Investigation of peripeduncular-hypothalamic pathways involved in the control of Lordosis in the rat

Brain Research, 253 (1982) 287-302

287

Elsevier Biomedical Press

Investigation of Peripeduncular-Hypothalamic Pathways Involved in the Control of Lordosis in the Rat HECTOR S. L6PEZ and HUGO F. CARRER Instituto de Investigaci6n Mddica Mercedes y Martin Ferreyra, Casilla de Correo 389, 5000 Cordoba (Argentina)

(Accepted June 1st, 1982) Key words: sexual behavior - - lordosis - - evoked potential - - lesion - - female rat - - midbrain - - ventromedial hypothalamus

Single shock stimuli applied to the peripeduncular nucleus (PPN) elicited a complex evoked response in the hypothalamic ventromedial nucleus (VMN). An early component and a late component could be distinguished in the evoked response on the basis of their different latency, threshold, site of maximal amplitude, frequency-responsecharacteristics and also because restricted lesions eliminated specificallythe short- or the long-latencycomponents. Transection of the dorsal supraoptic pathway immediately in front of the PPN suppressed all the VMN-evoked responses. The same lesion eliminated lordotic responses in ovariectomized rats treated with estradiol benzoate and progesterone. Similar lesions placed more dorsally had no effect on either the sexual behavior or the evoked response. Evidence was obtained suggesting that the short-latency component is generated by activity that reaches the VMN directly through the ventral supraoptic commissure, while the long-latencyresponse involves substations in the amygdala and the bed nucleus of the stria terminalis. The effect of lesions on the performance of lordosis may be attributed to the disruption of ascending and/or descending neural impulses circulating between the PPN and the VMN, with relay stations in the amygdala and the bed nucleus of the stria terminalis. INTRODUCTION During coitus the female rat adopts a reflex postural attitude called lordosis. This behavior characterizes the estrous female and is dependent on the levels of circulating ovarian steroids, although in ovariectomized animals a large dose of estrogen is sufficient to allow lordotic responses in the absence of progesterone secretion (for review see ref. 26). Notable progress has been made in recent years on the knowledge of the neural structures which control the execution of lordosis in response to mounts by the male. On the sensory side of the reflex is the perineal region which mechanically stimulated by the male during copulation 11 originates essential afferent activity carried by the pudendal nerves 19. Sexually significant peripheral stimuli reach the spinal medulla, ascend through the ventrolateral columns is and reach the peripedtmcular nucleus (PPN) in the midbrain 6. From here on some of the structures involved have been identified but their functional role and the pathways involved have not 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

been elucidated. It has been determined that lesions of the ventromedial hypothalamic nucleus (VMN) 7, 16, the habenula 25, the anterodorsal hippocampus 5 and the anterior part of the medial amygdaloid nucleus 22 produce marked decrements in sexual behavior. These findings have generally been interpreted as indicating that those structures play a facilitatory or permissive role in the control of lordotic responses. On the other hand, lesions of the medial preoptic area 86, the lateral septum-bed nucleus of the stria terminalis region14, 29 and the posterior pole of the lateral amygdaloid nucleus 22 have the opposite effect, making it more probable that ovariectomized rats will respond with lordosis when primed with doses of estrogen that do not sustain such responses in non-lesioned animals. These results have been interpreted as indicating that these structures exert an inhibitory influence on lower sensory and/or motor links of the reflex chain 2. In the case of the VMNS, 38, the septat region 45, the medial preoptic area 30 and the medial and lateral

288 amygdaloid nuclei 22 the proposed explanations have received additional support from the fact that activation of these structures by electrical or electrochemical stimulation has the opposite effect to lesions. In order for the function of this complex system to be described in terms of neuron operations a complete schema of the intervening neuronal groups and connecting pathways should be obtained. The experiments to be described were designed to investigate where and how does neural activity originated in the PPN reach the higher brain structures involved in the control of lordosis. MATERIALS AND METHODS Experiments were performed in adult white albino female rats. Evoked potential studies were done in a total of 28 animals anesthetized with urethane (1 g/kg body weight) and placed in a stereotaxic apparatus. The recording electrode consisted of a pair of twisted stainless steel wires 150 # m in diameter, isolated with varnish except for the cut end. Stimulating electrodes were bipolar concentric made with a piece of 24-gauge cannula and a 50/~m diameter wire of stainless steel. Occasionally a pair of twisted wires similar to the recording electrode was used as stimulating electrode. The recording electrode was connected to a high impedance differential preamplifier with variable pass band. The output from this amplifier could be displayed in a storage oscilloscope, photographed or stored in magnetic tape using an FM recorder. To study evoked field potentials a pass band of 1-100 Hz was used. The output from the amplifier could be fed to a signal averager module to obtain averaged evoked potentials (AEP) which were then inscribed with an x-y plotter. When observing multiunit or unit responses, the pass band was changed to 300-3000 Hz. A histogram module was used to obtain post-stimulus time histograms (PSTH) of the evoked responses. Stimt, lating monophasic square pulses of 0.1-1 mA and 0.5 ms duration at 0.4 Hz were obtained from isolation units using a two-channel pulse generator. The effect of different types of lesions on the evoked responses was studied. After control records of the evoked response were obtained, a rectangular

