMMA is
NG -monomethyl-L-arginine; SNP is sodium nitroprusside and ODQ a guanylate cyclase inhibitor. Details are
found in [38,39,44,45,175–185].

A facilitatory role of NO in sexual behavior has been also reported to occur in the me-
dial preoptic area of male mice [186], although some controversy exists on this point [187],
in line with the existence of differences in sexual behavior and its neural control between
male mice and rats (see Section 1).

3.1.1. NO Facilitates Erectile Function and Sexual Behavior by Acting Intercellularly as a

Retrograde Messenger in the Medial Preoptic Area of Male Rats
The discovery that NO facilitates copulation by increasing dopamine release in the
medial preoptic area was soon followed by numerous studies aimed at identifying the
mechanism by which NO induces this effect. These studies show that NO facilitates penile
reflexes (e.g., penile erections induced by simulation of the genitalia) and dopamine release
in the medial preoptic area of male rats by acting as a retrograde messenger to increase
glutamic acid neurotransmission, which is responsible for the increase in dopamine release
induced by copulation [178,181,182]. In fact, it is generally accepted that the increase in
extracellular dopamine that occurs in the medial preoptic area during sexual activity is
mediated by glutamic acid released from neuronal efferents originating in the medial amyg-
dala and the bed nucleus of the stria terminalis, which are activated by sexual mainly olfac-
tory (pheromones and others) stimuli [177]. However, how glutamic acid released in the
medial preoptic area stimulates dopamine release from the dopaminergic nerve endings of
incertohypothalamic neurons is not completely understood. Experimental evidence shows
that glutamic acid acts on NMDA receptors coupled to voltage-dependent Ca2+ channels
in medial preoptic yet to be identified cells containing NO synthase. The NMDA receptor-
coupled voltage-dependent Ca2+ channels are linked through their carboxy-terminal tail
via a PSD-95 protein–protein interaction domain to NO synthase [188], thereby coupling
NO synthase with the NMDA receptor (a review on this coupling in the medial preoptic
area is found in [179]). The mechanisms by which NO facilitates dopamine release from
incertohypothalamic dopaminergic nerve endings is unknown. One possibility is that
NO synthase, once activated by the increased Ca2+ influx, increases the production of
NO, which is released in the extracellular space and travels as a retrograde messenger
to dopaminergic nerve endings. Here the available data suggest the involvement of a
NO-cGMP pathway, e.g., that NO activates guanylate cyclase, which in turn increases
cGMP levels to sustain dopamine release and copulation. Accordingly, the infusion by
reverse dialysis of 8-Bromo-cGMP, which mimics cGMP, increases, and of ODQ, which
inhibits guanylate cyclase, decreases extracellular dopamine in the medial preoptic area,
respectively. ODQ also blocked the increase in extracellular dopamine levels induced
Biomolecules 2021, 11, x 26 of 42

Biomolecules 2021, 11, 1866 25 of 40

cellular dopamine levels induced by the NO donor sodium nitroprusside, while the
competitive NO synthase inhibitor L-NMMA was ineffective in blocking the
8-Bromo-cGMP-induced increase in dopamine, in line with a site of action for cGMP lo-
by the
cated NO donortosodium
downstream nitroprusside,
NO. Finally, while thefacilitated,
8-Bromo-cGMP competitive NOODQ
while synthase inhibitor
inhibited cop-
L-NMMA was ineffective in blocking the 8-Bromo-cGMP-induced increase
ulation of male rats with a sexually receptive female rat (Table 10) (Figure 5) [182]. in dopamine,
in line with
However, a site
other of action
actions of NO forreleased
cGMP located
in thedownstream
extracellulartospace
NO. Finally, 8-Bromo-cGMP
in addition to those re-
called above cannot be ruled out. For instance, NO might also facilitate dopaminefemale
facilitated, while ODQ inhibited copulation of male rats with a sexually receptive release
rat (Table 10) (Figure 5) [182]. However, other actions of NO released in the extracellular
by inhibiting the dopamine transporter (DAT) on dopaminergic nerve endings, contrib-
space in addition to those recalled above cannot be ruled out. For instance, NO might also
uting to a further increase in dopamine content in the extracellular space and to prolong
facilitate dopamine release by inhibiting the dopamine transporter (DAT) on dopaminergic
its action on dopaminergic receptors [178]. Moreover, NO might also travel back to glu-
nerve endings, contributing to a further increase in dopamine content in the extracellular
tamatergic
space and synapses anditsact
to prolong thereon
action to dopaminergic
sustain glutamic acid release,
receptors possibly by
[178]. Moreover, NOactivating
might
a NO-cGMP signaling pathway similar to that described above in the dopaminergic
also travel back to glutamatergic synapses and act there to sustain glutamic acid release,
nerve endings.
possibly The twoamechanisms
by activating might also
NO-cGMP signaling be notsimilar
pathway mutually exclusive
to that describedand co-operate
above in the
both to maintainnerve
dopaminergic dopamine
endings.release and mechanisms
The two copulation. might
Further studies
also be notare necessary
mutually to ver-
exclusive
ify and
this co-operate
possibility.both to maintain dopamine release and copulation. Further studies are
necessary to verify this possibility.

