Multiple vasodilator pathways from the pelvic plexus to the penis of the rat.

The main penile or cavernous nerve is usually regarded as the most important vasodilator projection in the rat. Although other descending pathways have been described, there is little detailed information on their importance. In this present report, we provide topographic and quantitative information on lateral and ventral penile branches and examine the vasodilator fibers which join the pudendal neurovascular bundle. Seventeen Sprague-Dawley rats were used. The techniques included injection of dye in the penis to label neurons in the pelvic plexus in combination with transection of the main penile nerve (MPN). NADPH diaphorase (NADPH-d) histochemistry was used to assess the effects of transection of vasodilator pathways on innervation of the penis and for in situ staining of the pelvic plexus. Distinct clusters of penile neurons are aggregated at the origin of several nerve tracts leaving the posterior margin of the major pelvic ganglion (MPG). Multiple NADPH-d+ fiber bundles coursed over the anterior surface of the prostate to reach the penis. Branches from these tracts joined the pudendal neurovascular bundle proximal to the hilum of the penis and provided innervation to the artery throughout its course in the pudendal canal. Consistent with the presence of multiple penile pathways, transection of the MPN reduced, but did not eliminate retrograde labeling of penile neurons in the MPG and only modestly decreased NADPH-d+ fibers in the penis. This study confirms that there are multiple pathways by which vasodilator fibers reach the penis. If a similar allocation of vasodilator output is present in man, preservation of finer branches of the pelvic plexus would be important in surgical procedures on the prostate.

Preganglionic fibers in the rat hypogastric nerve project bilaterally to pelvic ganglia.

Stimulation of the hypogastric nerve (HGN) often evokes bilateral responses in some pelvic organs. Retrograde labeling studies indicate that axons of postganglionic neurons often cross to the opposite side. However, there is little information available as to whether preganglionic fibers in the HGN have a contralateral projection to pelvic ganglia. A retrograde tracer was injected into the left major pelvic ganglion (MPG) in rats receiving various lesions of preganglionic nerves (HGN and pelvic nerve, PN). The lumbar spinal cord was then examined for location and number of dye-filled neurons. In a second approach, the incidence of synaptophysin immunoreactivity (SN-IR) perineuronal profiles (baskets) was examined in the MPG and in the accessory pelvic ganglia (APG) after nerve lesions. Labeled neuronal profiles were found in spinal cord nuclei (Lumbar1-2) after dye injection of the MPG in animals with an intact contralateral HGN. Cutting both HGNs virtually eliminated dye labeling in the lumbar cord, as did severing commissural branches (CB) between pelvic ganglia (leaving the contralateral HGN intact). Some SN-IR baskets were found in the left APG when only the contralateral HGN was intact, but baskets were rare when all four preganglionic nerves were cut. It could not be determined whether the HGN projects to the contralateral MPG, since SN-IR baskets were numerous in the MPG even when all four nerves were cut. This study has shown that some preganglionic fibers in the HGN synapse on neurons in contralateral pelvic ganglia. Both the APG and MPG receive contralateral innervation, but it is likely that neurons in the APG are the primary target of this input. Thus, in addition to crossing postganglionic fibers, a portion of the bilateral control of pelvic tissues is accomplished by preganglionic fibers which target autonomic neurons in contralateral ganglia.

Cytochrome oxidase staining in the major pelvic ganglion of the male rat.

Cytochrome oxidase staining was used as a marker of metabolic activity in neural elements in the rat major pelvic ganglion. Many neurons in the ventral pole of the ganglion have little cytochrome oxidase activity, while neurons in other locations show gradations in staining intensity. Punctate staining around principal neurons may represent preganglionic terminals, since it was greatly reduced after denervation of the ganglion. Image analysis was used to compare neuronal size to staining intensity. There was a negative correlation between cell size and staining intensity; the largest neurons were only lightly stained for cytochrome oxidase, while the medium and the small neurons showed a full range of metabolic activity. To study metabolic activity of an identified neuronal population, the seminal vesicles were injected with a retrograde tracer. The largest seminal vesicles neurons (1500 to 3200 microns2) had low enzyme activity, whereas the majority of neurons to this organ were smaller with gradations in staining. These results are indicative of the metabolic activity of the autonomic innervation to various pelvic tissues. Cytochrome oxidase histochemistry should prove valuable in assessing the demands placed on autonomic ganglia in differing functional and dysfunctional states.