blade made from a 2 mm wide piece of razor blade was allowed to descend into different parts of the brain so as to interrupt the connections between the PPN, the amygdala and the VMN. Using a stereotaxic apparatus the blade was lowered into the brain in a step-wise fashion, at the same time observing the evoked responses. In this way it was possible to determine the approximate vertical position of the fibers whose interruption produced modifications in the shape and/or size of the evoked responses. Also the effect of electrocoagulation lesions in the pathways involved was studied; they were produced through tungsten electrodes isolated except for the tip. This method was preferred when studying the effect of lesions placed dose to the recording electrode, to avoid the distortions that mechanical displacement of the tissue, produced by movement of a knife, may cause. To study the effect of a local anesthetic on the evoked potential, a cannula-electrode assembly was vsed to inject 2/~1 o f a 2 °4 solution of xylocaine. The assembly consisted of a 24-gauge guide-cannula and two 50/~m diameter wires cemented along the sides which could be used as recording or stimulating electrodes. The tips of the wires protruded approximately 300 ¢tm below the cannula and were separated approximately 300/~m from each other. Inside the guide cannula a piece of 30-gauge stainless steel cannula (injector) could be inserted so that the tip of this inner cannula would be flush with the lower end of the guide cannula. The inner cannula was used to inject the solutions by means of a 5 #1 syringe and a piece of flexible catheter attached to it. Pilot experiments showed that the injector could be removed and re-inserted without apparent change in the evoked response recorded from the wires. After the effect of different lesions on the evoked response was determined, the effect of some of the lesions on the sexual behavior of female rats was studied. Approximately 2-month-old animals were ovariectomized and kept in air-conditioned quarters with inverted lighting schedule: 12 h light-12 h dark, lights on at 21.00 h. Three weeks after castration all animals received a priming dose of 100 #g estradiol benzoate (EB) and 48 h later 2 mg progesterone (P) per kg body weight. Hormones were dissolved in olive oil; injections were given sc before the lights went off. Six to 9 h after P administration sexual

289 behavioral tests were made by bringing the female to the home cage o f trained male rats. Ten mounts were allowed and the number o f lordotic responses were recorded. A lordotic quotient (LQ ~-- number lordosis × 100/number mounts) was obtained for each animal. Rats showing a LQ of less than 80 were discarded. Three weeks after the preliminary behavioral test the animals were injected with either 10 or 100 #g of EB and 2 mg P/kg body weight and behaviorally tested with the same schedule as outlined above. The animals were divided into 3 groups and different lesions were made in each group. One group received a transection of the brainstem immediately in front of the PPN; this lesion included the dorsal supraoptic pathway and was made with the same knife and stereotaxic coordinates as used in the recording experiments (AP 3.2; V --3.5; L 3.5 outer edge). A second group received a transection of the tectal region made with the same knife as the preceding group. The AP and L coordinates were the same, but the knife was descended only to the level of the medial geniculate body (V 0), immediately above the dorsal supraoptic pathway, so that its fibers were spared. Both lesions were made bilaterally. In the third group the knife was descended in the midline so as to transect the periaqueductal gray (AP 3.0; V --3.0). After the animals had recuperated from surgery, and never before 3 weeks had elapsed from the last hormone treatment, animals were again treated with the same doses of EB and P and tested for sexual behavior with the same schedule as before. The LQs obtained for each rat before and after the lesions were compared and the differences were statistically evaluated using Wilcoxon's matched-pairs signed-ranks test 40. At the termination of recording experiments and after all behavioral testing was made, the animals were sacrificed and the brain fixed in l0 ~ formalin. Brains were serially cut in a freezing microtome and studied microscopically to determine placement of recording and stimulating electrodes. Size and placement of lesions were evaluated from direct drawings of the outer limits of the lesions on plates of the stereotaxic atlas of K6nig and Klippe117. RESULTS

