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Participation of Paraventricular Nucleus of Hypothalamus in Central Regulation of Penile Erection in the Rat
0022-5347B71158 1-0238$03.00/0
Vol. 158, 238-244, July 1997 Printed i n U.S.A.
THEJOIVISAI. OF UHOUIGY Copyright 0 1997 by AAIKHI(,LY UROUK;I( hi. A..IX.IATION. 1w
PARTICIPATION OF PARAVENTRICULAR NUCLEUS OF HYPOTHALAMUS IN CENTRAL REGULATION OF PENILE ERECTION IN THE RAT KUANG-KUO CHEN, SAMUEL H. H. CHAN, LUKE S. CHANG
AND
JULIE Y. H. CHAN*
From the Dtcwion of Urologv, Department of Surgery, and Department of Medical Research, Veterans General Hospital-Taipec. and the Department of Urologv. and Center for Neurosclence, Natronal Yang-Mmg University, Taipei, T a i u m , Republic of China
ABSTRACT
Purpose: To investigate the possible participation of the paraventricular nucleus of hypothalamus in central regulation of penile erection. Materials and Methods: Male adult Sprague-Dawley rats were anesthetized and maintained with pentobarbital sodium. The intracavernous pressure (ICP) was used as a n experimental index for penile erection, and was recorded alongside systemic arterial pressure and heart rate. The effect on ICP of electrical (30-s train of 30-120 d, 40-160 Hz, 1-ms rectangular pulses) or chemical (L-glutamate, 0.5 nmoV50 nl.) activation of the paraventricular nucleus of hypothalamus (PVN)o r hippocampal formation was evaluated. Results: Electrical activation of the PVN elicited both multiple and single episodes of elevation in ICP, along with visible erection and ejaculation. The former pattern exhibited a n increase in ICP that was more sustained, with higher peak amplitude and longer latency. Chemical stimulation of neuronal perikarya in the PVN also resulted in similar patterns of rise in ICP and visible erection. These effects were, nonetheless, not accompanied by significant alterations in systemic arterial pressure and heart rate. Activation of the hippocampal formation, a s we reported previously, similarly elicited multiple and single episodes of increase in ICP. These erectile responses, however, were substantially reduced or eliminated upon electrolytic lesion of the ipsilateral PVN. Conclusion: These observations suggest that the PVN may be an important nucleus that participates in central neural regulation of penile erection in the rat. Furthermore, a n efferent pathwayk) from the hippocampal formation to PVN may constitute part of the neural circuitry in the forebrain in the regulation of erectile functions. KEY
WORDS: paraventricular nucleus of hypothalamus, hippocampal formation, intracavernous pressure, penile erection
During the past two decades, studies which focused on the vascular organization of the penis have greatly advanced our understanding of the hemodynamics and pharmacology of penile erection.'-4 As such, it is now recognized that penile erection is the result of a n increase in arterial blood inflow to the penis, with subsequent distension of sinusoids and restriction of venous outflow in the corpora cavernosa.2 Intracavernous administration of smooth muscle relaxants, such a s papaverine hydrochloride,"" prostaglandin El,".fi or a-adrenergic blocker, p h e n t ~ l a m i n einduces ,~ penile erection in human and animals. Although the vascular mechanisms of penile erection are well established, the mechanism of neurologic regulation of penile erection is, on the other hand, less documented.8.9 I t is generally considered that penile erection is regulated by opposing peripheral sympathetic and parasympathetic neural outflows to penile vascular smooth muscles via the action of acetylcholine and noradrenaline..',g--'' Whereas adrenergic neurotransmission mediates contraction of corporal smooth Accepted for publication February 18, 1997. * Requests for reprints: Department of Medical Research, Veterans General Hospital-Taipei, Taipei 11217, Taiwan, Republic of
China. Supported by grants NSC82-0115-B-075-125,NSC83-0412-B-075105 and NSC84-2331-B-075-026from the National Science Council, VGH-267 from the Veterans General Hospital-Taipei. and DOH83HR-221 from the National Health Research Institute, Taiwan, Republic of China.
muscles and causes detumescence, cholinergic neurotransmission promotes smooth muscle relaxation and penile erection. In addition, non-adrenergic non-cholinergic neural mechanisms are also involved in the regulation of the erectile process.2.4-10 One possible mediator of the non-adrenergic non-cholinergic system is nitric oxide.12 Our current knowledge with regard to central neural control of penile erection is sparse at best.8 MacLean and P100g13 reported that penile erection was observed after electrical stimulation of the paraventricular nucleus of hypothalamus (PVN) in the squirrel monkey. Intraventricular administration of apomorphine or oxytocin was reported to induce visible penile erection in rats,4.*4-'" with the PVN proposed to be the site of action.16-18 In these investigations, penile erection was assessed visually by counting the number of erectile episodes. Our laboratory has established recently a rat mode1,6 which uses the intracavernous pressure (ICP) in anesthetized rat as the index for a more sensitive, objective and quantitative assessment of penile erection. Utilizing this experimental index, we have demonstrated that t h e hippocampal formation is a n important locus in the forebrain for neural regulation of penile erection.19 The present study was undertaken to further explore the participation of the PVN as a neural substrate in central regulation of the erectile process, and its relationship with hippocampal formation in the promotion of penile erection. Our results are consistent with the notion that the PVN may
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PVN AND PENILE ERECTION be an important brain nucleus in the elicitation of penile erection in the rat. Furthermore, an efferent pathway(s)from the hippocampal formation to PVN may constitute part of the neural circuitry in the forebrain that subserve regulation of erectile functions. MATERIALS A N D M E T H O D S
Animals. Male, adult Sprague-Dawley rats (200-300 gm.) anesthetized with pentobarbital sodium (50 mg./kg., i.p., with 10 mg./kg./hr. i.v. supplements) were used. Routine surgical preparations included cannulation of the left femoral artery and vein for the measurement of systemic arterial pressure and continuous infusion of supplemental anesthetic. Systemic arterial pressure was monitored via a pressure transducer (Gould 23ID) and a pressure processor amplifier (Gould 13-4615-52). Heart rate was determined using a biotachometer amplifier (Gould 13-4615-65) triggered by the arterial pulses. An endotracheal tube was established for a patent airway. The head of the rat was placed in a stereotaxic headholder (Kopf goo), with the rest of the body positioned on a heating pad and elevated to a suitable position. All data were collected from animals with a maintained rectal temperature of 37C, with a steady mean systemic arterial pressure above 90 mm. Hg throughout the experiment. Recording of intracavernous pressure. Measurement of ICP, which was used as the experimental index for penile erection, was essentially the same as described previously.6.9,19.20In brief, the lower part of the body was slightly rotated for adequate exposure of the genital area. The penis was degloved to expose both corpora cavernosa. A 26-gauge needle, which was connected to a pressure transducer (Gould 23ID) via a PE-20 tubing filled with saline, was carefully inserted into one of the corpus cavernosa. The ICP thus recorded was monitored alongside systemic arterial pressure and heart rate on a polygraph (Gould ES 1000). Intracavernous administration of saline (250 pl.) was routinely executed a t the beginning of the experiment to ascertain the lack of leakage. At the end of each experiment, papaverine (400 pg. ), a vasoactive agent most commonly used in clinical management of impotence,2 was delivered intracavernously to ensure that the recording needle for ICP has been properly lodged into the corpus cavernosum throughout the experiment, and the cavernous tissue still manifested adequate responsiveness. Electrical activation of the PVN or the hippocampal formation. Electrical stimulation of the PVN or hippocampal formation was delivered using a stainless-steel bipolar concentric electrode (Rhodes Medical SNE-100). The stereotaxic coordinates for PVN were 1.5-2.0 mm. posterior to the bregma, 6.0-7.0 mm. below the dural surface and 0.5-1.0 mm. lateral to t h e midline. The coordinates for hippocampal formation were 1.8-2.6 mm. posterior to the bregma, 3.0-4.5 mm. below the dural surface and 0.5-1.5 mm lateral to the midline. The PVN or hippocampal formation was electrically activated by cathodal rectangular current pulses (30-s train of 1-ms pulses, at 30-120 pA and 40-160 Hz), using a Grass S88 stimulator coupled with a constant-current isolation unit (Grass PSIU6). For experiments in which both PVN and hippocampal formation were stimulated, the electrodes were routinely positioned on the same side. The effects of electrid stimulation of PVN and/or hippocampal formation on ICP, systemic arterial pressure and heart rate were continuously and simultaneously monitored. At the same time, the penis was carefully observed for the occurrence of visible erection and ejaculation. Chemical activation of the PVN. To ensure that the response in ICP to electrical activation of the PVN was not due to stimulation of fiber of passage to this hypothalamic nucleus, L - g l u w a t e (0.5 nmol), was microinjected into the PVN for p*wd activation.21 Microinjection of this
excitatory amino acid was carried out with a glass micropipette (tip diameter: 50-80 pm.), which was connected to a 1-p1. Hamilton microsyringe. The stereotaxic coordinates used were the same a s for electrical stimulation. A total volume of 50 nl. was delivered to the PVN over 1-2 rnin. to allow for full diffusion of the chemical. l'%Evans blue was added to the microinjection solution to aid in histological identification. The temporal effects of chemical activation of the PVN on ICP, systemic arterial pressure and heart rate were monitored for a t least 60 min. Electrolytic lesion of the PVN. For this series of experiments, a bipolar concentric stainless-steel electrode (Rhodes Medical SNE-100) was used to identify a site each in the ipsilateral hippocampal formation and PVN that induced significant elevation in ICP upon electrical activation. An electrolytic lesion of the same site in the PVN was subsequently carried out, using a n anodal current of 1 mA for 10 s. The effects of electrical stimulation of the individual sites in the PVN and hippocampal formation, using exactly the same stimulus parameters, were again evaluated after lesioning the PVN. Histology. At the conclusion of each experiment, the brain was removed and fured in 30% sucrose in 10% formaldehydesaline for at least 48 hr. 25-pm. frozen sections stained with either cresyl violet or neutral red were used for histological verification of the location of stimulation, microinjection or electrolytic lesion sites in the PVN or hippocampal formation. Statistical analysis. Data were presented as means z S.E.M. The effects of electrical activation of the PVN on the peak amplitude, duration or the onset latency of the rise in ICP were statistically assessed using one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls multiple range test for a posteriori comparison of means. p < 0.05 was considered statistically significant. RESULTS
Effect of electrical activation of the PVN on ICP. In agreement with our previous findings,".'S the resting ICP in our animals was 4.6 % 0.5 mm. Hg (n = 35). Upon electrical stimulation of PVN, there was a significant elevation in ICP, which exhibited at least five response characteristics. First, there were two basic response patterns: multiple episodes (fig. 1, A ) and single episode (fig. 1, B ) of rise in ICP. In some
200,
.