Figure 5. A hypothetical mechanism to explain how NO facilitates penile reflexes and copulation
Figure 5. Arats
in male hypothetical mechanism
by increasing dopamine torelease
explaininhow NO facilitates
the medial preopticpenile reflexes by
area induced and copulation
glutamic acidin
male rats byfrom
released increasing dopamine
glutamatergic release
synapses in the medial
of neurons preoptic
originating in thearea induced
amygdala andby in glutamic acid re-
the bed nucleus
leased from
of the striaglutamatergic synapses
terminalis activated of neurons
by sexual stimulioriginating
(e.g., those in the amygdala
produced and inreceptive
by a sexually the bed female
nucleus
of the
rat) stria terminalis activated
and controlling dopamineby sexual
release stimuli
from (e.g., those produced
incertohypothalamic by a sexually
dopaminergic receptive
neurons. Briefly,fe-
male rat) andformed
NO—once controlling dopamine release
by yet unidentified from incertohypothalamic
neurons/cells dopaminergic
in the medial preoptic area—is releasedneurons.
in the
Briefly,
extracellular space and acts as a retrograde intercellular messenger on (i) dopaminergicarea—is
NO—once formed by yet unidentified neurons/cells in the medial preoptic synapsesre-
leased in the extracellular
to potentiate space and
dopamine release and acts as a retrograde
facilitate copulation, intercellular messenger
and (ii) glutamatergic on (i) dopaminer-
synapses to sustain
gic glutamic
synapsesacid to potentiate dopamine release and facilitate copulation, and (ii) glutamatergic
release facilitating dopamine release, by activating a NO-cGMP signaling pathway.
syn-
apses to sustain glutamic acid release facilitating dopamine release, by activating a NO-cGMP
NO may also facilitate dopamine release from dopaminergic synapses by inhibiting the dopamine
signaling pathway. NO may also facilitate dopamine release from dopaminergic synapses by in-
transporter (DAT), thus allowing dopamine to persist in the extracellular space and act for longer
hibiting the dopamine transporter (DAT), thus allowing dopamine to persist in the extracellular
times on its receptors. (+) = activation; (-) = inhibition; (???) = unknown neurotransmitters. Details
space and act for longer times on its receptors. (+) = activation; (-) = inhibition; (???) = unknown
are found in [38,39,44,45,175–185].
neurotransmitters. Details are found in [38,39,44,45,175–185].
3.2. PVN
3.2. PVN
The medial preoptic area is not the only brain area in which increased NO activity has
Thedemonstrated
been medial preoptic areasexual
during is notbehavior
the onlyinbrain
malearea
rats.inExperimental
which increased NO shows
evidence activity
hasthat
been
thisdemonstrated
also takes placeduring sexual
in the PVN. In behavior in male
fact, as recalled rats.(i)Experimental
above, in the PVN, butevidence
not in
the amygdala
shows and takes
that this also in the place
bed nucleus of the stria
in the PVN. terminalis
In fact, (twoabove,
as recalled areas involved
(i) in theinPVN,
sexual
but
Biomolecules 2021, 11, 1866 26 of 40