The evoked response

An evoked response was observed in the ventro-

A 5340~

Fig. 1. The early component of the potential evoked in the ventromedial hypothalamus by stimuli applied to the PPN. All records were obtained from the same rat in 4 successive descents. Each trace represents the averaged evoked potential of 16 responses obtained at the location indicated by the corresponding dots in the schematic drawings of the hypothalamus. The AP coordinate of the atlas plate 17 is indicated. The largest amplitude evoked potential is marked*. Stimulus intensity was 250/~A for A5340 ,urn and A4620 pro; 750/~A for A3990/~m and A3290 pm. Calibration marks are 100 pV and 30 ms.

medial hypothalamus when single shock stimuli were applied to the PPN. In one experiment the anteroposterior coordinate of the recording electrode was varied in 4 different descents and the evoked response was studied as the recording electrode was lowered through the hypothalamus, 0.7 mm from the midline. In every penetration the largest potentials were obtained wh.en the electrode was at a V and AP position coincident with the VMN (Fig. 1). In 2 other experiments the L coordinate was changed in 0.7 mm steps and again the largest potentials were recorded when the electrode was within the VMN (Fig. 2).

290

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Fig. 2. The early and the late components of the potential evoked in the VMN by stimuli applied to the PPN. Records are averaged evoked potentials of 16 responses at each location indicated by the dots; the AP coordinate is indicated below. The record where the late component showed the largest amplitude is marked*. Stimulus intensity for all records was 850/tA. Two types of response were obtained from the VMN, depending on the recording site. The potential recorded from within or in sites close to the V M N consisted of a positive phase (P1) with mean latency to peak of 10 :~: 1 ins (mean ~: S.E.), a negative phase (N1) of 25 _-kz 2 ms latency and a positive phase (P2) with latency 52 ~z 4 ms (Fig. 3A). As the recording electrode was displaced along the V coordinate, the evoked potential showed a more complex waveform than the one just described (Figs. 2 and 3B). Along the downward going slope of the N1 phase, another negative-going phase appeared (N2) with a peak latency of 36 ~ 4 ms, followed by a larger positive going-(P3) phase with 47 ~ 4 ms latency, tb.en another negative (N3) wave with 65 ± 5 ms latency and a last, more variable phase (P4). This complex response was localized to a site approximately 200 #m long in the V coordinate. When the lower frequencies were elimi-

nated by filtering, 2 multiunitary responses could be obtained from the same recording site; one coincided with the N I wave of the early response flatency 13-22 ms) and the other was superimposed on the P3 wave (latency 30-57 ms) of the late response (Fig. 3B). Close examination of the records had already anticipated this result since several notches could be seen riding NI and P3. Small displacements of the recording electrode modified the number and size of these multiunitary responses. In 14 experiments an eleetroeoagulating current was passed through the recording electrode to mark the site where this complex potential was observed and in every case the marking spot was found at the dorsolateral border of the VMN, precisely at the limit between the cellular core and the acellular shell of the nucleus. In what follows the early or short-latency component consisting of PI, N I and P2 will be called EC

291

A

B

1 Fig. 3. Field responses and multiunitary responses evoked in the VMN by stimulation of the PPN. A: the early or shortlatency response. B: complex response showing the early and the late components. From top to bottom: oscilloscope photograph of the response as seen with a 1-100 Hz passband; averaged evoked potential of 16 responses obtained with the same pass-band; oscilloscope photograph of the multiunitary response as seen with a 300-3000 Hz passband; post-stimulus time histogram of the multiunitary response, 128 stimuli at 0.4 Hz. Vertical calibration mark is 125 ~V for the field responses, horizontal calibration mark is 20 ms.

while the late or long-latency component consisting of N2, P3, N3 and P4, obtained f r o m the boundary of the V M N will be called LC. The maximal response amplitude was obtained when the stimulus was applied to the PPN. Electrode positions either above or below it produced smaller responses. When the electrode was descended in a more lateral position (3.0 m m instead of 2.5 m m from the midline) the LC showed a clear predominance over the EC. Here again the largest amplitude of the response was obtained when the P P N was stimulated. With a more