____-
200
mm
6mn
8 -
FIG. 1. Two response patterns, multiple episodes (A)and si e isode (B)of increased intracavernous pressure (ICP), eliciteii; ereetrical stimulation of paraventricular nucleus of hypothalamus. Arrows denote short duration pressure spikes at peak of ICP.Note lack of simultaneous change in systemic arterial preseure (SAP)and heart rate (HR) immediately before or during period of elevation of ICP in both instances.
240
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PVN AND PENILE ERECTION
incidences, several short duration pressure "spikes" were detected in the pressure peak of both episodes of rise in ICP (figs. 1, A and 3). The elevation in ICP during multiple episodal response was more sustained, and displayed higher peak amplitude and longer onset latency (see table). Histologic examinations, however, revealed no topographic distribution of sites of stimulation that elicited multiple or single episodes of elevation in ICP. These sites overlapped with each other in the magnocellular and parvocellular subnuclei of the PVN (fig. 2). Second, the elevation of ICP was related to the stimulus intensity a n d o r frequency of PVN activation. We found that at a fixed stimulus train pulse frequency and duration, increasing the stimulus intensity of PVN activation produced greater elevations in ICP (fig. 3, upper panels). Similarly, a t a fixed stimulus train pulse intensity and duration, there was heightened increase in ICP upon activation of PVN with higher stimulus frequencies (fig. 3, lower panels). Third, visible penile erection was observed in some animals during single or multiple episodes of increase in ICP (see table ), at a threshold pressure of approximately 40 mm. Hg. During these instances, tumescence of the glans and shaft of penis was observed at the initial phase of ICP elevation. As ICP further increased to above 40 mm. Hg, intense erection of the glans (cup) and of the shaft, as well as anteroflexion of the penis (flip) due to a straightening of the penis with disappearance of the flexura penis between the shaft and glans (erectionP were displayed. Some of these events were accompanied by ejaculation (see table ), demonstrated by expulsion of urethral contents and production of coagu-
30 s
zloo1 E
100,
,' ,A&
120 vA. 80
Hz. I ms
120 U A . 120
3v-
Comparison of characteristics of single and multiple episodes of increase in intracavernous pressure (ICP) upon electrical actwation of paraventricular nucleus of hypothalarri us ~~
Single Episode
Multiple Episodes ~~
Resting ICP lmm. Hg) Peak increase in ICP (mm. Hg)
4.7 2 0.5 (2-81 53.5 t 9.8
Onset latency Is)
28.8 2 7.5
(12-100) Duration lmin.1
..-
1 rns
FIG.3. Relationship between stimulus parameters of electrical stimulation of paraventricular nucleus of hypothalamus and response in intracavernous pressure (ICP). Arrows denote short duration pressure spikes at peak of ICP.
(2-801
rn
Hz.
7.4 2 2.7 ( 1.5-20)
-
~-
4.5 t 0.4 ( 3-7 77.7 t 8.3I 35-100 I 41.9 Z 11.6' 16-1601 13.7 I 4.7(2-55 I 10 111 4 110
,
Visible erection 6 111 Ejaculation following visible 3 16 erection Mean z S.E.M. and range. * p < 0.05, compared with single episode of increase in ICP, n per group. ~
-1.8
.2.1
2
ME 0
?'
mm
FIG.2. Diagrammatic representation of two rostral-caudal levels of paraventricular nucleus of hypothalamus (PVN) relative to bregma, illustrating sites upon which microinjection of L-glutamate ( A )or electrical stimulation elicited single (0) and multiple ( 0 ) episodes of increase in intracavernous pressure (ICP). Although active sites were bilaterally re resented, they are indicated on one side of diagram for clarity. &tes upon which microinjection of L-glutamate or electrical stimulation were ineffective were indicated by (W). Abbreviation: AHC, anterior hypothalamus centralis; AHP, anterior hypothalamus posterialis; ME, median eminence; PaMP, subnucleus parvocellularis medialis; PaLM, subnucleus mangocellularis; P a m , subnucleus parvocellularis posterialis; PaV, subnucleus parvocellularis ventralis; Pe, periventricular nucleus; SO, supraoptic nucleus; VMH, ventral medial hypothalamus; f, fornix: opt, optic tract; sox, supraoptic decussation; 3V, third ventricle.
= 11 animals
lated ejaculates (plugs). In two occasions, sperms were detected under microscopic examination in the urethral secretion. Fourth, the erectile effect upon PVN activation was not associated with significant changes in mean systemic arterial pressure (100.0 -t 5.0 to 96.3 t 5.1 mm. Hg, n = 22) or heart, rate (379.3 t 10.0 vs 379.3 % 9.9 bpm, n = 22) before and during the elevation in ICP (fig. 1).Fifth, activation of sites outside the contour of PVN (fig. 2) elicited no significant change in ICP. This was not attributed to end-organ failure, however, since the penis still responded positively to intracavernous injection of papaverine (0.4 mg., 250 PI.) with an increase in ICP (52.6 2 7.0 mm. Hg, n = 6). Effect of chemical activation of PVN on ICP. We also ascertained with chemical activation that penile erection induced by electrical stimulation of the PVN was due mainly to perikaryal activation of the PVN, and not fibers of passage.21 As exemplified by fig. 4, microinjection of L-glutamate (0.5 nmol, 50 nl.) into the PVN induced a multiple episodal rise in ICP, with an onset latency of 62 s and a peak effect of 96 mm. Hg. This elevated ICP persisted for approximately 40 min before returning to the baseline level. Once again, visible penile erection was observed during multiple episodes of increase in ICP. Similar results were obtained in 4 experiments. In addition to multiple episodes, microinjection of L-glutamate into the PVN also elicited single episode of ICP elevation, accompanied by visible penile erection ( n = 3). Histologic examination demonstrated that the injection sites were located in both magnocellular and parvocellular subnuclei of the PVN (fig. 2).