behavior) sexually potent male rats have NO synthase messenger RNA levels twice higher
than those found in sexually impotent rats (e.g., rats that do not copulate when put together
with a sexually receptive female rat) [41]; (ii) neuronal NOS protein expression measured
by Western blot analysis is reduced by about 40% in the PVN of streptozocin-treated rats (a
rat model of diabetes) that show a blunted erectile response to NMDA injected into the
PVN compared control rats, which is restored by restoring neuronal NOS within the PVN
by gene transfer using adenoviral transfection [189] and (iii) increased NO production also
occurs in the PVN of male rats when penile erection takes place in physiological contexts,
e.g., when male rats are put in the presence of an inaccessible receptive female rat and
during copulation [74]. The latter behavioral and biochemical effects are prevented by the
inhibition of NO synthase in the PVN (Table 4) [47,48]. However, at variance from the
medial preoptic area, in the PVN NO acts intracellularly to facilitate non-contact erections
and copulation and with a mechanism yet to be identified that does not involve a NO-
cGMP pathway. Accordingly, 8-Bromo-cGMP injected into the PVN does not induce penile
erection and inhibition of guanylate cyclase by methylene blue or NO scavenging from
the extracellular space in the PVN with hemoglobin does not impair non-contact erections
and copulation (Table 4, Figure 2) [47,48]. Further studies are necessary to identify the
mechanism by which NO activates oxytocinergic neurotransmission in the PVN to facilitate
non-contact erections and copulation in male rats.

3.3. Ventral Tegmental Area, Ventral Subiculum, Amygdala and Bed Nucleus of the
Stria Terminalis
To our knowledge, no data are available showing that increased NO production also
occurs in the ventral tegmental area, ventral subiculum, amygdala and bed nucleus of
the stria terminalis during sexual behavior despite the increase in NO production that
occurs in these areas after the injection of oxytocin at a dose that induces penile erection;
an increase that is prevented by the inhibition of NO synthase in these areas. However,
a little experimental evidence supporting that this may occur in the ventral tegmental
area is available. Accordingly, sildenafil and tadalafil, two phosphodiesterase type V
inhibitors (clinically used for the therapy of erectile dysfunction) injected into the ventral
tegmental area increase the number of non-contact erections in both groups of male rats
that have been previously screened for their ability to show or not, respectively, this sexual
response [163]. Interestingly, the facilitatory effect on non-contact erection by these drugs
takes place with a concomitant increase in extracellular dopamine in the nucleus accumbens
dialysate, the dopamine increase being higher in male rats screened for not showing non-
contact erections than in those showing the sexual responses [163]. This finding is in line
with the fact that these compounds injected in the ventral tegmental area are expected
to increase the endogenous cGMP levels due to their ability to markedly and selectively
inhibit phosphodiesterase activity that inactivates cGMP. It is tempting to speculate that
an increase in NO production similar to that found after oxytocin injection in the ventral
tegmental area at a dose that induces penile erection in male rats [47,48] also occurs in this
area when penile erection occurs in physiological contexts (i.e., presence of an inaccessible
receptive female rat and during copulation). Further experiments are necessary to verify
this hypothesis.

4. Central NO and Female Sexual Behavior

As already described for male sexual behavior in Section 3, female sexual behavior is
also organized in an anticipatory phase, which includes motivation towards and searching
for an adequate partner for copulation [59,60,166,173], and a consummatory phase, whose
main components include the manifestation of lordosis (see below), vaginal lubrication,
clitoris erection and possibly orgasm in the majority of female mammals [190], except that
human primates, including women, do not show lordosis during sexual intercourse. Female
sexual behavior also has been extensively studied in female rats (although studies on other
rodents and animal species are available). Briefly, in female rats, the anticipatory phase is
characterized by proceptive behaviors such as hops/darts and solicitation episodes, while
Biomolecules 2021, 11, 1866 27 of 40

the consummatory phase is characterized mainly by lordosis, i.e., the assumption of a