The sensory branch of the pudendal nerve is the major route for adrenergic innervation of the penis in the rat.

BACKGROUND: Multiple pathways have been proposed for the course of adrenergic fibers to the penis and, although it is generally recognized that the pudendal nerve (PudN) is the most important, there is little quantitative information available. METHODS: We used image analysis of catecholamine histofluorescence to quantify the effect of various nerve lesions on the adrenergic innervation of the rat penis. In addition to the denervation studies and as a direct test of whether penile adrenergic fibers traversed the pelvic plexus, penile neurons in the sympathetic chain were first labeled with a retrograde dye placed in the penis. The cavernous nerve of these animals was later exposed to another dye with different spectral characteristics. RESULTS: Interruption of the sensory branch of the PudN reduced adrenergic innervation of cavernosal smooth muscle by 86% (+/- 2.5%). Vascular fibers of the deep penile and helicine arteries were also severely reduced but not entirely eliminated. Interruption of the motor branch of the PudN had a lesser and more variable effect on penile adrenergic innervation: a 21.2% (+/- 6.8%) decrease in cavernosal muscle innervation but no obvious affect on vasomotor fibers. Combining the nerve lesions with phenol degeneration of perivascular fibers of the pudendal vessels further reduced but did not entirely eliminate adrenergic fibers in the cavernosal muscle and penile vessels. CONCLUSIONS: The dramatic reduction of adrenergic innervation of the penis after section of the PudN, especially the sensory branch, and the absence of double-labeled neurons in the sympathetic chain suggest that the PudN nerve is the major, if not the exclusive, pathway by which adrenergic fibers reach penile erectile tissue of the rat.

Denervation-induced changes in perineuronal plexuses in the major pelvic ganglion of the rat: immunohistochemistry for vasoactive intestinal polypeptide and tyrosine hydroxylase and histochemistry for NADPH-diaphorase.

To characterize further the injury response of autonomic ganglia, we have examined the effect of chronic denervation on perineuronal plexuses that are immunoreactive for vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase (TH) or that stain for nicotinamide adenine dinucleotide phosphate (NADPH) in the rat major pelvic ganglion, and their relationship to an identified sub-population of neurons in the ganglion (the penile neurons). Penile neurons contain VIP and NADPH diaphorase (NADPH-D) but lack TH. VIP-immunoreactive (VIP-IR) and TH-IR perineuronal plexuses (baskets) are rare in the rat major pelvic ganglion and those present are not associated with penile neurons. A small increase in VIP-IR baskets occurs 2 weeks after proximal interruption of the pelvic nerve, but TH-IR baskets increase five-fold. The emergent VIP-IR and TH-IR baskets enclose TH-negative neurons, none of which are penile ganglion cells. These changes remain up to 4 weeks after denervation. Interrupting the pelvic nerve nearer the margin of the major pelvic ganglion results in a rapid, more dramatic increase in VIP-IR, in cell bodies and beaded fibers, than that seen with the more proximal lesion. About 27% of neurons in the ventral pole of the ganglion are enveloped by NADPH-D perineuronal baskets. The incidence of NADPH-D baskets falls to less than 1% after acute interruption of the pelvic and hypogastric nerves, but their frequency returns to control levels in chronically denervated ganglia. The rapid, vigorous changes in peptide (VIP) fibers after the pelvic nerve is cut close to the major pelvic ganglion may be attributable to the interruption of axons of postganglionic neurons and to preganglionic nerve fibers, whereas the slowly developing changes in VIP-IR and TH-IR fibers after more proximal lesions may represent the more modest effects of true decentralization. The source and significance of the VIP-IR, TH-IR, and NADPH-D baskets that appear in chronically denervated ganglia remain unclear.

Selective upregulation of arterial endothelial nitric oxide synthase in pulmonary hypertension.