24 1
PVN AND PENILE EHECTlON
E
10 min
u loo
1
PVN
lesion
After
PVN
lesion
3 r s
701
" 01 0
Before
-
-
Hippocarnpus
PVN
FIG. 6. Single episodes of change in intracavernous pressure (ICP) upon electrical stimulation of CA1 area in hippocampal formation and paraventricular nucleus of hypothalamus (PVN)before and after electrolytic lesion of ipsilateral PVN. Note single episode of increase in ICP was eliminated upon electrical stimulation of hippocampal formation after lesion of PVN. 4 0 min
FIG.4. Time-course changes in intracavernous pressure (ICP), systemic arterial pressure ( S A P )and heart rate (HR) following microinjection of L-glutamate (0.5 nmol, 50 nl.) into paraventricular nucleus of hypothalamus. Note again lack of simultaneous hemodynamic changes before and during period of increased ICP.
Relationship between hippocampal formation and PVN in the elicitation of penile erection. Since the characteristics of PVN-induced elevation in ICP greatly resembled that elicited by hippocampal activation,'" we further delineated the relationship between these two forebrain nuclei in penile erection. Comparable to our previous findings,lS electrical activation of the granule cells at the dentate gyms (fig. 5 ) or pyramidal cells at the CA1 or CA3 field of the Ammon's horn (fig. 6 ) resulted in, respectively, multiple episodes or single episode of rise in ICP. Both patterns of elevation in ICP t o hippocampal activation were abolished after electrolytic lesion of the ipsilateral PVN (figs. 5 and 6 ) .At the same time, electrical activation of the same site in the PVN that was lesioned no longer induced significant effect on ICP. Complete elimination of penile erection elicited by stimulation of the hippocampal formation following lesioning the
A f f e r P V N lesion
30-5
a
0
1 HippocamPuS
PVN
FIG. 5. Multiple episodes of change in intracavernous pressure (ICP) upon electrical stimulation of dentate gyrus in hippcam a1 formation and paraventricular nucleus of hypothalamus (PVN) fore and after electrolytic lesion of ipsilateral PVN. Note multiple episodes of increase in ICP were eliminated u on electncal stimulatlon of hippocampal formation aRer lesion of bVN.
ipsilateral PVN (fig. 7 ) was demonstrated in 11out of 14 rats. Substantial reduction in amplitude a n d o r duration of erectile response to stimulation of the hippocampal formation was observed in the remaining animals. Thus, it appears that penile erection promoted by hippocampal formation and PVN took place in a serial fashion. D I S C U S S I 0N
Utilizing ICP as the experimental index for a quantitative evaluation of penile erection,",". 19.2" the present study demonstrated that PVN plays an important role in neural regulation of erectile functions. We found that both electrical and chemical stimulation of the PVN resulted in an increase in ICP. On the other hand, physical elimination of the ipsilatera1 PVN eliminated or significantly diminished the erectile response promoted by activation of the hippocampal formation. These observations suggest that a n ipsilateral efferent pathway(s) from hippocampal formation to PVN may constitute part of the neural circuitry in the forebrain that is involved in the regulation of penile erection. Our study benefited greatly by using the ICP as the experimental index for penile erection. This index allowed us to detect changes in ICP with a sensitivity of 2 mm. Hg.6 As a result, we were able to identify the minimal peak amplitude of ICP upon activation of the PVN to be 12 mm. Hg (see table). Although this is already more than 100% increase over the baseline value, it is still much below 40 mm. Hg,*9 which is the threshold ICP for visible erection, the basis for behavioral evaluations. Thus, ICP monitoring records not only the presence or absence of erectile events, but also the degree of erection attained.22By continuously monitoring the fluctuations in ICP, we were also able to define the latency, duration and temporal patterns of alterations in ICP following activation of the PVN and hippocampal formation. In addition, the demonstration that activation of the PVN elicited a n elevation in ICP without significant changes in systemic arterial pressure and heart rate immediately dissociated the erectile response from indirect, hemodynamic, influences. A hypothalamic-hippocampal oxytocinergic pathway has been proposed to mediate the penile erection induced by application of apomorphine to the PVN.16-18.23 The present study provides evidence to suggest that a novel hippocampalhypothalamic linkage may also participate in central neural regulation of penile erection. We found that activation of the PVN elicited multiple and single episodes of elevation in Icp that resembled those produced by hippocampal activation.1s
L-
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PVN AND PENILE ERECTION
hypothalamic areas that subserve regulation of penile erection cannot be ruled out. In t h e preliminary experiments, however, we found t h a t lesioning the anterior hypothalamus, which did not significantly alter ICP upon electrical stimulation, resulted in no appreciable effect on t h e elevation in ICP in response to stimulation of the hippocampal formation, I t is noteworthy t h a t we found no topographic distribution of stimulation sites in t h e subnuclei of PVN in the expression of the two patterns of increase in ICP (fig. 2). This was in contrast to our previous finding'" in t h e hippocampal formation, viz, pyramidal cells in CA1 and CA3 areas a r e responsible for the single episode, a n d granule cells in dentate gyrus evoke multiple episodes. These differential results were interpreted to suggest a convergence of efferent projections from the CA1 and/or CA3 areas and dentate gyrus of the hippocampal formation to t h e PVN. At t h e same time, they also indicate a n integrative role for t h e PVN in t h e control of erectile functions by the forebrain. The identification of multiple and single episodes of rise in ICP to PVN activation is intriguing. Since the PVN serves as a n integrative area for autonomic and endocrine functions, it i s tempting to speculate that both neural and hormonal mechanisms may be involved in the elicited erectile process. In support of t h e neural mechanisms, a direct descending projection from the PVN to t h e spinal nucleus of t h e bulhocavernosus (SNB)and the dorsolateral intermediolateral nucleus (CLN)in lumbosacral region of t h e spinal cord h a s b w n established.","6.27 Axons from motoneurons of t h e SNB and CLN innervate t h e striated penile muscles, t h e bulbospongiosus (BS) and t h e ischiocavernosus (IC) muscles, t h a t are essential for penile erection in t h e rat.H.zH-31 Multiple cBpisodes of the rise in corpus spongiosum penis (CSP) pressure, another experimental index for objective quantification of penile erections,2' were found to coincide directly with rhythmical contraction of the IC and BS muscle^.^.^^,^^ Based on combined retrograde tracing and immunohistochemical staining, Wagner and Clemens32 demonstrated t h a t neurons FIG. 7. Diagrammatic representation of 3 levels of hypothalamus i n t h e PVN that project to the SNB contain neurophysin, the with reference to bregma, showing location of largest extent of ipsi- coproduct of oxytocin and vasopressin. Functionally, the lateral lesion of PVN region, which eliminated increase in ICP to descending oxytocinergic pathways from the PVN to the electrical stimulation of hippocampal formation. AH,anterior hypothalamic area; Arc, arcuate nucleus; DC. dorsomedial cap of para- lumbosacral spinal cord has been reported'" to mediate ventricular nucleus of hypothalamus; LH, lateral hypothalamic area; apomorphine-induced penile erection. Whether hormonal ME, median eminence; MP, medial parvocellular subnucleus of para- mechanisms are also involved i n the exhibition of multiple or ventricular nucleus of hypothalamus; PM, magnocellular subnucleus single episode of ICP increase to PVN activation, is not well of paraventricular nucleus of hypothalamus; PV, periventricular nucleus of hypothalamus; RCh, retrochiasmatic area; SO, supraoptic documented. However, serum level of oxytocin was shown to nucleus; VMH, ventromedial hypothalamic nucleus; f, fornix; opt, increase after sexual behavior in animal and human.:':' .Ii optic tract; sox, supraoptic decussation; 3V, third ventricle. Another important issue is the connection between the PVN and sympathetic and/or parasympathetic outflows involved in the initiation of penile erection. In this regard, The increase in ICP by PVN and hippocampal stimulation a p a r t from a direct connection to the sympathetic pregangliwas accompanied by visible penile erection and ejaculation i n onic neurons i n the spinal cot-d,9>10.36-3"neurons in t h e I'VN some circumstances. Lesioning t h e ipsilateral PVN pre- project to the dorsolateral funiculus indirectly via t h e reticvented penile erection induced by electrical stimulation of ular formation in pons a n d medulla.s6.87,3g Fibers originated the hippocampal formation. This suggested functional link from PVN neurons also project to t h e parasympathetic between hippocampal formation and PVN in the regulation preganglionic neurons i n the lumbosacral spinal cord."-"' w9 of penile erection is supported by the presence of neuronal Thus, it is possible that activation of t h e PVN excites the pathways from the hippocampus that exert a n excitatory descending fibers to the spinal cord, which in t u r n elicits (e.g. glutamatergic) influence on PVN neuron^.^.'^.'^ To- penile erection by modulating the peripheral autonomic outgether with the proposed participation of a hypothalamic- flows to tissues t h a t are engaged in t h e erectile processc,s.n.9 hippocampal oxytocinergic pathway i n apomorphine-induced Compare to t h e relatively short duration of penile erection penile erection,'" our present findings suggest t h a t a recip- reported previo~sly,~.9.20.22 the duration of increase in ICP rocal connections between the PVN and hippocampal for- following electrical or chemical activation of PVN w a s long in mation may constitute a neural circuitry in the forebrain the present study. This observation was not uncommon, since t h a t regulates penile erection. The significance of the we reported previously that electrical or chemical activation hippocampal-hypothalamic glutamatergic pathways t h a t a r e of the hippocampal formation induces a n elevation of ICP involved in the regulation of penile erection, however, re- t h a t lasts for at least 30 min.19 The symptomatology of penile quires further investigation. erection following microinjection of apomorphinelx or osytoThe experimental design for our lesion experiments called cin17 into the PVN begins within 5 min postinjection and for lesioning only loci in t h e PVN on which electrical stimu- lasts for more t h a n 60 min. As suggested above of a reciprocal lation elicited rise in the ICP. As such, the possibility for connections between t h e PVN and hippocampal formation, if formation to other is possible that such a long duration of rise'in ICP by Pm efferent projections from the hiDDOCamDal - ..