posture characterized by arching of the back and lateral moving of the tail, which occurs
when the male touches the flanks and/or perineal region of the female and which is aimed
to facilitate intromission of the male penis into the vagina for ejaculation [166,173]. Most
important, lordosis occurs only when female rats are sexually receptive, that is when they
are in the estrous phase of the estrous cycle, and is strictly dependent from the marked
increase in circulating estradiol that occurs in this phase of the cycle. In fact, at variance
from male rats, female rat sexual behavior is under the control of the cyclic variations of
ovarian hormones, estradiol and progesterone, which allow female rats to become sexually
receptive and able to engage in copulation with a male only after the increase in estradiol
levels ([191] and references therein). In general, female sexual responses are activated and
mediated by the same neural circuitry shown schematically in Figure 1 [192], although
it has to be integrated with the neural pathways that lead to the lordotic posture only
when the male touches the flanks and/or perineal region of a receptive female. Briefly,
the lordosis reflex is activated by the sensory tactile stimuli secondary to the touch of
female perineal regions, which send sensory inputs that travel in spinal nerves to the
lumbosacral L1-S1 spinal tract, and from here to the mesencephalic periaqueductal gray
matter. This brain region coordinates striated back muscles activity together with other
brain regions and in particular with the sexually dimorphic ventromedial nucleus of the
hypothalamus, which usually inhibits the lordosis reflex, except when circulating ovarian
estradiol levels are increased, as it occurs in sexually receptive females in the estrous phase
of the cycle [191,193–195] and Section 6).
As already reported for male sexual behavior, numerous neurotransmitters and neu-
ropeptides are also involved in female sexual behavior and NO was only added to this list
in 1995. However, at variance from male sexual behavior, the role of central NO in female
sexual behavior has received much less attention, as revealed by the much lower number
of studies on NO and female sexual behavior compared to those of NO and male sexual
behavior that appear in Pub Med and Google Scholar medlines. These earlier studies
show that neurons containing NO synthase in the rat hypothalamus stimulate the pulsatile
release of LH-RH in vivo and in vitro, which also stimulate female sexual behavior as
measured by the lordotic posture assumed by the female when the male touches the female
rat’s flanks, [196,197]. In this study L-NAME, but not D-NAME, injected into the third
cerebral ventricle was found able to reduce and sodium nitroprusside to increase the num-
ber of lordosis episodes induced by progesterone when administered to estrogen-primed
ovariectomized rats. Since sodium nitroprusside also facilitates lordosis in the absence of
progesterone and this response is blocked by a LH-RH antiserum, this leads to suggest
that progesterone causes the release of NO, which in turn stimulates LH-RH release that
facilitates lordosis and sexual behavior in female rats [196–198]. The facilitatory effect of
NO on lordosis behavior is apparently mediated by a NO-cGMP pathway, since inhibition
of guanylate cyclase by ODQ given into the third ventricle significantly decreased lordosis
quotient in ovariectomized rats primed with estradiol and progesterone [199]. One of the
hypothalamic areas in which NO may act to facilitate lordosis is the ventromedial nucleus,
as suggested by the increase in neuronal NO synthase mRNA that occurs in this but not in
the arcuate or supraoptic nucleus following short-term estrogen treatment [200]. Recent
studies in female mice suggest that NO synthase containing neurons in the ventrolateral
part of the ventromedial nucleus modulating lordosis, but not mating preference, may
be under the control of kisspeptin neurons originating in the hypothalamic anteroventral
periventricular area [201,202]. NO seems also play a role in the higher expression of NO
synthase that occur in the medial preoptic area of sexually experienced female rats in a
paced mating test with a male rat when compared to naive female rats [203]. Whether
mechanisms similar to those identified in female rats and mice are operative also in the
central nervous system in women is unknown, although it is tempting to speculate that this
preclinical evidence may prove useful for the therapy of female sexual dysfunctions [141].
Biomolecules 2021, 11, 1866 28 of 40

