Covergent effects of lordosis-relevant somatosensory and hypothalamic influences on central gray cells in the rat mesencephalon

EXPERIMENTAL

NEUROLOGY

70, 269-281 (1980)

Covergent Effects of Lordosis-Relevant Somatosensory Hypothalamic Influences on Central Gray Cells in the Rat Mesencephalon

and

YASUO SAKUMA AND DONALD W. PFAFF' The

Rockefeller

University, Received

New February

York,

New

York

10021

4, 1980

In female rats anesthetized with urethane, extracellular recordings were made from neurons in the mesencephalic central gray. Their responses to somatosensory stimuli and the hypothalamic electrical stimulation were tested with or without estrogen treatment. Effects of stimuli which potentiate or disrupt the lordosis reflex were examined. Of the 156 cells tested 32.7% were transsynaptically activated from the gigantocellular nucleus of the medulla. Pressure on the skin had excitatory effects on cutaneous-responsive cells, some of which were found to have welldefined receptive fields on the perineum. Noxious stimuli produced either excitation or inhibition. One-third of the cells changed their discharge rate after stimulation of the ventromedial nucleus of the hypothalamus or the medial preoptic region by trains of pulses at 10 Hz. Facilitation prevailed in the responses to the ventromedial hypothalamic stimulation. Convergences of somatosensory and hypothalamic effects were commonly observed; 55.2% of the cells responded to at least two different stimuli. Directions of responses by individual cells to the various somatosensory and hypothalamic stimuli were frequently consistent with their effects on lordosis. In these cells estrogen treatment resulted only in a subtle shift toward excitatory responses. Central gray neurons recorded here, probably shortaxoned, may help to integrate hypothalamic outputs with somatosensory influences on natural behavior patterns, including lordosis in the female rat.

INTRODUCTION Cells in the dorsal mesencephalon are thought to participate in the supraspinal control mechanism for the lordosis reflex of female rats (36, Abbreviations: CG-central gray, POA-medial preoptic region; VMN-ventromedial nucleus, NGc-gigantocellular nucleus; EEG-electroencephalogram. i Supported by National Institutes of Health grant HD-05751 and by an institutional grant from the Rockefeller Foundation for the study of reproductive biology. Dr. Sakuma is on leave from the University of Gunma Medical School, Maebashi, Japan. The authors thank Mr. N. Brodyn for his excellent technical assistance. 269 OO14-4886/80/110269-13$02.00/O Copyright All rights

Q 1980 by Academic Press, Inc. of reproduction in any form reserved

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37). This reflex, a dorsiflexion of the vertebral column, is elicited in sexually receptive female rodents by somatosensory stimuli (30). Results from stimulation and lesion experiments (37, 38) suggest that the mesencephalic central gray (CG) may help to integrate somatosensory and motor components of a reflex loop for lordosis. Projections to the CG by branches of the paleospinothalamic system (24, 43) would provide any somatosensory information involved. The lordosis reflex is dependent on estrogen (30). Estrogen has been thought to modulate outputs of forebrain and hypothalamic structures known to accumulate radioactive estradiol (29). Of these structures, attention has been focused on the medial preoptic region (POA) and the ventromedial nucleus of the hypothalamus (VMN). Local implants of estrogen in these regions potentiated lordosis responses (1, 9, 22). Neuronal electrical activity in these structures undergoes changes after estrogen administration (5, 21, 42). Electrical stimulation of the VMN facilitated lordosis, whereas stimulation of the POA had the opposite effect (31). These effects are hypothesized to operate through a tonic, hormonesensitive bias exerted on the CG (31, 32). Recent anatomic studies revealed heavy projections from the POA and VMN which terminate in the CG (7, 8, 19) It is possible, therefore, that influences from these structures could intersect somatosensory influences in and around the CG. The present recording experiments were designed to examine cellular responses in the CG of female rats to various inputs, including those which trigger lordosis (cutaneous pressure) or potentiate it (VMN electrical stimulation), as well as those which disrupt lordosis (pain) or suppress it (POA stimulation). We found response properties of some local neurons (i.e., neurons without long descending axons) in and around the CG to somatosensory and hypothalamic influences that were consistent with the possible involvement of these neurons in the control of the lordosis reflex in female rats. METHODS Recordings were made in 33 Sprague-Dawley albino female rats under light urethane anesthesia (1.2 g/kg body weight, given intraperitoneally 0.5 g/ml). The rats were ovariectomized and either untreated or implanted with an estrogen pellet to bring them into behavioral receptivity (confirmed before the anesthetic agent was administered). The animal was tracheotomized and secured in a stereotaxic frame, with the incisor bar 5 mm above the center of the ear bars. A craniotomy was made in the parietal area and the dura was removed. The exposed cerebral cortex was covered with warm agar-saline (4%). Rectal temperature was maintained between 35 and 38°C. The electroencephalogram (EEG) was