PVN AND 1’ENII.E EIIECTION s t i m u l a t i o n m a y involve a s u b s e q u e n t activation of‘ t h e hippocampal formation. A d i r e c t association of g l a n s erections w i t h s h o r t d u r a t i o n p r e s s u r e “spikes” of CSP p r e s s u r e p e a k has been s h o w n , c o n c u r r e n t with BS m u s c l e bursts.”‘),’” Thus, i t is t e m p t i n g t o s p e c u l a t e t h a t the s h o r t d u r a t i o n p r e s s u r e “spikes“ visible during the p e a k of b o t h single and m u l t i p l e episodes of ICP following PVN s t i m u l a t i o n (figs. 1 and 3) m a y be associated w i t h c o n t r a c t i o n s of the striated penile muscles, i n p a r t i c u l a r the BS muscles. In s o m e a n i m a l s , s t r a i g h t e n i n g of the p e n i s w a s i n d e e d observed d u r i n g p r o m i n e n t i n c r e a s e in ICP that c o m p r i s e d s e v e r a l short d u r a t i o n p r e s s u r e spikes. S i n c e w e did n o t s i m u l t a n e o u s l y record the electromyogram of the striated penile muscles, the validity of t h i s speculation a w a i t s f u r t h e r clarification. In addition, the p a t t e r n of i n c r e a s e s in ICP following PVN a c t i v a t i o n r e s e m b l e s that recorded during ex copula reflexes (e.g., u r e t h r o g e n i t a l reflex) in anesthetized m a l e rats.”.’o Under b o t h conditions, m u l t i p l e episodes of rise i n ICP, accomp a n i e d by d e t e c t a b l e penile erection and ejaculation, w e r e displayed. The mean a m p l i t u d e of p e a k i n c r e a s e during erectile e v e n t s w e r e also similar. Finally, the flaccid state of p e n i s u n d e r b o t h conditions w a s c h a r a c t e r i z e d by relatively low and stable ICP p r e s s u r e w i t h no s p o n t a n e o u s p r e s s u r e p e a k s . On the o t h e r h a n d , e x copula reflex fails to p r o d u c e s i n g l e episodal i n c r e a s e s in ICP. As s u c h , it is our feeling that a l t h o u g h s i m i l a r p e r i p h e r a l mechanisms m a y be responsible for reflexive and PVN-induced penile erection, a d d i t i o n a l s u p r a s p i n a l m e c h a n i s m s are involved in the latter responses. C o m p a r i n g t o the p e r i p h e r a l autonomic m e c h a n i s m s , o u r c u r r e n t understanding of the physiologic role of the central nervous system in the r e g u l a t i o n of penile erection i s f a r f r o m comprehensive. B y utilizing ICP as an experimental i n d e x for a m o r e sensitive and q u a n t i t a t i v e assessment of penile erection,6.20 the p r e s e n t s t u d y r e v e a l e d that the PVN m a y be an important f o r e b r a i n nucleus that mediates penile erection. We also demonstrated the existence of a hippocampalhypothalamic pathway in the neural circuitry for the regulation of erectile functions. REFERENCES
1. Lue, T. F., Takamura, T., Schmidt, R. A,, Palubinskas, A. J . and Tanagho, E. A,: Hemodynamics of erection in the monkey. J. Urol., 130: 1237, 1983. 2. Lue, T. F. and Tanagho, E. A,: Physiology of erection and pharmacological management of impotence. J . Urol., 137: 829, 1987. 3. Vardi, Y. and Siroky, M. B.: A canine model for hemodynamic study of isolated corpus cavernosum. J . Urol., 1 3 8 663, 1987. 4. Argiolas, A. and Melis, M. R.: Neuromodulation of penile erection: a n overview of the role of neurotransmitters and neuropeptides. Prog. Neurobiol., 47: 235, 1995. 5. Sarosdy, M. F., Hudnall, C. H., Erickson, D. R., Hardin,T. C. and Novicki, D. E.: A prospective double-blind trial of intracorporeal papaverine versus prostaglandin E l in the treatment of impotence. J. Urol., 141: 551, 1989. 6. Chen, K. K., Chan, J . Y. H., Chang, L. S., Chen, M. T. and Chan, S.H. H.: Intracavernous pressure as a n experimental index in a rat model for the evaluation of penile erection. J. Urol., 147: 1124, 1992. 7. Sidi. A. A. and Chen. K. K.: Clinical experience with vasoactive intracavernous pharmacotherapy fortreatment of impotence. World J. Urol., 5: 156, 1987. 8. Meisel, R. L. and Sachs, B. D.: The physiology of male sexual behavior. In: The Physiology of Reproduction. Edited by E. Knobil and J . D. Neill. New York: Raven Press, vol. 2, chapt. 35, pp. 3-105, 1994. 9. Giuliano.. F.., RamDin. 0.. Bernabe. J . and Rousseau, J.-P.: Neural control of penile erection in t h e rat. J . Auton. Nerv. Syst., 55: 36, 1995. 10. de Groat, W. C. and Steers, W. D.: Neuroanatomy and neurophysiology of penile erection. In: Contemporary Management of Impotence and Infertility. Edited by E. A. Tanagho, T. F.