medial position (L 2.0 mm) the LC could no longer be evoked and the EC decreased in size. To rule out the possibility that the potentials observed were due to artifact or current spread, the effect of a lethal dose of sodium pentobarbital was studied; the results are shown in the sequential records of Fig. 4A. An increase-decrease-no response sequence was observed. The LC was very sensitive to high stimulus frequency; already at 0.6 Hz a notable decrease in the amplitude of N2 and P3 was apparent (Fig. 4B and C). At 2 Hz the LC disappeared and only the EC was observed; EC could follow relatively high stimulus frequencies albeit at 40 Hz also this response decreased in amplitude. Threshold stimulus for the EC was lower than for the LC. In order to gain some understanding of the consequences (facilitation or inhibition) of the excitation of the neuronal system activated by the stimulus, the effect of paired shocks to the P P N was studied. Both stimuli were delivered through the same electrode placed in tbe PPN, separated by varying time intervals. Fig. 5A shows the results of one experiment in which both stimuli were of the same suprathreshold intensity for both responses. A clear inhibition of the LC can be seen in the test response, with intervals ranging from 40 to 900 ms. As can be seen in Fig. 5B also the multiunitary response was inhibited in the test response. However, when the stimuli were subthreshold for the LC, a clear facilitation of the LC was observed (Fig. 5C) in the test response. This facilitation lasted at least 50 ms.

Effect of lesions and xylocaine injections on the evoked response Data from neuroanatomical studies indicated 2 possible pathways for the conduction of PPN-hypothalamic activity: (1) the perive~tricular system of fibers1°; and (2) the lateral system of fibers. The latter is described by Jones et al. 15 as a longitudinal brindle which follows the dorsal aspect of the optic tract (this part will be referred to as the dorsal supraoptic pathway - - DSO) which at the level of the hypothalamus gives off a bundle of fibers that descend medially into Meynert's commissure (this part will be referred to as the supraoptic commissure pathway - - SOC). Transections of these pathways were made at different locations and with different methods in an attempt to establish which is involved

292 A AF[ER

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Fig. 4. A: effect of a lethal dose of pentobarbital on the V M N evoked response. B: averaged evoked potentials of 16 responses taken with different stimulus frequencies as indicated to the left of each record. C: the first 4 responses obtained with stimulus frequencies as indicated to the left of each record. Note decrement of late component with higher frequency. Calibration marks are 200/iV and 13 ms for B; 150 #V and 15 ms for C.

in the conduction of the early and late components of the PPN-hypothalamic-evoked response. The effect of injecting a local anesthetic, xylocaine, at selected sites along these pathways was also studied. The effect of a frontal transection placed so as to interrupt the DSO immediately in front of the PPN was studied in 9 animals. The rectangular knife described in Methods was lowered in the brain at AP 3.2 with the medial edge at 1.5 m m from the midline. The knife was gradually allowed to descend so that the evoked potential could be seen to decrease and disappear as the DSO fibers were transected and not before. More medial lesions involving the medial forebrain bundle or the periaqueductal gray had no effect on the evoked response (Fig. 6); in these cases the knife was lowered at the same AP coordinate as in the preceding experiments as well as in more anterior positions to make sure that fibers travelling from the P P N to the hypothalamus were included in the lesion. To study the effect of sectioning the medially

directed fibers (SOC) that course from DSO towards the V M N and the BNST 15, a large lesion was made at the lateral limit of the hypothalamus. An electrocoagulation lesion was made through a pair of tungsten electrodes placed 2 m m apart in a parasagittal plane immediately above the hypothalamic fissure. The electrodes were isolated with varnish except for the terminal 1 mm. After the electrodes were in place a control AEP was obtained; then 120 mC were passed through the electrodes. AEPs were obtained at several times after this lesion, but the EC and the LC were no longer observed. Upon histological examinatioo of the brain we found that the lesion variably affected structures around the hypothalamic fissure, i.e. optic tract, medial amygdaloid nucleus, ventral supraoptic commissure, ventral amygdalofugal fibers and ventral-most fibers of the cerebral peduncle. Fibers entering or leaving the hypothalamus along its ventrolateral aspect had been interrupted from A5340 to A3430. The following experiments were made in an at-

293 B

A

250

100 msec

msec

c

500 msec

C 900 msec

J Fig. 5. Paired shock analysis of the VMN-evokedresponse. A: conditioning (C) and test (T) stimuli are suprathreshold for the late component (900 ffA); note inhibition of late component in the test response; conditioning-test intervals are indicated to the left of each record; calibration marks are 100 ffV and 42 ms for the upper record, 17 ms for the lower records. B: averaged evoked potential (upper part) and post-stimulus time histogram (lower part) showing inhibition of the late component in the test response; conditioning-test interval was 100 ms. C: facilitation of the late component by a conditioning stimulus. Both stimuli were of the same intensity (500 ffA) subthreshold for the late component. tempt to determine whether different pathways and/or substations were responsible for the production of the EC and the LC. Neuroanatomical data obtained in the monkey 15 and the rat4,10, ~0 suggested 3 possibilities : (1) a direct projection to the V M N or indirect projections with substations in (2) the amygdala or (3) the bed nucleus of the stria terminalis (BNST). These 3 possibilities were explored. Using either a blade knife or electrocoagulation, tbe more medially situated fibers of the DSO were interrupted at the level of the posterior pole of the VMN.