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recorded from the frontal cortex through a pair of monopolar stainless-steel screw electrodes on the dura, and an electrocardiogram was monitored throughout the recording session. Coaxial bipolar electrodes were constructed from 28-gauge hypodermic tubings as outer barrels and nickel-chromium wire of 120 pm diameter as active electrodes. Except for the cut ends they were insulated by Epoxylite, and had DC resistance of approximately 3Ofi. These electrodes were inserted into the medial preoptic region (POA), the ventromedial nucleus of the hypothalamus (VMN), and the gigantocellular nucleus of the medullary reticular formation (NGc), and were fixed to the skull with dental cement. Stereotaxic coordinates for these operations are reported elsewhere (39). Extracellular potentials were recorded using glass micropipets filled with 0.5 M sodium acetate solution. Pontamine sky blue 6BX was added to make a 2% solution of dye to allow marking the end of each recording track. The dc resistance of the pipet was 10 to 30 ma, 20 MIR being an optimal value. Potentials were recorded between the electrode and a chloride-silver plate under the skin in the temporal area. A conventional amplification and display system was used for the recording. Electrical stimulation with an amplitude of 750 @ or less with negative rectangular pulses of 0.2 or 0.5 ms duration was applied to the NGc. Stimulation was continuous at 0.5 Hz while systematic exploration was made in the deep tectal region and the CG. Characteristics of the antidromic responses obtained by this stimulation (criteria: constant latency, ability to follow high frequency, collision), particularly those in the central gray, were described in recent communications (39,40). The cells in this study either showed orthodromic responses to, or failed to respond to the NGc stimulation, which with these parameters did not interfere with changes in the cortical EEG. When a spontaneously active neuron was encountered, it was tested for responsiveness to somatosensory and hypothalamic electric stimulation. The effects of two different types of somatosensory stimuli, noxious and pressure on the skin, were examined. The noxious stimulus was produced by pinching the tail or limbs with forceps. Desynchronization of the cortical EEG was considered to indicate the effectiveness of the stimuli. The cutaneous pressure stimuli were applied on various portions of the animal’s trunk. Special emphasis was placed on the effects of pressure on the flanktailbase-perineum area, which is sufficient to induce lordosis in the freemoving sexually receptive animal. If responses to pressure stimuli were found, an attempt was made to determine receptive field characteristics of that particular neuron. Electrical stimulation of the VMN and POA was accomplished by either repetitive pulses at 0.5 Hz or by a train of pulses at 10 Hz, with each pulse

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lasting 0.2 ms and being 250 pa in amplitude. Some neurons which showed orthodromic responses were further tested with rates to 200 Hz. The parameters for train stimulation were selected on the basis of its behavioral effects (31). After completion of each penetration, iontophoretic deposit of the dye was made to facilitate localization of the recording sites. At the end of each recording session, the animal was administered an overdose of Nembutal and perfused through the heart with 10% Formalin. Frozen serial sections (100 pm) were made in the frontal plane. The sections which contained the dye spots were stained with cresyl violet, and those with lesions caused by the penetration of stimulating electrodes were stained with 1~x01 fast blue and cresyl violet. Recording and stimulation sites were then determined from analysis of the stained sections. RESULTS Recordings were made from 156 neurons in the mesencephalic CG and adjacent structures in untreated or estrogen-primed female rats. These cells had resting discharge rates from 0.1 to 50 Hz, with the largest population in the range of 0.5- 1.0 Hz (Fig. 1). The anatomic distributions

Total n = 156 Ovariectomized Ovariectomized + Estrogen

Resting Discharge FIG. 1. Distributions estrogen-treated and

Rate (Hz)

of resting discharge rates of central gray ovariectomized nontreated female rats.

neurons

in ovariectomized

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FIG. 2. Diagrams offrontal sections through the mesencephalon, with large dots indicating the positions of cells driven transsynaptically from the gigantocellular nucleus of the medulla (NGc); inverted triangles, cells inhibited by NGc stimulation; small dots, nonresponsive cells. Distance posterior to bregma (mm) is shown at the top right of each diagram. CG-mesencephalic central gray, K-inferior colliculus, ML-medial lemniscus, MLF-medial longitudinal fascicle, MR-median raphe, LL-lateral lemniscus, P-pons, PC-cerebral peduncle, SCP-superior cerebellar peduncle, III-oculomotor nucleus, V-mesencephalic nucleus of trigeminal nerve.