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Lur and H. I). Mc(’lurc. Ihltimore: Williams & Wilkins. pp. :1-27. 1988. 1I . Steers. W. D.: Ncwrai control of pc*nilrrrection.Sem. Urol.. 8: 66. 1990. 12. Rajfer. J., Aronson. W. J . . Bush. P. A,. Dorey. F. J . and Ignarro, I,. -1.: Nitric oxide as a nwdiator of relaxation of the corpus cavernosum in response to nonndrcnergic. noncholinergic n m rotransrnission. New Engl. .J. Med., 3 2 6 90. 1992. 13. MacLean. P. D. and Pliwg. L). W.: Cerebral representation of penile erection. J . Neurophysiol.. 2 5 29. 1962. 14. Argiolas. A.. Melis. M . H. and Gessa, G. I,.: Intraventricular oxytocin induces yawning and penile crcction in rats. Eur. J . Pharmacol.. 117: 395, 1985. 15. Argiolas, A.. Melis. M . R. and Gessa. G. L.: Oxytocin: an extremely potent inducer of penile erection and yawning in male rats. Eur. J. Pharmacol., 130 265, 1986. 16. Argiolas, A., Melis, M. R., Mauri. A. and Gessa, G . L.: Paraventricular nucleus lesion prevents yawning and penile erection induced by apomorphine and oxytocin hut not by ACTH in rats. Brain Res., 421: 349, 1987. 17. Melis, M. R., Argiolas, A. and Gessa. G. L.: Oxytocin-induced penile erection and yawning: site of action in the brain. Brain Res., 3 9 8 259, 1986. 18. Melis, M. R., Argiolas, A. and Gessa. G. L.: Apomorphineinduced penile erection and yawning: site of action in brain. Brain Res., 415: 98, 1987. 19. Chen, K. K., Chan, J. Y. H., Chang, L. S..Chen. M. T. and Chan, S. H. H.: Elicitation of penile erection following activation of the hippocampal formation in the rat. Neurosci. Lett.. 141: 218, 1992. 20. McKenna, K. E., Chung. S.K. and McVary, K. T.: A model for the study of sexual function in anesthetized male and female rats. Am. J . Physiol., 261: R1276, 1991. 21. Goodchild, A. K.. Dampney, R. A. L. and Bandler, R.: A method for evoking physiological responses by stimulation of cell bodies, but not axons of passage, within localized regions of the central nervous system. J. Neurosci. Meth.. 6 351, 1982. 22. Schmidt, M. H., Valatx. J.-L., Sakai, K., Debilly, G. and Jouvet, M.: Corpus spongiosurn penis pressure and perineal muscle activity during reflexive erection in the rat. Am. J . Physiol., 2 6 9 R904, 1995. 23. Melis, M. R., Stancampiano, R. and Argiolas, A.: Hippocampal oxytocin mediates apomorphine-induced penile erection and yawning. Pharmacol. Biochem. Behav.. 42: 61, 1992. 24. Saphier, D. and Feldman, S.: Effects of septa1 and hippocampal stimuli on paraventricular nucleus neurons. Neuroscience, 20: 749, 1987. 25. Tribollet, E. and Dreifuss, J . J.: Localization of neurones projecting to the hypothalamic paraventricular nucleus area of the rat: A horseradish peroxidase study. Neuroscience, 6 1315.1981. 26. Wagner, C. K. and Clemens, L. G.: Projections of the paraventricular nucleus of the hypothalamus to the sexually dimorphic lumbosacral region of the spinal cord. Brain Res.. 539 254, 1991. 27. Wagner, C. K., Sisk, C. L. and Clemens, L. G.: Neurons in the paraventricular nucleus of the hypothalamus that project to t h e sexually dimorphic lower lumbar spinal cord concentrate “H-estradiol in the male rat. J. Neuroendocrinol.. 5 545, 1993. 28. Sachs, B. D.: Role of striated penile muscles in penile reflexes, copulation, and induction of pregnancy in the rat. J. Reprod. Fertil., 66:433, 1982. 29. Wallach, S. J. R. and Hart, B. L.: The role of the striated penile muscles of the male rat in seminal plug dislodgement and deposition. Physiol. Behav.. 31: 815, 1983. 30. Elmore, L. A. and Sachs, B. D.: Role of the bulbospongiosus muscles in sexual behavior and fertility in the house mouse. Physiol. Behav., 44: 125, 1988. 31. Schmidt, M. H. and Schmidt, H. S.: The ischiocavernosus and bulbospongiosus muscles in mammalian penile rigidity. Sleep. 1 6 171, 1993. 32. Wagner, C. K and Clemens, L. G.: Neurophysin-containing pathway from the paraventricular nucleus of the hypothalamus to a sexually dimorphic motor nucleus in lumbar spinal cord. J . Comp. Neurol., 336 106, 1993. 33. Carmichael, M. S., Humbert, R., Dixen. J.. Talmisano, G.. Greenleaf, W. and Davidson, J. M.: Plasma oxytocin increases in the human sexual response. J. Clin. Endoninol. Metab., 6(: 27, 1987.
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34. Melis, M. R., Argiolas, A., Stancampiano, R. and Gessa, G . L.: Effect of apomorphine on oxytocin concentration in different brain areas and plasma. Eur. J. Pharmacol., 182: 101, 1990. 35. Ogawa, S., Kudo, S., Kitsunai, Y. and Fukuchi, S.: Increase in oxytocin secretion a t ejaculation in male. Clin. Endocrinol., 1 3 95, 1980. 36. Saper, C. B., h w y , A. D., Swanson, L. W. and Cowan, W. M.: Direct hypothalamo-autonomic connections. Brain Res., 117: 305, 1976. 37. Hosoya. T.: The distribution of spinal projection neurons in the
hypothalamus of the rat, studied with t h e HRP method. Exp. Brain Res., 4 0 79. 1980. 38. Luiten, P. G. M., ter Horst. G. J., Karst, H. and Steffens, A. B.: The course of paraventricular hypothalamic efferents to autonomic structures in medulla and spinal cord. Brain Res., 329: 374, 1985.
39. Schwanzel-Fukuda, M., Morrell, J. T. and Pfaff, D. W.: Localiza. tion of forebrain neurons which project directly to the medulla and spinal cord of the r a t by retrograde tracing with wheat germ agglutinin. J. Comp. Neurol., 2 2 6 1, 1984.
Vol. 158, 238-244, July 1997 Printed i n U.S.A.
THEJOIVISAI. OF UHOUIGY Copyright 0 1997 by AAIKHI(,LY UROUK;I( hi. A..IX.IATION. 1w
PARTICIPATION OF PARAVENTRICULAR NUCLEUS OF HYPOTHALAMUS IN CENTRAL REGULATION OF PENILE ERECTION IN THE RAT KUANG-KUO CHEN, SAMUEL H. H. CHAN, LUKE S. CHANG
AND
JULIE Y. H. CHAN*
From the Dtcwion of Urologv, Department of Surgery, and Department of Medical Research, Veterans General Hospital-Taipec. and the Department of Urologv. and Center for Neurosclence, Natronal Yang-Mmg University, Taipei, T a i u m , Republic of China
ABSTRACT
Purpose: To investigate the possible participation of the paraventricular nucleus of hypothalamus in central regulation of penile erection. Materials and Methods: Male adult Sprague-Dawley rats were anesthetized and maintained with pentobarbital sodium. The intracavernous pressure (ICP) was used as a n experimental index for penile erection, and was recorded alongside systemic arterial pressure and heart rate. The effect on ICP of electrical (30-s train of 30-120 d, 40-160 Hz, 1-ms rectangular pulses) or chemical (L-glutamate, 0.5 nmoV50 nl.) activation of the paraventricular nucleus of hypothalamus (PVN)o r hippocampal formation was evaluated. Results: Electrical activation of the PVN elicited both multiple and single episodes of elevation in ICP, along with visible erection and ejaculation. The former pattern exhibited a n increase in ICP that was more sustained, with higher peak amplitude and longer latency. Chemical stimulation of neuronal perikarya in the PVN also resulted in similar patterns of rise in ICP and visible erection. These effects were, nonetheless, not accompanied by significant alterations in systemic arterial pressure and heart rate. Activation of the hippocampal formation, a s we reported previously, similarly elicited multiple and single episodes of increase in ICP. These erectile responses, however, were substantially reduced or eliminated upon electrolytic lesion of the ipsilateral PVN. Conclusion: These observations suggest that the PVN may be an important nucleus that participates in central neural regulation of penile erection in the rat. Furthermore, a n efferent pathwayk) from the hippocampal formation to PVN may constitute part of the neural circuitry in the forebrain in the regulation of erectile functions. KEY
WORDS: paraventricular nucleus of hypothalamus, hippocampal formation, intracavernous pressure, penile erection
During the past two decades, studies which focused on the vascular organization of the penis have greatly advanced our understanding of the hemodynamics and pharmacology of penile erection.'-4 As such, it is now recognized that penile erection is the result of a n increase in arterial blood inflow to the penis, with subsequent distension of sinusoids and restriction of venous outflow in the corpora cavernosa.2 Intracavernous administration of smooth muscle relaxants, such a s papaverine hydrochloride,"" prostaglandin El,".fi or a-adrenergic blocker, p h e n t ~ l a m i n einduces ,~ penile erection in human and animals. Although the vascular mechanisms of penile erection are well established, the mechanism of neurologic regulation of penile erection is, on the other hand, less documented.8.9 I t is generally considered that penile erection is regulated by opposing peripheral sympathetic and parasympathetic neural outflows to penile vascular smooth muscles via the action of acetylcholine and noradrenaline..',g--'' Whereas adrenergic neurotransmission mediates contraction of corporal smooth Accepted for publication February 18, 1997. * Requests for reprints: Department of Medical Research, Veterans General Hospital-Taipei, Taipei 11217, Taiwan, Republic of
China. Supported by grants NSC82-0115-B-075-125,NSC83-0412-B-075105 and NSC84-2331-B-075-026from the National Science Council, VGH-267 from the Veterans General Hospital-Taipei. and DOH83HR-221 from the National Health Research Institute, Taiwan, Republic of China.