Typically the EC but not the LC disappeared or was greatly reduced. Fig. 7 shows tbe results of one experiment. If the lesion extended further laterally, comprising all of the DSO, the whole response was suppressed. These results suggested that the EC is caused by activity coursing in the fibers that project directly from the PPN to the VMN, while the LC is produced by impulses reaching the V M N through some indirect route. The possible participation of the amygdala was explored studying the effect of xylocaine injections and lesions in the posterior part of

294

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2 Hz BEFORE

BEFORE LESION

A

B

AFTER LESION

A 2970

AFTER

B

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Fig. 6. The effect of mesencephalic transections on the VMN evoked response. A: the knife was descended along the shaded area, no change was observed in the averaged evoked potentials taken after introducting the knife to the base of the brain. B: the same knife introduced in a more lateral position suppressed the evoked response after the dorsal supraoptic pathway was transected (middle and lower records). Calibration marks 200/~V and 20 ms.

the lateral a m y g d a l o i d nucleus (AL), a structure which p a r t i c i p a t e s in the c o n t r o l o f sexual behavior 22 a n d receives profuse projections f r o m the P P N 31. T o o u r k n o w l e d g e the effects o f x y l o c a i n e on the e v o k e d responses r e c o r d e d at the site o f injection have n o t been studied. The injection o f 2 #1 o f artificial c e r e b r o s p i n a l fluid zs at the rate o f 0.1 #1 every 20 s h a d no effect on the r e c o r d e d activity. Fig. 8 shows that xylocaine injected locally at the same rate caused a c o m p l e t e b u t t r a n s i t o r y suppression o f the response e v o k e d in the V M N when the P P N was stimulated. A f t e r the response h a d recuperated, a second injection h a d the same effect. W h e n the recording electrode was displaced 700 # m f r o m the site o f injection a clear response was observed. W h e n the c a n n u l a - e l e c t r o d e assembly was p l a c e d in A L ( A P 4.2; V - - 2 ; L 5.2) an e v o k e d potential was observed when the P P N was stimulated. W h e n xylocaine was injected in A L , the e v o k e d potential

A 4380

Fig. 7. Effect of a lesion interrupting the medial part of the dorsal supraoptic pathway. A and B show the control records at 2 different stimulation frequencies so that in B only the early component can be observed. C and D were taken after making the lesion shown in F; note suppression of the early component in both records whereas the late component can still be observed at 0.2 Hz. E: post-stimulus time histogram of the differentiated record taken at 0,2 Hz after the lesion, the early multiunitary response has been suppressed. F: the shaded area indicates the lesion at the plane of maximum size, the arrowhead indicates placement of recording electrode. Calibration marks: 175 #V, 20 ms. r e c o r d e d locally as well as the L C p o r t i o n o f the potential e v o k e d in the V M N by P P N s t i m u l a t i o n were suppressed, whereas the EC was n o t affected (Fig. 9). N o change was o b s e r v e d in the size or shape o f the e v o k e d potentials when 2 #1 o f artificial c e r e b r o s p i n a l fluid were injected at the same site. A lesion o f a p p r o x i m a t e l y 900 # m in d i a m e t e r placed in the same site in A L c o m p l e t e l y a n d irreversibly e l i m i n a t e d the L C ; the E C was n o t affected. The electrodes placed a l o n g the c a n n u l a c o u l d also be used to stimulate A L , e v o k i n g a field potential response in the V M N . In one e x p e r i m e n t in which injection in A L p r o d u c e d suppression o f the L C an identical injection m a d e 800 # m m o r e lateral h a d no effect o n the P P N - V M N - e v o k e d response. A n e v o k e d p o t e n t i a l c o u l d also be r e c o r d e d f r o m the B N S T when the P P N was stimulated. Minutes