5. Can Central NO Have a Role in Strategies Aimed to Improve Erectile Function and
Sexual Behavior in Humans?
The studies reviewed so far show that central NO is involved in the control of erectile
function and sexual behavior at several levels, from the PVN and the medial preoptic
area to the limbic system and the spinal cord. However, it is difficult sustaining that the
facilitatory role of this neurotransmitter/neuromodulator may be selectively activated at
the central level to improve penile erection in men with erectile dysfunction of central
origin (psychogenic erectile dysfunction) either physiologically or pharmacologically. This
is due to the main role of NO at the penile level in the relaxation of cavernous corpora
smooth muscles. In fact, such selectivity is difficult to obtain, as this requires the use of
an administration route yet to be discovered that allows a drug (such as a NO synthase
inhibitor or a NO donor) to cross the blood–brain barrier and reach the central nervous
system without reaching the systemic blood circulation, or the synthesis of drugs that
differentially release NO in the brain, cavernous corpora and vascular tissues. This is a
very important point because NO is one of the main vasorelaxing modulators present in
all vascular tissues, and this may cause severe complications (e.g., marked hypotension).
The achievement of a selective action at central level is also very important because penile
neuronal NO synthase, its variants and its regulatory protein inhibitor are present and
co-localized not only at the local level in penile nerves and pelvic ganglia, but also in
the hypothalamic (PVN and medial preoptic area) and spinal cord regions involved in
the control of penile erection [204]. Irrespective of the above criticism, several studies
investigated the use of compounds that are supposed to increase NO levels or activate NO
synthase to improve erectile function in men. These compounds include EGb 761 (a Ginkgo
biloba extract), ginsenoside Rg1 (isolated from Panax ginseng) and Lycium Barbarum
Polysaccharide, obtained by the Lycium Barbarum berries extensively used in Chinese
medicine, and are used as food supplements. In rats these compounds have been found able
to improve non-contact erections and sexual behavior, sometimes accompanied by increases
in NO synthase expression in the hypothalamus, the medial preoptic area and the spinal
cord [205–207]. However, the most widely used food supplement by men seeking natural
treatment and/or self-medication for erectile dysfunction is L-arginine, the physiological
NO synthase substrate. Several studies show an improving effect of L-arginine on erectile
function in men. In a recent study, L-arginine, given daily at the dose 2.500 mg for 12 weeks,
was found able to improve erectile function in men with low to moderate, but not a severe
grade of erectile dysfunction, as determined by the International Index of Erectile Function-
Erectile Function (IIEF-EF) questionnaire and in particular from the change in IIEF-EF
score and in per-patient percentage of “yes” responses to the Sexual Encounter Profile
Questionnaire Question 3 from baseline to after treatment, with an effect comparable to that
of the phosphodiesterase inhibitor tadalafil given at the dose of 5 mg [208]. The improving
effect of L-arginine on erectile dysfunction in men revealed by this and other studies is
in line with the animal studies reviewed before, which show that this amino acid is able
to reverse the inhibitory effect of competitive NO synthase inhibitors such L-NAME and
L-NMMA on penile erection and copulatory behavior not only when given systemically
but also in the PVN and the medial preoptic area, although it induces only modest effects
when given alone [38,41,85] (see Sections 2.2.2 and 3.1). Conversely, classic clinically used
NO donors have produced contrasting and even negative results in double-blind crossover
trials when tested for the treatment of human erectile dysfunction [133]. As recalled above,
this may be due to the fact that NO is a potent vasorelaxing modulator present in all vascular
tissues, and this may cause severe complications (e.g., marked hypotension). Whether this
picture is going to change with the use of the new NO donors under development cited
above (Sections 2.2.1 and 2.2.2) [138–140] is still unknown.

6. Concluding Remarks
The studies reviewed suggest a key role of NO in the PVN for the control of erectile
function and of both the PVN and the medial preoptic area in male rat sexual behavior.
Biomolecules 2021, 11, 1866 29 of 40

In the PVN NO is synthesized by NO synthase located in the cell bodies of oxytocinergic