muscles and causes detumescence, cholinergic neurotransmission promotes smooth muscle relaxation and penile erection. In addition, non-adrenergic non-cholinergic neural mechanisms are also involved in the regulation of the erectile process.2.4-10 One possible mediator of the non-adrenergic non-cholinergic system is nitric oxide.12 Our current knowledge with regard to central neural control of penile erection is sparse at best.8 MacLean and P100g13 reported that penile erection was observed after electrical stimulation of the paraventricular nucleus of hypothalamus (PVN) in the squirrel monkey. Intraventricular administration of apomorphine or oxytocin was reported to induce visible penile erection in rats,4.*4-'" with the PVN proposed to be the site of action.16-18 In these investigations, penile erection was assessed visually by counting the number of erectile episodes. Our laboratory has established recently a rat mode1,6 which uses the intracavernous pressure (ICP) in anesthetized rat as the index for a more sensitive, objective and quantitative assessment of penile erection. Utilizing this experimental index, we have demonstrated that t h e hippocampal formation is a n important locus in the forebrain for neural regulation of penile erection.19 The present study was undertaken to further explore the participation of the PVN as a neural substrate in central regulation of the erectile process, and its relationship with hippocampal formation in the promotion of penile erection. Our results are consistent with the notion that the PVN may
238
239
PVN AND PENILE ERECTION be an important brain nucleus in the elicitation of penile erection in the rat. Furthermore, an efferent pathway(s)from the hippocampal formation to PVN may constitute part of the neural circuitry in the forebrain that subserve regulation of erectile functions. MATERIALS A N D M E T H O D S
Animals. Male, adult Sprague-Dawley rats (200-300 gm.) anesthetized with pentobarbital sodium (50 mg./kg., i.p., with 10 mg./kg./hr. i.v. supplements) were used. Routine surgical preparations included cannulation of the left femoral artery and vein for the measurement of systemic arterial pressure and continuous infusion of supplemental anesthetic. Systemic arterial pressure was monitored via a pressure transducer (Gould 23ID) and a pressure processor amplifier (Gould 13-4615-52). Heart rate was determined using a biotachometer amplifier (Gould 13-4615-65) triggered by the arterial pulses. An endotracheal tube was established for a patent airway. The head of the rat was placed in a stereotaxic headholder (Kopf goo), with the rest of the body positioned on a heating pad and elevated to a suitable position. All data were collected from animals with a maintained rectal temperature of 37C, with a steady mean systemic arterial pressure above 90 mm. Hg throughout the experiment. Recording of intracavernous pressure. Measurement of ICP, which was used as the experimental index for penile erection, was essentially the same as described previously.6.9,19.20In brief, the lower part of the body was slightly rotated for adequate exposure of the genital area. The penis was degloved to expose both corpora cavernosa. A 26-gauge needle, which was connected to a pressure transducer (Gould 23ID) via a PE-20 tubing filled with saline, was carefully inserted into one of the corpus cavernosa. The ICP thus recorded was monitored alongside systemic arterial pressure and heart rate on a polygraph (Gould ES 1000). Intracavernous administration of saline (250 pl.) was routinely executed a t the beginning of the experiment to ascertain the lack of leakage. At the end of each experiment, papaverine (400 pg. ), a vasoactive agent most commonly used in clinical management of impotence,2 was delivered intracavernously to ensure that the recording needle for ICP has been properly lodged into the corpus cavernosum throughout the experiment, and the cavernous tissue still manifested adequate responsiveness. Electrical activation of the PVN or the hippocampal formation. Electrical stimulation of the PVN or hippocampal formation was delivered using a stainless-steel bipolar concentric electrode (Rhodes Medical SNE-100). The stereotaxic coordinates for PVN were 1.5-2.0 mm. posterior to the bregma, 6.0-7.0 mm. below the dural surface and 0.5-1.0 mm. lateral to t h e midline. The coordinates for hippocampal formation were 1.8-2.6 mm. posterior to the bregma, 3.0-4.5 mm. below the dural surface and 0.5-1.5 mm lateral to the midline. The PVN or hippocampal formation was electrically activated by cathodal rectangular current pulses (30-s train of 1-ms pulses, at 30-120 pA and 40-160 Hz), using a Grass S88 stimulator coupled with a constant-current isolation unit (Grass PSIU6). For experiments in which both PVN and hippocampal formation were stimulated, the electrodes were routinely positioned on the same side. The effects of electrid stimulation of PVN and/or hippocampal formation on ICP, systemic arterial pressure and heart rate were continuously and simultaneously monitored. At the same time, the penis was carefully observed for the occurrence of visible erection and ejaculation. Chemical activation of the PVN. To ensure that the response in ICP to electrical activation of the PVN was not due to stimulation of fiber of passage to this hypothalamic nucleus, L - g l u w a t e (0.5 nmol), was microinjected into the PVN for p*wd activation.21 Microinjection of this
excitatory amino acid was carried out with a glass micropipette (tip diameter: 50-80 pm.), which was connected to a 1-p1. Hamilton microsyringe. The stereotaxic coordinates used were the same a s for electrical stimulation. A total volume of 50 nl. was delivered to the PVN over 1-2 rnin. to allow for full diffusion of the chemical. l'%Evans blue was added to the microinjection solution to aid in histological identification. The temporal effects of chemical activation of the PVN on ICP, systemic arterial pressure and heart rate were monitored for a t least 60 min. Electrolytic lesion of the PVN. For this series of experiments, a bipolar concentric stainless-steel electrode (Rhodes Medical SNE-100) was used to identify a site each in the ipsilateral hippocampal formation and PVN that induced significant elevation in ICP upon electrical activation. An electrolytic lesion of the same site in the PVN was subsequently carried out, using a n anodal current of 1 mA for 10 s. The effects of electrical stimulation of the individual sites in the PVN and hippocampal formation, using exactly the same stimulus parameters, were again evaluated after lesioning the PVN. Histology. At the conclusion of each experiment, the brain was removed and fured in 30% sucrose in 10% formaldehydesaline for at least 48 hr. 25-pm. frozen sections stained with either cresyl violet or neutral red were used for histological verification of the location of stimulation, microinjection or electrolytic lesion sites in the PVN or hippocampal formation. Statistical analysis. Data were presented as means z S.E.M. The effects of electrical activation of the PVN on the peak amplitude, duration or the onset latency of the rise in ICP were statistically assessed using one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls multiple range test for a posteriori comparison of means. p < 0.05 was considered statistically significant. RESULTS
Effect of electrical activation of the PVN on ICP. In agreement with our previous findings,".'S the resting ICP in our animals was 4.6 % 0.5 mm. Hg (n = 35). Upon electrical stimulation of PVN, there was a significant elevation in ICP, which exhibited at least five response characteristics. First, there were two basic response patterns: multiple episodes (fig. 1, A ) and single episode (fig. 1, B ) of rise in ICP. In some
200,
.
____-
200
mm
6mn
8 -
FIG. 1. Two response patterns, multiple episodes (A)and si e isode (B)of increased intracavernous pressure (ICP), eliciteii; ereetrical stimulation of paraventricular nucleus of hypothalamus. Arrows denote short duration pressure spikes at peak of ICP.Note lack of simultaneous change in systemic arterial preseure (SAP)and heart rate (HR) immediately before or during period of elevation of ICP in both instances.
240
-
PVN AND PENILE ERECTION
incidences, several short duration pressure "spikes" were detected in the pressure peak of both episodes of rise in ICP (figs. 1, A and 3). The elevation in ICP during multiple episodal response was more sustained, and displayed higher peak amplitude and longer onset latency (see table). Histologic examinations, however, revealed no topographic distribution of sites of stimulation that elicited multiple or single episodes of elevation in ICP. These sites overlapped with each other in the magnocellular and parvocellular subnuclei of the PVN (fig. 2). Second, the elevation of ICP was related to the stimulus intensity a n d o r frequency of PVN activation. We found that at a fixed stimulus train pulse frequency and duration, increasing the stimulus intensity of PVN activation produced greater elevations in ICP (fig. 3, upper panels). Similarly, a t a fixed stimulus train pulse intensity and duration, there was heightened increase in ICP upon activation of PVN with higher stimulus frequencies (fig. 3, lower panels). Third, visible penile erection was observed in some animals during single or multiple episodes of increase in ICP (see table ), at a threshold pressure of approximately 40 mm. Hg. During these instances, tumescence of the glans and shaft of penis was observed at the initial phase of ICP elevation. As ICP further increased to above 40 mm. Hg, intense erection of the glans (cup) and of the shaft, as well as anteroflexion of the penis (flip) due to a straightening of the penis with disappearance of the flexura penis between the shaft and glans (erectionP were displayed. Some of these events were accompanied by ejaculation (see table ), demonstrated by expulsion of urethral contents and production of coagu-
30 s
zloo1 E
100,
,' ,A&
120 vA. 80
Hz. I ms
120 U A . 120
3v-
Comparison of characteristics of single and multiple episodes of increase in intracavernous pressure (ICP) upon electrical actwation of paraventricular nucleus of hypothalarri us ~~
Single Episode
Multiple Episodes ~~
Resting ICP lmm. Hg) Peak increase in ICP (mm. Hg)
4.7 2 0.5 (2-81 53.5 t 9.8
Onset latency Is)
28.8 2 7.5
(12-100) Duration lmin.1
..-
1 rns
FIG.3. Relationship between stimulus parameters of electrical stimulation of paraventricular nucleus of hypothalamus and response in intracavernous pressure (ICP). Arrows denote short duration pressure spikes at peak of ICP.