295

BEFORE

2'AFTER XYLOCAINE

rat 4/A of the xylocaine solution were injected in the lateral ventricle. No change was observed in the V M N response. In 2 rats in which a lesion was made in the BNST by electrocoagulation through the same electrodes used to stimulate or record, the LC disappeared completely and this change was irreversible (Fig. 10). The EC showed no change either after xylocaine injection or after the lesion. If the electrodes in BNST were used for stimulation and evoked potential was observed in the VMN. No changes were observed in the VMN-evoked responses after xylocaine injections were made in the periaqueductal gray at the level of the meso-diencephalic junction, ipsilateral to the recording and stimulating electrodes. Effect o f lesions on the lordotic response

45' AFTER XYLOCAINE 2' AFTER SECOND XYLOCAINE INJECTION Fig. 8. Effect of xylocaine injected locally in the VMN on the potential evoked by PPN stimulation. Calibration marks are 150/~V and 20 ms. after 2 / A of xylocaine were injected in the BNST the LC disappeared and then gradually returned to normal (Fig. 10); a second injection had the same effect. To rule out that the changes observed may be due to xylocaine diffusing into the ventricles, in one

Table I shows that after transection of the lateral brainstem at the level of the DSO receptivity was no longer observed, even in animals primed with a very large dose of EB. On the other hand, a lesion made with the same AP and L coordinates, which was not deep enough to disrupt the DSO, had no effect on sexual receptivity. In animals bearing a lesion in the periaqueductal gray, a moderate and transitory decrease of sexual receptivity was produced, since only in animals primed with the lower dose of EB was a significant reduction observed. A second test performed in the same animals 90 days after the lesion showed that the LQ had recuperated to normal levels (82 zk 11). After priming the animals with 100 #g EB/kg body weight LQ was normal. Out of 37 animals with transection of the DSO immediately in front of the PPN, 9 died during the post-operative period. Also 11 out of 38 animals

TABLE I Effect of brainstem transections on the receptive behavior of ovariectomized rats under different doses o/estradiol benzoate

Rats in all groups received 2 mg progesterone/kg body weight (b.w.) 48 h after estrogen injection. Group

Tectal lesion Periaqueductal gray lesion DSO lesion

n

15 13 9 15 12

Estrogen (pg/kg b.w.)

Lordosis quotient (mean ~ S.E.) Prelesion

Postlesion

10 10 100 10 100

100 98 ± 1 100 98 4- 2 100

98 ~ 65 ± 93 ± 3± 2±

* P < 0.05; ** P < 0.001 versus pre-lesion score.

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Fig. 9. Effect of xylocaine injection in the lateral amygdala. A: two minutes after injection the late component disappears to reappear approximately 45 min after injection. Second, third and fourth records are photographs of oscilloscope traces; the rest are averaged evoked potentials. B: injection of xylocaine in the amygdala eliminated the response to PPN stimulation evoked in the amygdala site itself as well as the late component of the V M N response; however, it did not affect the response evoked in the VMN when the injection site in AL was stimulated. Calibration marks are 150 yV and 20 ms. C: placement if recording (~k), stimulation

and injection(@) sites. with transection of the periaqueductal gray died, whereas all 10 animals with tectal lesion survived until the termination of the experiments. No attempt was made to force-feed arty of the operated rats; during the first 2-3 weeks after surgery animals with DSO and periaqueductal transections lost some weight, but later on they gained weight normally. No evidence of motor deficit was evident, but no rigorous neurological evaluation was attempted. Histological examination of the brain of lesioned animals showed tissue loss and gliosis of varying extents. In animals with periaqueductal gray transection, the lesion occupied the middle portion of the brainstem at the level of the posterior commissure (A2970 to A2420) and extended approximately 1

mm beyond each side of the midline, reaching the base of the brain. Animals with transections of the DSO showed lesions of similar size, placed at the same AP plane as the preceding group, but in a lateral position, the inner border of the lesion usually traversing across the middle of the medial lemniscus, and extending 2 mm laterally therefrom; the brainstem was transected to the base of the brain, but touched only minimally the basal part of the hemispheres, at the level of the subiculum. Special attention was paid to the state of the PPN; in several animals in which evidence was found of the lesion having extended to encroach upon this nucleus, the corresponding behavioral data were not taken into account. Lesions of the tectum occupied similar AP

297

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AFTER

Fig. 10. The effect of lesion and xylocaine injection in the bed nucleus of the stria terminalis. A: the lower record was obtained 5 rain after xylocaine injection, the late component was suppressed whereas the early response remained unmodified. B: PSTH of the differentiated response (128 sweeps) show that the multiunitary response corresponding to the late component was also suppressed. C: the xylocaine effect was transitory, the upper record was taken 45 rain after the injection. An electrocoagulation lesion made in the same site irreversibly eliminated the late component. D : microphotograph of a coronal section of the brain showing the lesion in BNST.