We have previously demonstrated that arterial, but not venous, vasodilatory responses to endothelium-derived nitric oxide (EDNO)-dependent agonists are enhanced in lungs isolated from rats with chronic hypoxia (CH)-induced pulmonary arterial hypertension. These data suggest that CH is associated with increased endothelial nitric oxide synthase (eNOS) activity within the pulmonary arterial vasculature. In addition, the correlation of increased pulmonary arterial pressure with selectively enhanced arterial responsiveness to EDNO-mediated agonists suggests that arterial hypertension, rather than hypoxia per se, is a contributing factor in this response. Therefore, we hypothesized that 1) CH selectively upregulates eNOS within the pulmonary arterial vasculature and 2) monocrotaline (MC)-induced pulmonary arterial hypertension selectively enhances pulmonary arterial dilation to EDNO-dependent dilators and upregulates arterial eNOS. We examined the responses to the EDNO-dependent dilators arginine vasopressin and ionomycin in U-46619-constricted isolated perfused lungs from control and MC-treated rats. Microvascular pressure was assessed by the double-occlusion technique, allowing calculation of segmental resistances. Lungs from MC-treated rats exhibited augmented arterial dilation to arginine vasopressin compared with control lungs. However, the responses to ionomycin were not different between the two groups. Quantitative immunocytochemistry was used to compare pulmonary eNOS immunoreactivity in vessels from control, CH, and MC-treated rats. eNOS staining was more intense in the arteries of CH and MC-treated rats compared with those of control animals, whereas CH and MC treatment had no effect on eNOS staining in veins. We conclude that pulmonary arterial hypertension, or altered vascular mechanical forces associated with hypertension, may be responsible for the augmented EDNO-dependent arterial dilation and upregulation of arterial eNOS in lungs from CH and MC-treated rats.

The pelvic plexus: innervation of pelvic and extrapelvic visceral tissues.

The pelvic plexus is an association of neurons that govern visceral tissues involved in eliminative and reproductive functions. It is the singular site in the autonomic nervous system where sympathetic and parasympathetic neurons occur in the same ganglia. Within the plexus, ganglia are not randomly positioned; sympathetic neurons tend to occur more ventrally while parasympathetic neurons are located more dorsally, both in accordance with the location of their target tissues and the entry point of their corresponding preganglionic nerve tracts. For example, the vas deferens and seminal vesicle are ventral in position and thus are innervated by more ventrally located pelvic neurons. Neurochemical studies of pelvic ganglia indicate that there are some characteristic associations of putative neurotransmitters which are based on target organ distribution and in part, dictated by the variety of target tissues within each organ. Penile neurons comprise a uniform population in that they are cholinergic and also may release vasoactive intestinal polypeptide (VIP) and nitric oxide. In contrast, target tissues of the internal genitalia are more diverse, requiring adrenergic and nonadrenergic innervation and a complementary neuropeptide. Preganglionic innervation may also be coded and although sympathetic and parasympathetic fibers are cholinergic, they may differ in respect to neuropeptides and nitric oxide. Sensory neuron collaterals may also influence principal neurons as do intrinsic neurons such as small intensely fluorescent cells. Transmission through pelvic ganglia may be simple as is apparent in penile innervation, or shows a greater integrative capacity, as exemplified by the innervation of the urinary bladder. The extent of interaction of sympathetic and parasympathetic pathways at the level of the pelvic plexus remains largely unknown.

Neural and endothelial nitric oxide synthase activity in rat penile erectile tissue.

NADPH-diaphorase (NADPH-D) activity and immunoreactivity for neural and endothelial nitric oxide synthase (nNOS and eNOS, respectively) were used to investigate nitric oxide (NO) regulation of penile vasculature. Both the histochemical and immunohistochemical techniques for NOS showed that all smooth muscles regions of the penis (dorsal penile artery and vein, deep penile vessels, and cavernosal muscles) were richly innervated. The endothelium of penile arteries, deep dorsal penile vein, and select veins in the crura and shaft were also stained for NADPH-D and eNOS. However, the endothelium of cavernous sinuses was unstained by both techniques. Fewer fibers were seen in the glans penis, those present being associated with small blood vessels and large nerve bundles near the trabecular walls. All penile neurons in the pelvic plexus, located by retrograde transport of a dye placed in the corpora cavernosa penis, were stained by the NADPH-D method. Essentially similar results were obtained with an antibody to nNOS. These data suggest that penile parasympathetic neurons comprise a uniform population, as all seem capable of forming nitric oxide. However, in contrast to the endothelium of penile vessels, the endothelium lining the cavernosal spaces may not be capable of nitric oxide synthesis.

The effect of chronic decentralization on the enkephalin immunoreactive plexus around penile ganglionic neurons.