of these neurons are presented in Fig. 2, with their responses to electric stimulation of the gigantocellular nucleus of the medullary reticular formation (NGc) indicated. This manuscript deals with cells which do not project directly to the NGc. The characteristics of the cells which were antidromically driven by NGc stimulation were presented elsewhere (39, 40). Electric stimulation of the NGc, which contains ascending fibers of the paleospinothalamic system (24, 43), activated 51 (32.7%) of 156 neurons TABLE

1

Responses to Various Stimuli by Central Grey Neurons” Number and percentage of cells tested Stimulation

Total No. of cells

Excitation

Inhibition

No response

NGc Noxious Cutaneous pressure VMN POA

156 156 136 112 105

51 (32.7)” 46 (29.5) 60 (44.1) 34 (30.4) 18 (17.1)

7 (4S)b 36 (23.1) 4 (2.9) 8 (7.1) 18 (17.1)

98 (62.8)” 74 (47.4) 72 (52.9) 70 (62.5) 69 (65.7)

a For abbreviations see text. b Percentage of total number of cells.

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transsynaptically. These responses to NGc stimulation had a relatively stable latency with fluctuations in each individual response of less than 5 ms, and almost always a one-to-one response to each stimulus. The responsive cells were situated in the lateral region of the CG; cells in the environs of the cerebral aqueduct showed no response to NGc stimulation. Inhibition by NGc stimulation at 0.5 Hz was seen in seven (4.5%) cells. Responses of these neurons to somatosensory stimuli are summarized in Table 1. Facilitation prevailed in the responses to pressure stimulation on the skin. With this stimulus, 44.1% of the cells were facilitated whereas only 2.9% were inhibited. Noxious stimuli facilitated 29.5% and inhibited 23.1% of the neurons tested. The responses to cutaneous touch were not routinely associated with changes in the cortical EEG, nor were neuronal responses to this stimulus a function of the EEG state. Although the noxious stimuli inevitably desynchronized the EEG, the resting discharge rate of a majority (78.7%) of cells accelerated by noxious stimuli was not correlated with EEG activity. Receptive fields for pressure stimulation were determined in 21 neurons (Fig. 3). One group of 12 cells was characterized (Fig. 3A) by receptive fields limited to the perineum, sometimes extending to the flanks. The boundary of this type of receptive field was well defined, and tended to be bilaterally symmetrical. The cells with this type of field had resting discharge rates of less than 1 Hz, and were nonresponsive to the noxious stimuli. A second group (N = 5) of cells had larger receptive fields residing exclusively on the back (Fig. 3B). The boundary of the field was fairly

FIG. 3. Representative examples of three types of receptive field found for cells activated by pressure on the skin. Diagrams of sections through the mesencephalon are shown to indicate cell loci. A-well-defined receptive field on the perineum; B-large receptive field limited to the dorsal surface of the trunk; C-receptive field with indistinct boundary covering large area. Ipsi(Contra): ipsi(contra-) lateral to the recording site. For abbreviations, see Fig. 2.

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distinct, and was almost completely symmetrical. The resting discharge rate of these cells varied from 2.0 to 4.0 Hz, and all the cells were inhibited by noxious stimuli. Finally, some cells (N = 4), with the largest receptive fields, responded to stimuli on both the ventral and dorsal surfaces of the trunk (Fig. 3C). The boundary of the receptive field was indistinct. These cells had resting discharges of about 2 Hz, and three of them inhibited by the noxious stimuli. As shown in Fig. 3, the cells with the larger receptive fields were found at the dorsal boundary of the CG. Electrical stimulation of the ventromedial nucleus of the hypothalamus (VMN) and the medial preoptic area (POA) was applied to 112 and 105 cells, respectively. Among the 34 cells facilitated by trains of stimuli to the VMN, 21 responded in a time-locked fashion to each stimulus, with mean latencies between 5 and 21 ms, and fluctuations in each cell’s response of less than 5 ms. The response to each VMN stimulus followed on a one-toone basis to 30 Hz, but aborted at 50 Hz. In 8 of 18 cells facilitated by train stimulation of the POA, each stimulus was followed by responses with mean latencies of 8 to 30 ms, with fluctuations of less than 5 ms. The results summarized in Table 1 show the effects of stimulus trains at 10 Hz, which lasted 5 s. With these parameters, effects persisted after the end of stimulation. Facilitatory responses for VMN stimulation were more frequent than inhibitory responses. POA stimulation induced facilitatory and inhibitory responses in equal numbers of cells. Convergent effects of stimulation with different modalities on the same cell were commonly seen (Fig. 4). Among 105 cells recorded with noxious or pressure stimuli of the NGc, VMN, or POA, 55.2% responded to at least

30 -

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ONumber

of Effectwe

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FIG. 4. Histogram showing response patterns by cells with responses to stimuli in different modalities. Electrical stimulation of the gigantocelhdar nucleus of the medulla (NGc) was given at 0.5 Hz; ventromedial nucleus (WAN) and medial preoptic region (POA) were stimulated at 10 Hz for 5 s.