298

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Fig. 11. Schematic representation of the proposed pathways for ascending impulses from the PPN and the effect of their interruption at several places on the potentials evoked in the VMN. The waveform traced with dotted line represents the part of the evoked potential suppressed by the lesion indicated in the schematic representation of a horizontal section of the brain. Since direct connections from AL to VMN have not been described, the pathway is interrupted by a question mark.

299 and L positions as the preceding ones but reached only to the medial geniculate body. DISCUSSION The results described above indicate that the PPN and the VMN are functionally related, since a complex evoked response was observed in the VMN when single shocks were applied to the PPN. In this evoked response 2 components (referred to as LC and EC) could be distinguished on the basis of their different latency, threshold, sites of maximal amplitude and frequency response characteristics. Since destruction of the more medial fibers included in the DSO at the level of the VMN eliminated the EC but preserved the LC, while a lesion or xylocaine injection in AL or BNST had the opposite effect, it may be proposed that at least 2 pathways exist for the interaction between PPN and VMN. We found that a transection immediately in front of the PPN eliminated both evoked responses, so it may be proposed that both pathways run anteriorly along the lateral aspect of the brainstem. A large parasagittal lesion affecting the fibers that course transversely towards the hypothalamus and BNST had similar effects. These data suggest that the pathways for the generation of LC and EC overlap along the DSO and the SOC. According to Jones et al.15 the efferents from the PPN are placed dorsally along the optic tract and then divide as they run anteriorly: some fibers turn laterally to reach several amygdaloid nuclei including the AL while others turn medially to reach the hypothalamus and the BNST. Moreover, HRP injections in the VMN retrogradely filled neurons in the PPN4,10. These results would be compatible with the idea that the EC depends on the integrity of fibers emerging rostrally from the PPN within the DSO and entering the hypothalamus as part of the ventral supraoptic commissure (Fig. 11). The pathway for the LC is different from the one proposed for the EC. Anatomical data sustain the contention that PPN originated activity may reach the VMN by way of the AL and BNST. There is solid evidence showing that AL and BNST receive afferences from the PPN15, ~1, there being no doubt that BNST projects to VMN4,10. The connection from AL to VMN would seem to be indirect, since

direct projections have not been fotmd4,10, although it has repeatedly been observed that stimulation in AL generates evoked responses in VMN12,27,37. It may be argued that the suppressive effect observed after the electrocoagulating lesions in AL and BNST was due to the predominance of some inhibitory influence triggered by the irritative effect of the electrical lesion. This is not the case since temporary inactivation of these regions with xylocaine had the same effect. This method also allowed us to study the effect of suppressing more than one structure in the same animal. It appears that the inactivation of neural elements produced with this method does not extend farther than 800 #m, since at this distance from an injection site normal activity was recorded and furthermore the response evoked in the VMN was suppressed by a xylocaine injection in AL but was not affected by an identical injection made 800 /~m more lateral. The possibility of a generalized effect caused by increased cranial pressure or diffusion through the ventricles was also ruled out. Sustaining the hypothesis of an indirect pathway with substations in AL and BNST are the findings that PPN stinmli also evoked field potentials in those structures. Moreover, as xylocaine was applied, the local response and the VMN response disappeared, to reappear gradually in both sites at the same time. It is not possible with the available information to determine whether activity from AL reaches the VMN directly or through the BNST. The dependence of the LC on the functional integrity of both the BNST and AL supports that possibility, albeit a mechanism such as temporal or spatial summation of both inputs could also account for the results obtained. Fig. 11 represents schematically the proposed pathways and the effect of their transection on the evoked responses. VMN activity evoked by PPN stimuli may be the experimental counterpart of a functional relation significant for the control of sexual behavior. This possibility is suggested by the fact that the lesion frontal to the PPN which completely eliminated VMN responses also suppressed lordotic responses in ovariectomized rats primed with EB plus P. Moreover, the large parasagittal lesion affecting the fibers that course transversely towards the hypothalamus and BNST which also eliminated VMN responses has been shown to render female rats