neurons projecting to extra-hypothalamic brain areas and mediating this sexual response.
Accordingly, NO synthase is activated by agents supposed to induce penile erection by
acting on these neurons in the PVN by increasing Ca2+ influx in their cell bodies (Figure 2).
In fact, the activation of NO synthase is necessary for the induction of the sexual response
induced by dopamine agonists, oxytocin, NMDA, hexarelin peptide analogues and VGF-
derived peptides, since penile erection does not occur when the enzyme has been previously
inhibited, for instance by NO synthase inhibitors injected in the PVN. Once formed, NO in
the PVN activates yet unidentified intracellular targets, apparently different from guanylate
cyclase (i.e., ADP-ribosyl-transferases and other iron-containing enzymes [4,12,26]), which
in turn lead to the activation of oxytocinergic neurons mediating the appearance of the
sexual response by releasing oxytocin in sites distant from the PVN, i.e., the ventral
tegmental area, hippocampus, amygdala, ventral medulla and the spinal cord [40,53,54,56,
57]. Interestingly, NO seems to act as an intracellular rather than an intercellular messenger,
since penile erection is not prevented by NO scavenging from the PVN extracellular space
with hemoglobin [96,97,103,104,143]. The pro-erectile effect of NO in the PVN is further
confirmed by the ability of several NO donors to induce penile erection when injected into
this nucleus. Additionally, in this case, a NO-cGMP pathway seems not to be involved,
as the erectile response by NO donors is not abolished by NO scavenging or guanylate
cyclase inhibition. Despite the importance of a normal function of NO in the PVN in the
expression of penile erection, there are several pieces of evidence that NO is also involved
in the control of this sexual response in sites different from the PVN. Among these are
the ventral tegmental area, the ventral subiculum of the hippocampus, the posteromedial
cortical nucleus of the amygdala, the bed nucleus of the stria terminalis (Figure 4) and even
the ventral medulla and the spinal cord. In the majority of these areas NO is involved in the
induction of penile erection induced by oxytocin. Accordingly, oxytocin injected into these
areas induces penile erection which occurs with a concomitant increase in NO production
measured by the increase in NO2 − and NO3 − found in the dialysate collected from these
brain areas. At variance from the PVN, in the ventral tegmental area oxytocin induces penile
erection by activating mesolimbic/mesocortical dopaminergic neurons co-expressing both
NO synthase and guanylate cyclase, thus activating a NO-cGMP pathway in the same
neurons in which NO is formed and acts as an intracellular messenger. Accordingly, (i)
the NO synthase inhibitor S-methyl-thio-citrulline injected in this area before oxytocin
antagonized both oxytocin-induced penile erection and the concomitant increase in NO
production that occurs in the ventral tegmental area dialysate, and (ii) the active cGMP
analogue 8-Bromo-cGMP induces penile erection, while the inhibitor of guanylate cyclase
ODQ prevents oxytocin-induced penile erection without antagonizing the increase in NO
production that occurs in the ventral tegmental area, in line with the hypothesis that in
dopaminergic cell bodies cGMP acts downstream to NO [47,49–51]. As for the increased NO
production that occurs in the ventral subiculum of the hippocampus or in the posteromedial
cortical nucleus of the amygdala that leads to penile erection after the oxytocin injection in
these two brain areas, the available data support the hypothesis that NO is produced in the
cell bodies of glutamatergic neurons from which it is released in the extracellular space and
acts as a intercellular messenger to activate glutamatergic neurons projecting directly or
indirectly to the ventral tegmental area. Accordingly, oxytocin-induced penile erection and
the concomitant increase in NO production that occur in the ventral subiculum are abolished
not only by the prior blockade of oxytocinergic receptors and NO synthase inhibition but also
by NO scavenging with hemoglobin. Further experiments are necessary to verify if a similar
mechanism also takes place in the posteromedial cortical nucleus of the amygdala and/or
in the bed nucleus of the stria terminalis. Two other sites in which NO may be involved in
the control of erectile function are the ventral medulla and the spinal cord. Accordingly, NO
synthase has been identified in these structures [52,127]. Since penile erection induced by
5HT1C agonists is prevented dose dependently by NO synthase inhibitors given i.c.v. but
not in the PVN [83], this raises the possibility that NO may be involved in the expression of
Biomolecules 2021, 11, 1866 30 of 40