(2-801
rn
Hz.
7.4 2 2.7 ( 1.5-20)
-
~-
4.5 t 0.4 ( 3-7 77.7 t 8.3I 35-100 I 41.9 Z 11.6' 16-1601 13.7 I 4.7(2-55 I 10 111 4 110
,
Visible erection 6 111 Ejaculation following visible 3 16 erection Mean z S.E.M. and range. * p < 0.05, compared with single episode of increase in ICP, n per group. ~
-1.8
.2.1
2
ME 0
?'
mm
FIG.2. Diagrammatic representation of two rostral-caudal levels of paraventricular nucleus of hypothalamus (PVN) relative to bregma, illustrating sites upon which microinjection of L-glutamate ( A )or electrical stimulation elicited single (0) and multiple ( 0 ) episodes of increase in intracavernous pressure (ICP). Although active sites were bilaterally re resented, they are indicated on one side of diagram for clarity. &tes upon which microinjection of L-glutamate or electrical stimulation were ineffective were indicated by (W). Abbreviation: AHC, anterior hypothalamus centralis; AHP, anterior hypothalamus posterialis; ME, median eminence; PaMP, subnucleus parvocellularis medialis; PaLM, subnucleus mangocellularis; P a m , subnucleus parvocellularis posterialis; PaV, subnucleus parvocellularis ventralis; Pe, periventricular nucleus; SO, supraoptic nucleus; VMH, ventral medial hypothalamus; f, fornix: opt, optic tract; sox, supraoptic decussation; 3V, third ventricle.
= 11 animals
lated ejaculates (plugs). In two occasions, sperms were detected under microscopic examination in the urethral secretion. Fourth, the erectile effect upon PVN activation was not associated with significant changes in mean systemic arterial pressure (100.0 -t 5.0 to 96.3 t 5.1 mm. Hg, n = 22) or heart, rate (379.3 t 10.0 vs 379.3 % 9.9 bpm, n = 22) before and during the elevation in ICP (fig. 1).Fifth, activation of sites outside the contour of PVN (fig. 2) elicited no significant change in ICP. This was not attributed to end-organ failure, however, since the penis still responded positively to intracavernous injection of papaverine (0.4 mg., 250 PI.) with an increase in ICP (52.6 2 7.0 mm. Hg, n = 6). Effect of chemical activation of PVN on ICP. We also ascertained with chemical activation that penile erection induced by electrical stimulation of the PVN was due mainly to perikaryal activation of the PVN, and not fibers of passage.21 As exemplified by fig. 4, microinjection of L-glutamate (0.5 nmol, 50 nl.) into the PVN induced a multiple episodal rise in ICP, with an onset latency of 62 s and a peak effect of 96 mm. Hg. This elevated ICP persisted for approximately 40 min before returning to the baseline level. Once again, visible penile erection was observed during multiple episodes of increase in ICP. Similar results were obtained in 4 experiments. In addition to multiple episodes, microinjection of L-glutamate into the PVN also elicited single episode of ICP elevation, accompanied by visible penile erection ( n = 3). Histologic examination demonstrated that the injection sites were located in both magnocellular and parvocellular subnuclei of the PVN (fig. 2).
24 1
PVN AND PENILE EHECTlON
E
10 min
u loo
1
PVN
lesion
After
PVN
lesion
3 r s
701
" 01 0
Before
-
-
Hippocarnpus
PVN
FIG. 6. Single episodes of change in intracavernous pressure (ICP) upon electrical stimulation of CA1 area in hippocampal formation and paraventricular nucleus of hypothalamus (PVN)before and after electrolytic lesion of ipsilateral PVN. Note single episode of increase in ICP was eliminated upon electrical stimulation of hippocampal formation after lesion of PVN. 4 0 min
FIG.4. Time-course changes in intracavernous pressure (ICP), systemic arterial pressure ( S A P )and heart rate (HR) following microinjection of L-glutamate (0.5 nmol, 50 nl.) into paraventricular nucleus of hypothalamus. Note again lack of simultaneous hemodynamic changes before and during period of increased ICP.
Relationship between hippocampal formation and PVN in the elicitation of penile erection. Since the characteristics of PVN-induced elevation in ICP greatly resembled that elicited by hippocampal activation,'" we further delineated the relationship between these two forebrain nuclei in penile erection. Comparable to our previous findings,lS electrical activation of the granule cells at the dentate gyms (fig. 5 ) or pyramidal cells at the CA1 or CA3 field of the Ammon's horn (fig. 6 ) resulted in, respectively, multiple episodes or single episode of rise in ICP. Both patterns of elevation in ICP t o hippocampal activation were abolished after electrolytic lesion of the ipsilateral PVN (figs. 5 and 6 ) .At the same time, electrical activation of the same site in the PVN that was lesioned no longer induced significant effect on ICP. Complete elimination of penile erection elicited by stimulation of the hippocampal formation following lesioning the
A f f e r P V N lesion
30-5
a
0
1 HippocamPuS
PVN
FIG. 5. Multiple episodes of change in intracavernous pressure (ICP) upon electrical stimulation of dentate gyrus in hippcam a1 formation and paraventricular nucleus of hypothalamus (PVN) fore and after electrolytic lesion of ipsilateral PVN. Note multiple episodes of increase in ICP were eliminated u on electncal stimulatlon of hippocampal formation aRer lesion of bVN.