300 unreceptive2°,21,3a, 35. In a recent publication Edwards and Pfeifle13 report that lordotic responses were also suppressed when a sagittal cut lateral to the VMN on one side of the brain was combined with a lesion of the peripedtmcular region on the other side of the brain, indicating that the integrity of a lateral pathway connecting the VMN and midbrain is essential for the induction of sexual receptivity. We may conclude then that the interruption of these pathways in the DSO or the SOC not only eliminates the VMN responses but makes the animals incapable of sexual behavioral performance. One important question remains to be settled: is the behaviorally meaningful activity travelling towards the VMN as our results suggest or in the opposite direction? Anatomical data indicate that reciprocal connections between the PPN and the VMN are superimposed along the DSO and the SOC 9,15,24,39 so that both ascending and descending fibers were interrupted by the lesions in question. As a matter of fact, work in progress (Masco and Carter, unpublished observations) shows that VM N stimulation produces evoked responses in the PPN. The interpretation best fitting the available data would be that both ascending and descending impulses travelling along the DSO and SOC between the PPN and the VMN are essential for the normal execution of lordotic responses. The fact that parts of the evoked response depended on the activation of the AL and BNST supports the idea that VMN activity evoked by PPN stimulation may be related to the neural control of lordosis, since experimental data in.dicate that AL and BNST also participate in this function. Indeed, lesion of the posterior part of AL increased the probability of lordotic responding in ovariectomized estrogen-progesterone-primed rats, whereas electrochemical stimulation had tbe opposite effect22. The participation of the BNST in the control of sexual behavior has not been sufficiently investigated. It has been demonstrated that stimulation in the region of the BNST was most effective in suppressing lordotic responses 4s and interruption of the fibers coursing between this region and the ventromedial hypothalamus has repeatedly been shown to increase sensitivity to estrogen 42-44. Lesions of the basal septum which included at least parts of the BNST greatly increased sexual behavioral re-

sponses, although the behavioral effects did not correlate with the involvement of BNSTIl,z.% These findings have been interpreted as indicating that AL and perhaps BNST participate in the control of sexual behavior originating activity which ultimately results in behavioral suppression. Since lesions in AL and BNST suppress inhibitory influences on sexual behavior as well as the LC, this part of the evoked response may be related to the arrival of such behavior-suppressing activity to the VMN. This hypothesis is supported by the fact that the LC could be recorded exclusively from the periphery of the VMN, where inhibitory interneurons receiving amygdaloid-originated activity are situated ~7. Activity originated in AL and BNST synapsing on these inhibitory interneurons may suppress the ~ctivity of effector cells having a facilitatory or permissive influence on the performance of lordotic responses, It was evident that the activity evoked in the VMN by stimuli applied to the PPN included firing of neurons. These responses were contained within the field potential and in several occasions could be seen to constitute a population spike. Work in progress using microelectrodes (Lopez and Carrer, unpublished results) shows that coincident with the EC and the LC unit responses can be orthodromically driven from the PPN. The different spatial distribution of the responses raises the possibility that the EC and the LC are generated by different populations of neurons. Further support for this possibility comes from experiments using paired shock stimuli, since a subthreshold stimulus facilitated the generation of LC whereas a suprathreshold stimulus produced a pronounced and long-lasting inhibition of the response. Neither effect was observed for the EC response, In behavioral experiments we found that periaqueductal gray lesions moderately and transitorily decreased sexual receptivity. In the work of de Olmos and Carrer 1°, a column of HRP-filled neurons was fotmd ascending from the PPN towards the periaqueductal gray and periventricular system to end in the ventromedial hypothalamus. This pathway offered an alternative route for PPN impulses to reach the VMN. Complete section of the periaqueductal gray did not affect the responses evoked in the VMN by PPN stimuli, so it appears unlikely that PPN-generated activity ascends near the midline.

301

O n the other h a n d S a k a m a a n d Pfaff 3s have obt a i n e d evidence suggesting that the V M N is the origin o f activity descending t h r o u g h the periaqueductal gray to reach b r a i n s t e m centers responsible

P P N has been shown to receive activity generated i n the p u d e n d a l nerves which carry the single most i m p o r t a n t sensory i n p u t for the elicitation of lordosis 6. Since all these structures as well as the V M N

for the m o t o r control o f lordotic responses, a propo-

c o n t a i n cells that concentrate estrogen a2,41 it is very

sition which could explain the behavioral effects

likely that they represent sites of sensory--endocrine integration for the control o f reproductive pro-

observed by us. C o n c e r n i n g the physiological significance o f the circuits investigated here, it should be r e m e m b e r e d that A L a n d B N S T are connected to the olfactory

cesses. ACKNOWLEDGEMENTS

system, receiving sensory afferences that have been

Supported by the Consejo N a c i o n a l de Investiga-

shown to play an i m p o r t a n t role in the behavioral a n d endocrine aspects o f r e p r o d u c t i o n 1,3, while the

ciones Cientificas y T6cnicas of Argentina. H.S.L.

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