penile erection induced by these compounds in these two areas [80]. Likewise, it is possible
that NO synthase inhibitors prevent ACTH-induced penile erection [41] by acting in a yet
undiscovered brain area.
In the medial preoptic area, NO is formed in NO synthase-containing neurons/cells
that are activated to facilitate the release of dopamine by the nerve endings of glutamatergic
neurons originating in the amygdala and the bed nucleus of the stria terminalis, which are
stimulated by sexual (mainly olfactory, i.e., pheromones) stimuli [178,179,183–185]. Glu-
tamic acid activates dopamine release by acting on NMDA receptors coupled to voltage-
dependent Ca2+ channels in still unknown medial preoptic neurons/cells. The increased
Ca2+ influx activates NO synthase to increase NO production. Once released, NO in turn
travels as a retrograde messenger to dopaminergic synapses and possibly also to the gluta-
matergic synapses, where it activates guanylate cyclase to increase cGMP levels to facilitate
dopamine release to improve copulation and sustain glutamic acid release to maintain
dopamine release (Figure 5) [39,178–185]. Accordingly, in male rats (i) NO synthase in-
hibitors, when injected into the medial preoptic area, abolish both dopamine and glutamic
acid release that occur in the medial preoptic area during copulation and impair sexual
behavior; (ii) 8-Bromo-cGMP facilitates dopamine release and copulation, while ODQ
inhibits glutamic acid and dopamine release and copulation as well (Table 10) [181–183].
NO facilitates sexual behavior in male rats by acting not only in the medial preoptic area,
but also in the PVN. Accordingly, (i) NO production increases in the PVN when penile
erection occurs in physiological contexts; that is, when male rats show non-contact erec-
tions in the presence of an inaccessible sexually receptive female or when it occurs during
copulation, and (ii) inhibition of NO synthase in the PVN prevents non-contact erections
and impairs copulation. However, at variance from the medial preoptic area, in the PVN
NO facilitates sexual behavior by a yet undiscovered mechanism not involving cGMP [74].
The results reviewed in this work show also that NO plays a key role in the control
of erectile function at central level by acting as a common mediator of several neuro-
transmitters and neuropeptides that control this sexual function. This occurs at level of
the PVN, which is considered a sort of integration center between the central and the
peripheral autonomous nervous systems [209], and at the level of the limbic system, mainly
in the ventral tegmental area, which is, among others, strictly connected with the hip-
pocampus, the amygdala, the bed nucleus of the stria terminalis and the medial preoptic
area [53,54,56,57]. Most importantly, the activation of mesocorticolimbic dopaminergic
neurons by oxytocin injected in the ventral tegmental area leads to the release of dopamine
in the nucleus accumbens [47–49]. This in turn causes an increase in dopamine release from
incertohypothalamic neurons that impinge on oxytocinergic neurons in the PVN sending
projections back to the ventral tegmental area, the hippocampus, the amygdala, the bed
nucleus of the stria terminalis, the medial preoptic area and the spinal cord leading to
penile erection [57,144]. Together these findings support the hypothesis that NO plays a
main role at several levels in a complex neural circuit that interconnects these brain areas
and contains the neuronal systems responsible of the anticipatory phase (mediating sexual
motivation and reward) with those responsible of the consummatory phase (mediating
copulation and sexual performance) of sexual behavior (Figure 6) [53–55]. In view of
the existence of a similar distribution of central oxytocinergic neurons between male and
female rats, it is feasible to assume that the neural circuit described above is also involved
in female sexual behavior, although with some differences between male and female rats.
The most important difference regards the role of the medial preoptic area, which exerts
inhibitory control on female rat sexual behavior rather than facilitatory as extensively
reported for male rat sexual behavior (see Section 3). In fact, at variance from the ventro-
medial nucleus of the hypothalamus, the activation of the medial preoptic area in sexually
receptive (estrogen-primed) female rats inhibits rather than facilitates the lordotic reflex,
while the opposite is seen after lesions of this brain area [191,210]. The medial preoptic area
inhibitory effect on lordosis is apparently mediated by the activation of neurons projecting
to the ventral tegmental area and from here to the periaqueductal gray matter [210], which
Biomolecules 2021, 11, 1866 31 of 40

receives the sensory inputs produced by tactile stimulation of the female flanks and/or
perineal regions from the lumbosacral L1-S1 spinal tract to coordinate the striated muscles
activity leading to the lordotic posture [191,193–195]. Irrespective of the different role the
medial preoptic area may have in male and female rat sexual behavior, and of the fact that
lordosis is mediated by neural pathways different from those that mediate erectile function
and ejaculation in males and clitoris erection and vaginal lubrication in females [190],
it is reasonable to assume that this neural circuit and its components, including NO, not
only contribute to the consummatory aspects of sexual behavior (erectile function and
copulation in males and lordosis in females), but also in the same time activate mesolim-
bic/mesocortical dopaminergic neurons providing a neural substrate for explaining the
motivational and rewarding properties of sexual activity (Figure 6). In agreement with this
possibility, the central administration of NO synthase inhibitors decreases the percentage of
copulating male rats and impairs the indexes of sexual activity in sexually experienced and
naive male rats [38,41,175]. More intriguing and relevant to the mechanisms controlling
erectile function and sexual behavior considered in this review, the NO synthase messenger
RNA expression is found to be in the PVN of sexually potent male rats about twice that of
impotent male rats [41]. In view of the importance of the PVN in the expression of penile
erection, it was suggested in 1995 that NO was deeply involved in the control of male
sexual behavior and that the inhibitory effect of NO synthase inhibitors given centrally on
male copulatory performance might be due to the inhibition of erectile function secondary
to the inhibition of central oxytocinergic transmission at the PVN level [211]. However,
since the activation of NO synthase facilitates erectile function and sexual behavior by
acting not only in the PVN, but also in many other brain areas (i.e., the ventral tegmental
area, ventral subiculum of the hippocampus, amygdala-posteromedial cortical nucleus,
bed nucleus of the stria terminalis, medial preoptic area and spinal cord) (see above), it is
evident that the role on NO in erectile function and sexual behavior is more diffused across
the brain than was thought in the 1990s. Although difficult to translate to humans, the
increased knowledge obtained in laboratory animals, mainly rodents, on the sexual role
Biomolecules 2021, 11, x 33 of 42
of NO at central level will contribute to finding new strategies for the therapy of sexual
dysfunctions and other sexual disorders in humans.