Target organ responses to sympathetic nerve stimulation are altered following partial decentralization of the pelvic plexus in the rat. One possible explanation for the new responses is that nerve injury has led to a reorganization of synaptic connections within pelvic ganglia. Since one measure of synaptic influence is the occurrence of a pericellular plexus of varicose fibers around autonomic ganglion cells, the present study has used immunocytochemistry for enkephalin (ENK), a peptide present in nerve fibers in the pelvic plexus, to follow changes in the innervation of penile ganglionic neurons after interruption of preganglionic pathways. Penile ganglion cells were located by the injection of the tracer Fluorogold into the penile crura. Four days after lesion of the pelvic nerve, innervation of penile neurons falls from 76% to 20%. This number increases however, to 31% in chronically (6 weeks) lesioned animals. In the totally decentralized ganglia, ENK immunoreactive (IR) fibers enclose fewer than 12% of the penile neurons 4 days after nerve lesion. However, this value rises to 35% in the chronically decentralized pelvic ganglion. Therefore, recovery of an enkephalin plexus occurs irrespective of whether the pelvic nerve, or both the hypogastric and pelvic nerve have been cut. Although these findings suggest sprouting within partially decentralized ganglia, the similar incidence of an ENK plexus in ganglia subjected to chronic partial or total decentralization indicates that preganglionic fibers are not responsible for the emergent fibers.

Expression of nitric oxide synthase by syncytiotrophoblast in human placental villi.

The endogenous biosynthesis of nitric oxide (NO) is increased during gestation. To begin our investigation of a possible tissue source (or sources), we examined the placenta. We postulated that analogous to the endothelium of blood vessels, the syncytiotrophoblast (STr) cell layer that lines the intervillous blood space of the human placenta would express NO synthase. Our results show that human placental villi express a calcium- and calmodulin-sensitive form of NO synthase, located mainly in the microsomal cell fraction. By in situ hybridization using a riboprobe generated from human endothelial NO synthase cDNA, we observe NO synthase mRNA expression in STr. The STr also shows NADPH-diaphorase staining, indicating the presence of NO synthase, and most likely other flavin-containing enzymes involved in sex steroid metabolism. NO synthase activity was also detected in the villi of a complete mole placenta (which lacks fetal vessels), further supporting a trophoblastic origin. Our findings suggest a previously unrecognized role for STr-derived NO in placental function.

NADPH diaphorase innervation of the rat anococcygeus and retractor penis muscles.

NADPH diaphorase histochemistry was used to determine whether the rat anoocccygeus (AC) and retractor penis (RP) muscles are innervated by nerves capable of synthesizing nitric oxide. In both tissues, muscle fascicles were enclosed by a varicose plexus of NADPH diaphorase positive (ND+) fibers. Perikarya of neurons on the surface of the AC muscle were also intensely stained for NADPH diaphorase. Many AC-RP ganglion cells in the pelvic plexus, located by the retrograde tracer Fluorogold, also stained for the enzyme. However, a significant population of AC-RP neurons in this location remained unstained. These results provide further evidence that nitric oxide may be an important neurotransmitter in these tissues.

The vasculature of the rat penis: a scanning electron microscopic and histologic study.

As a basis for understanding the mechanism of erection in an animal model frequently used in research in reproductive biology, the angioarchitecture of the penis of the rat has been described using scanning electron microscopy. Study of the penile vasculature of the rat indicates that the corpora cavernosa penis and the corpus spongiosum are independent erectile tissues, each with its own arterial and venous vessels. The large vascular spaces and abundant smooth muscle of the penile crura are compatible with its role in regulating blood flow to more distal penile tissues. Helicine arteries of the crura, but not the parent deep penile artery or arteries elsewhere, have muscular cushions in their walls. The venous drainage of the penile crura is via subtunical veins which are thought to be compressed during erection to elevate pressure within the penis. Large, paired cavernous veins drain the shaft of the penis. A unique method for inhibiting blood flow from the penis is indicated by the division of the cavernous veins into smaller channels prior to joining the subtunical venous plexus. Erectile tissue in the bifid origins of the corpus spongiosum has abundant cavernous muscle, while in the remainder of the corpus spongiosum little smooth muscle lines the cavernous spaces. The cavernous spaces on either side of the urethra coalesce to form vessels, each of which communicates with cavernous spaces in the glans. In addition, a bypass of the glans is effected by communication of these vessels directly with the deep dorsal vein. The apparent absence of muscular pads in vessels of the spongiosum, the relative paucity of cavernous smooth muscle, and the ample venous drainage provided by the deep dorsal vein may account for the lack of a venous occlusive mechanism similar to that of the corpora cavernosa penis.

Distribution and origin of neuropeptide Y-immunoreactive fibers in the penis of the rat.