ipsilateral PVN (fig. 7 ) was demonstrated in 11out of 14 rats. Substantial reduction in amplitude a n d o r duration of erectile response to stimulation of the hippocampal formation was observed in the remaining animals. Thus, it appears that penile erection promoted by hippocampal formation and PVN took place in a serial fashion. D I S C U S S I 0N
Utilizing ICP as the experimental index for a quantitative evaluation of penile erection,",". 19.2" the present study demonstrated that PVN plays an important role in neural regulation of erectile functions. We found that both electrical and chemical stimulation of the PVN resulted in an increase in ICP. On the other hand, physical elimination of the ipsilatera1 PVN eliminated or significantly diminished the erectile response promoted by activation of the hippocampal formation. These observations suggest that a n ipsilateral efferent pathway(s) from hippocampal formation to PVN may constitute part of the neural circuitry in the forebrain that is involved in the regulation of penile erection. Our study benefited greatly by using the ICP as the experimental index for penile erection. This index allowed us to detect changes in ICP with a sensitivity of 2 mm. Hg.6 As a result, we were able to identify the minimal peak amplitude of ICP upon activation of the PVN to be 12 mm. Hg (see table). Although this is already more than 100% increase over the baseline value, it is still much below 40 mm. Hg,*9 which is the threshold ICP for visible erection, the basis for behavioral evaluations. Thus, ICP monitoring records not only the presence or absence of erectile events, but also the degree of erection attained.22By continuously monitoring the fluctuations in ICP, we were also able to define the latency, duration and temporal patterns of alterations in ICP following activation of the PVN and hippocampal formation. In addition, the demonstration that activation of the PVN elicited a n elevation in ICP without significant changes in systemic arterial pressure and heart rate immediately dissociated the erectile response from indirect, hemodynamic, influences. A hypothalamic-hippocampal oxytocinergic pathway has been proposed to mediate the penile erection induced by application of apomorphine to the PVN.16-18.23 The present study provides evidence to suggest that a novel hippocampalhypothalamic linkage may also participate in central neural regulation of penile erection. We found that activation of the PVN elicited multiple and single episodes of elevation in Icp that resembled those produced by hippocampal activation.1s
L-
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hypothalamic areas that subserve regulation of penile erection cannot be ruled out. In t h e preliminary experiments, however, we found t h a t lesioning the anterior hypothalamus, which did not significantly alter ICP upon electrical stimulation, resulted in no appreciable effect on t h e elevation in ICP in response to stimulation of the hippocampal formation, I t is noteworthy t h a t we found no topographic distribution of stimulation sites in t h e subnuclei of PVN in the expression of the two patterns of increase in ICP (fig. 2). This was in contrast to our previous finding'" in t h e hippocampal formation, viz, pyramidal cells in CA1 and CA3 areas a r e responsible for the single episode, a n d granule cells in dentate gyrus evoke multiple episodes. These differential results were interpreted to suggest a convergence of efferent projections from the CA1 and/or CA3 areas and dentate gyrus of the hippocampal formation to t h e PVN. At t h e same time, they also indicate a n integrative role for t h e PVN in t h e control of erectile functions by the forebrain. The identification of multiple and single episodes of rise in ICP to PVN activation is intriguing. Since the PVN serves as a n integrative area for autonomic and endocrine functions, it i s tempting to speculate that both neural and hormonal mechanisms may be involved in the elicited erectile process. In support of t h e neural mechanisms, a direct descending projection from the PVN to t h e spinal nucleus of t h e bulhocavernosus (SNB)and the dorsolateral intermediolateral nucleus (CLN)in lumbosacral region of t h e spinal cord h a s b w n established.","6.27 Axons from motoneurons of t h e SNB and CLN innervate t h e striated penile muscles, t h e bulbospongiosus (BS) and t h e ischiocavernosus (IC) muscles, t h a t are essential for penile erection in t h e rat.H.zH-31 Multiple cBpisodes of the rise in corpus spongiosum penis (CSP) pressure, another experimental index for objective quantification of penile erections,2' were found to coincide directly with rhythmical contraction of the IC and BS muscle^.^.^^,^^ Based on combined retrograde tracing and immunohistochemical staining, Wagner and Clemens32 demonstrated t h a t neurons FIG. 7. Diagrammatic representation of 3 levels of hypothalamus i n t h e PVN that project to the SNB contain neurophysin, the with reference to bregma, showing location of largest extent of ipsi- coproduct of oxytocin and vasopressin. Functionally, the lateral lesion of PVN region, which eliminated increase in ICP to descending oxytocinergic pathways from the PVN to the electrical stimulation of hippocampal formation. AH,anterior hypothalamic area; Arc, arcuate nucleus; DC. dorsomedial cap of para- lumbosacral spinal cord has been reported'" to mediate ventricular nucleus of hypothalamus; LH, lateral hypothalamic area; apomorphine-induced penile erection. Whether hormonal ME, median eminence; MP, medial parvocellular subnucleus of para- mechanisms are also involved i n the exhibition of multiple or ventricular nucleus of hypothalamus; PM, magnocellular subnucleus single episode of ICP increase to PVN activation, is not well of paraventricular nucleus of hypothalamus; PV, periventricular nucleus of hypothalamus; RCh, retrochiasmatic area; SO, supraoptic documented. However, serum level of oxytocin was shown to nucleus; VMH, ventromedial hypothalamic nucleus; f, fornix; opt, increase after sexual behavior in animal and human.:':' .Ii optic tract; sox, supraoptic decussation; 3V, third ventricle. Another important issue is the connection between the PVN and sympathetic and/or parasympathetic outflows involved in the initiation of penile erection. In this regard, The increase in ICP by PVN and hippocampal stimulation a p a r t from a direct connection to the sympathetic pregangliwas accompanied by visible penile erection and ejaculation i n onic neurons i n the spinal cot-d,9>10.36-3"neurons in t h e I'VN some circumstances. Lesioning t h e ipsilateral PVN pre- project to the dorsolateral funiculus indirectly via t h e reticvented penile erection induced by electrical stimulation of ular formation in pons a n d medulla.s6.87,3g Fibers originated the hippocampal formation. This suggested functional link from PVN neurons also project to t h e parasympathetic between hippocampal formation and PVN in the regulation preganglionic neurons i n the lumbosacral spinal cord."-"' w9 of penile erection is supported by the presence of neuronal Thus, it is possible that activation of t h e PVN excites the pathways from the hippocampus that exert a n excitatory descending fibers to the spinal cord, which in t u r n elicits (e.g. glutamatergic) influence on PVN neuron^.^.'^.'^ To- penile erection by modulating the peripheral autonomic outgether with the proposed participation of a hypothalamic- flows to tissues t h a t are engaged in t h e erectile processc,s.n.9 hippocampal oxytocinergic pathway i n apomorphine-induced Compare to t h e relatively short duration of penile erection penile erection,'" our present findings suggest t h a t a recip- reported previo~sly,~.9.20.22 the duration of increase in ICP rocal connections between the PVN and hippocampal for- following electrical or chemical activation of PVN w a s long in mation may constitute a neural circuitry in the forebrain the present study. This observation was not uncommon, since t h a t regulates penile erection. The significance of the we reported previously that electrical or chemical activation hippocampal-hypothalamic glutamatergic pathways t h a t a r e of the hippocampal formation induces a n elevation of ICP involved in the regulation of penile erection, however, re- t h a t lasts for at least 30 min.19 The symptomatology of penile quires further investigation. erection following microinjection of apomorphinelx or osytoThe experimental design for our lesion experiments called cin17 into the PVN begins within 5 min postinjection and for lesioning only loci in t h e PVN on which electrical stimu- lasts for more t h a n 60 min. As suggested above of a reciprocal lation elicited rise in the ICP. As such, the possibility for connections between t h e PVN and hippocampal formation, if formation to other is possible that such a long duration of rise'in ICP by Pm efferent projections from the hiDDOCamDal - ..
PVN AND 1’ENII.E EIIECTION s t i m u l a t i o n m a y involve a s u b s e q u e n t activation of‘ t h e hippocampal formation. A d i r e c t association of g l a n s erections w i t h s h o r t d u r a t i o n p r e s s u r e “spikes” of CSP p r e s s u r e p e a k has been s h o w n , c o n c u r r e n t with BS m u s c l e bursts.”‘),’” Thus, i t is t e m p t i n g t o s p e c u l a t e t h a t the s h o r t d u r a t i o n p r e s s u r e “spikes“ visible during the p e a k of b o t h single and m u l t i p l e episodes of ICP following PVN s t i m u l a t i o n (figs. 1 and 3) m a y be associated w i t h c o n t r a c t i o n s of the striated penile muscles, i n p a r t i c u l a r the BS muscles. In s o m e a n i m a l s , s t r a i g h t e n i n g of the p e n i s w a s i n d e e d observed d u r i n g p r o m i n e n t i n c r e a s e in ICP that c o m p r i s e d s e v e r a l short d u r a t i o n p r e s s u r e spikes. S i n c e w e did n o t s i m u l t a n e o u s l y record the electromyogram of the striated penile muscles, the validity of t h i s speculation a w a i t s f u r t h e r clarification. In addition, the p a t t e r n of i n c r e a s e s in ICP following PVN a c t i v a t i o n r e s e m b l e s that recorded during ex copula reflexes (e.g., u r e t h r o g e n i t a l reflex) in anesthetized m a l e rats.”.’o Under b o t h conditions, m u l t i p l e episodes of rise i n ICP, accomp a n i e d by d e t e c t a b l e penile erection and ejaculation, w e r e displayed. The mean a m p l i t u d e of p e a k i n c r e a s e during erectile e v e n t s w e r e also similar. Finally, the flaccid state of p e n i s u n d e r b o t h conditions w a s c h a r a c t e r i z e d by relatively low and stable ICP p r e s s u r e w i t h no s p o n t a n e o u s p r e s s u r e p e a k s . On the o t h e r h a n d , e x copula reflex fails to p r o d u c e s i n g l e episodal i n c r e a s e s in ICP. As s u c h , it is our feeling that a l t h o u g h s i m i l a r p e r i p h e r a l mechanisms m a y be responsible for reflexive and PVN-induced penile erection, a d d i t i o n a l s u p r a s p i n a l m e c h a n i s m s are involved in the latter responses. C o m p a r i n g t o the p e r i p h e r a l autonomic m e c h a n i s m s , o u r c u r r e n t understanding of the physiologic role of the central nervous system in the r e g u l a t i o n of penile erection i s f a r f r o m comprehensive. B y utilizing ICP as an experimental i n d e x for a m o r e sensitive and q u a n t i t a t i v e assessment of penile erection,6.20 the p r e s e n t s t u d y r e v e a l e d that the PVN m a y be an important f o r e b r a i n nucleus that mediates penile erection. We also demonstrated the existence of a hippocampalhypothalamic pathway in the neural circuitry for the regulation of erectile functions. REFERENCES
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