Figure 6. Nitric oxide produced by NO synthase (NOS) participates in a complex neural circuit that con-
Figure 6. Nitric oxide produced by NO synthase (NOS) participates in a complex neural circuit that
trols sexual motivation, reward and performance at central level together with other neurotransmitters
controls sexual motivation, reward and performance at central level together with other neuro-
transmitters and neuropeptides. This circuit interconnects the hypothalamus and its nuclei (para-
ventricular and ventromedial) with limbic areas (ventral tegmental area, hippocampus, amygdala,
bed nucleus of the stria terminalis) and with the ventral medulla and spinal cord. Among neural
pathways involved are (i) PVN oxytocinergic neurons that send their projections to the ventral
medulla and spinal cord, and to the ventral tegmental area, hippocampus, amygdala and bed nu-
Biomolecules 2021, 11, 1866 32 of 40

and neuropeptides. This circuit interconnects the hypothalamus and its nuclei (paraventricular and
ventromedial) with limbic areas (ventral tegmental area, hippocampus, amygdala, bed nucleus of the
stria terminalis) and with the ventral medulla and spinal cord. Among neural pathways involved are
(i) PVN oxytocinergic neurons that send their projections to the ventral medulla and spinal cord, and
to the ventral tegmental area, hippocampus, amygdala and bed nucleus of the stria terminalis; (ii)
mesolimbic/mesocortical dopaminergic neurons originating in the ventral tegmental area that send
projections to the nucleus accumbens, medial prefrontal cortex, bed nucleus of the stria terminalis,
hippocampus and amygdala; (iii) glutamatergic neurons that interconnect the hippocampus and
the amygdala with the ventral tegmental area, prefrontal cortex, bed nucleus of the stria terminalis,
medial preoptic area and PVN; (iv) neurons that project from the ventromedial nucleus of the hy-
pothalamus to the periaqueductal gray matter and from here to the spinal cord (dashed lines), which
control lordosis in estrous females and whose activity is modulated by different brain areas (medial
preoptic area, ventral tegmental area, lateral septum). When this circuit is activated by physiological
stimuli (i.e., pheromones released by sexually receptive females in males or by sexually potent males
in females and/or other sexual stimuli) or by drugs or peptides given in one of the areas of the
circuit, NO becomes involved at different sites, i.e., in the paraventricular and ventromedial nuclei
and medial preoptic area, which are involved more in sexual performance (penile erection and
copulation in males, lordosis in females), and the ventral tegmental area, hippocampus, amygdala
and bed nucleus of the stria terminalis, which act on mesolimbic/mesocortical dopaminergic neurons
that control sexual motivation, arousal and reward (light blue boxes indicate the brain areas of the
circuit containing NO synthase). Thus, changes in NO activity in these brain areas participate in
the modulation of both sexual motivation/arousal/reward and sexual performance (erectile func-
tion/lordosis/copulation). (?) = unknown neurotransmitters/neuropeptides. Details are found
in [4,16,17,26,47,49,53–57,64–70,96–99,104,143,144,151,152,160,174,179,183–185,190,192–194] and ref-
erences therein.

Author Contributions: Both authors have contributed to the organization and writing of the
manuscript. Both authors have accepted responsibility for the entire content of this manuscript and
approved submission. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by funding from the Department of Biomedical Sciences (FIR
2016-2020) to M.R.M. and A.A.
Conflicts of Interest: The authors declare no conflict of interest regarding this article.

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