The present study investigated the distribution of neuropeptide Y-immunoreactive fibers to the penis of the rat. In the corpora cavernosa penis, a dense plexus of fibers was associated with arteries, intrinsic cavernosal muscle, and veins, including the deep dorsal vein. In the corpus spongiosum, immunoreactive fibers were present around vascular smooth muscle and at the periphery of the acini of the paraurethral glands. Immunohistochemistry of penile neurons identified by retrograde tracer injection into the penis indicates that about 5% of the penile neurons in the pelvic plexus contained the neuropeptide while larger percentages of penile neurons in the sympathetic chains were immunoreactive for neuropeptide Y. Chemical and surgical sympathectomy greatly reduced the neuropeptide Y- and catecholamine-containing fibers in the erectile tissue but had no clear effect on the neuropeptide Y fibers around the paraurethral glands; a tissue that is not innervated by adrenergic fibers. It is concluded that (1) the widespread distribution of neuropeptide Y indicates that it may function in the control of penile blood flow, (2) with the possible exception of the paraurethral glands, the sympathetic chain is the most likely source of neuropeptide Y fibers in both erectile bodies of the penis, and (3) this peptide may play a role in the secretory functions of the paraurethral glands.

The ischiourethralis muscle of the rat: anatomy, innervation, and function.

The ischiourethralis (IU), a striated perineal muscle presumed to be involved in sexual reflexes, was studied in the rat. The paired muscle arises from the penile crus and the penile bulb and unites in a raphe over the deep dorsal vein of the penis. Retrograde tracing studies show that the muscle is innervated by neurons in the dorsolateral nucleus of the lumbar spinal cord, a pudendal nerve motor nucleus which also innervates the ischiocavernosus muscle. Excision of the IU muscle did not interfere with the ability of males to display normal copulatory behavior, nor did it affect significantly the number and intensity of reflexive erections. It nevertheless remains possible that the IU may contribute to intense glans erection by compressing the deep dorsal vein.

Cortical microstimulation thresholds adjacent to sensorimotor cortex injury.

The initial severe contralateral impairment of motor function after unilateral damage to a portion of sensorimotor (SM) cortex lessens within a few weeks after injury. In this study, two hypotheses proposed to explain recovery of behavioral function after cortical injury were tested: (1) Intact cortex adjacent to the injury reorganizes to take over the function of the destroyed area. (2) Intact SM cortex adjacent or connected to the injured area undergoes a transient shock (diaschisis), and as this dissipates, some behavioral recovery occurs. Using microstimulation of the cortex of the adult rat, movements evoked from areas near cortical injuries were studied at various times after undercut laceration, contusion, or suction ablation of an area of SM cortex. Stimulation areas were compared to those obtained from uninjured control animals and to the contralateral uninjured hemisphere. No evidence was obtained for any reorganization of stimulated motor responses in the injured hemisphere even in animals followed for as long as 475 days postinjury, suggesting other mechanisms underlying functional recovery. In intact cortex at some distance from contusion and laceration injuries, there was a marked elevation of thresholds to evoke movements that returned to normal by 9-15 days postinjury. Some intact hindlimb responses were observed after contusion injury that were absent in animals after 15 days postinjury, indicating a slow-growing lesion after this type of trauma. Surprisingly, no elevation in thresholds was noted for ablation injuries up to the edge of the cavity at any time postinjury, indicating that threshold changes near the boundary may be uncorrelated with functional recovery.

Innervation of the anococcygeus muscle of the rat.

The innervation of the anococcygeus muscle of the rat was investigated with regard to the histochemical features of nerve fibers within the muscle and to the location of the postganglionic autonomic neurons which are the source of these fibers. Acetylcholinesterase-positive fibers and catecholaminergic fibers are abundant in the anococcygeus as well as the related retractor penis muscle. Neuronal somata, either between muscle bundles of the anococcygeus or in the connective tissue sheath, are also acetylcholinesterase-positive. Nerve fibers and a minority of the ganglion cells in the anococcygeus and retractor penis muscles are immunoreactive for vasoactive intestinal polypeptide. Injection of the retrogradely transported dye Fluorogold into the anococcygeus muscle filled neurons in the abdomino-pelvic sympathetic chain, pelvic plexus and a small number of neurons in the inferior mesenteric ganglion. In the pelvic plexus, some neurons were located in the major pelvic ganglion but most were found along the main penile nerve and its branches to the anococcygeus muscle. Immunocytochemistry of these identified neurons indicates that about one half of them are positive for vasoactive intestinal polypeptide. These results raise the possibility that both acetylcholine and vasoactive intestinal polypeptide are important neurotransmitters in autonomic nerves to the anococcygeus muscle.

Penile erection in the rat: stimulation of the hypogastric nerve elicits increases in penile pressure after chronic interruption of the sacral parasympathetic outflow.

Penile erection, a vascular event mediated by the autonomic nervous system, is often adversely affected by injury to the spinal cord. To further characterize the laboratory rat as an animal model of penile erection and to investigate erectile responses following neural injury, the present study has examined pressor penile responses in intact rats and in animals deprived of sacral parasympathetic outflow. Increases in penile pressure result from graded stimulation of postganglionic parasympathetic fibers. The vasodilator response is insensitive to blockade with atropine, a cholinergic antagonist. Penile tumescence also results from stimulation of the pelvic nerve, but not the hypogastric nerve. However, beginning 3 days after unilateral interruption of the pelvic nerve, stimulation of the ipsilateral hypogastric nerve results in an increase in penile pressure. This novel response, which is blocked by a ganglionic antagonist, is maximally developed at 1 week post-lesion, is stable for at least 3 months and remains confined to the side of the lesion. These results suggest that the rat, although relatively small, can be used to obtain quantitative data on penile erection. Moreover, the model may lend itself to an analysis of the mechanisms of altered control of visceral tissues following injury to the nervous system.

Autonomic innervation of reproductive organs: analysis of the neurons whose axons project in the main penile nerve in the pelvic plexus of the rat.

An understanding of the composition of the various nerves of the pelvic plexus is essential in the design of studies to explore the autonomic control of pelvic visceral tissues. As a correlate of this interest, the present study was designed to determine the composition of the main penile nerve in the pelvic plexus of the laboratory rat, an animal commonly used for studies of reproductive physiology. Retrograde tracing studies indicate that the main penile nerve contains neurons which project to the penile crura, the corpus spongiosum, and the bulbourethral glands. The main penile nerve is the major source of neurons which innervate the corpus spongiosum and bulbourethral gland and contains about one-third of all parasympathetic neurons which project to the penile crura. Dye placed on the proximal cut end of the main penile nerve indicates that neurons in the parasympathetic region of the spinal cord (L6-S1) and to a lesser extent a sympathetic region of the cord, L1-L2, provide preganglionic innervation to ganglion cells in the main pelvic nerve. Processes of neurons in dorsal root ganglia L6-S1 and of neurons in the abdominopelvic sympathetic chain course in the main penile nerve to unknown destinations. In many respects this presumed postganglionic fiber tract is essentially a region of the pelvic plexus which subserves extrapelvic visceral tissues.

Parasympathetic nerves in penile erectile tissue of the rat contain choline acetyltransferase.

Choline acetyltransferase (ChAT), a biochemical marker of cholinergic neurons, was measured in the erectile tissue of intact rats and in rats in which postganglionic fibers from the pelvic plexus were interrupted. ChAT activity in the denervated erectile tissue fell by 56% compared to control tissues. Acetylcholinesterase positive (AChE+) nerves also fell by about 48%. Penile neurons distal to the lesion probably account for the residual ChAT activity and remaining AChE+ nerve fibers in erectile tissue. These results indicate that acetylcholine is an important neurotransmitter in the regulation of penile erection.

Autoradiographic evidence of muscarinic cholinergic receptors in the corpora cavernosa penis of the rat.

Recent studies have questioned the role of acetylcholine in the physiology of penile erectile tissue. The responsiveness of penile erectile tissue to acetylcholine would depend, in part, on the presence of cholinergic receptors on the smooth muscle. The specific binding of [3H]quinuclidinyl benzilate (QNB) to cholinergic receptors in sections of penile crura of the rat was analyzed by in vitro neurotransmitter autoradiography. Silver grain density measurements indicated that muscarinic cholinergic receptor binding sites are located almost entirely over the corpora cavernosa penis. Virtually no specific [3H]QNB binding was present in the tunica albuginea or adjacent skeletal muscle tissue. Within the erectile tissue, specific binding occurred both over the columns of intrinsic smooth muscle which form the walls of the cavernous spaces and around the more distal branches of the penile arteries. The high concentration of muscarinic cholinergic receptors in the corpora cavernosa penis is consistent with the suggestion that acetylcholine has an important, albeit undefined role in the function of penile erectile tissue.