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227-E

40 ki ," ;

30

POA

2

FIG. 5. Responses of a central gray neuron in an ovariectomized estrogen-treated female rat to pressure on the perineal skin (pressure), pinch of the tailbase (pain), electrical stimulation of the ventromedial nucleus of the hypothalamus (VMN), and of the medial preoptic region (POA). Each electrical stimulation was at 10 Hz, for 5 s. Inset A shows transsynaptic response of this neuron to pulses at 0.5 Hz in the gigantocellular nucleus of the medulla. Calibrations in A, 5 ms and 0.5 mV. Throughout this observation, changes in the discharge rate were independent of the frontal cortical EEG.

two kinds of stimuli. Only 13.3% of them responded specifically to one of the five stimuli, leaving 3 1.4% nonresponsive to any stimulus tested. The patterns of responses by cells which responded to four or five of the stimuli are shown in Fig. 5. Contrasting effects were exerted by noxious versus pressure stimulation, and by VMN versus POA stimulation. The most frequent combination of responses was: facilitation by pressure and VMN stimulation, with inhibition by noxious and POA stimulation. A representative example of this type of cell is shown (Fig. 5). Comparisons were made between the cells recorded in the ovariectomized untreated versus estrogen-treated animals with respect to resting discharge rate and responsiveness to various stimuli. The sample consisted of 94 cells from ovariectomized animals, and 62 cells from estrogen-treated animals. No distinct differences were seen in the frequency distribution of cells according to resting discharge rate (Fig. 1). The proportion of responsive neuronal pools to each stimulus in estrogen-treated rats was not significantly different from that found in ovariectomized animals ($ test). However, a shift toward excitatory response was found after each stimulus except NGc stimulation (Fig. 6). There were also no systematic differences seen in receptive field shape and size as a function of estrogen treatment. DISCUSSION More than 40% of the cells tested in this study responded to pressure stimulation of the body surface. Much of their receptive fields, especially

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Touch D*,hTn,,rn

-30 t

l-l

no variectomized 13 Ovariectomized + Estrogen

FIG. 6. Percentages of central gray neurons, in ovariectomized nontreated female rats, that responded to the various modalities abbreviations, see Fig. 5.

estrogen-treated or of stimulation. For

those of the flank and perineum, are included in the skin areas touched by the male during mating (30). Pressure on this skin area is sufficient to elicit lordosis in sexually receptive female rats (30). The responsive cells were located in the CG and its adjacent subtectal region. These are regions from which a large and prompt facilitation of the lordosis reflex is obtained by electrical stimulation in free-moving animals (36, 37). From observations of female rats with spinal transections, the anterolateral columns of the spinal cord have been linked to the transmission of lordosis-relevant somatosensory impulses (18). The major projection ascending from the anterolateral column passes through the medulla at the ventral portion of the NGc, and continues cephalad as far as the thalamic nuclei, innervating the mesencephalic region we recorded from as it goes (24,43). Therefore, orthodromic responses which were seen in 32.7% of the cells tested by NGc stimulation might be caused by activation of the fibers of the anterolateral system or by activation of NGc neurons which receive from those fibers. This possibility is consistent with the observation that approximately 70% of the cell responsive to NGc stimulation also responded to pressure or noxious stimuli, considered (15, 16) to be transmitted by the anterolateral system. Recent electrophysiologic studies in the mouse (10) and the cat (41), and anatomic studies in these and a number of other species (24, 34, 43) demonstrated the presence of a somatosensory zone in the CG, subtectal, and intercollicular regions of the dorsal mesencephalon. These mesencephalic regions appear to be involved in the integration of sensory inputs of different modalities in order to control orientation and localization of the animal to these sensory stimuli (14).

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Multimodal responses have often been recorded from the dorsal mesencephalon (3, 10, 41). Effective stimuli include somatic, visual, and auditory inputs. Differences in the responses of individual cells to tactile and nociceptive stimuli were noted in the cat mesencephalic reticular field (2). In the present study, contrasting effects of the pressure and noxious stimuli were seen in more than 44% of the cells which responded to both stimuli. Antagonism between nociceptive stimuli and vaginal probing has been seen in the ventrobasal complex of the rat thalamus (17), and in the medullary reticular field (13). These interactions are of interest, because electric stimulation of a part of the CG induces analgesia (25, 33). Cells described in this study do not include any which are antidromically driven from any of the three sites of electric stimulation: the NGc, VMN, and POA. A majority of cells which responded to noxious stimuli showed no correlation in their resting discharges with changes in the cortical EEG. CG cells serving as a relay in a polysynaptic pathway from the anterolateral system to the thalamic nuclei (4, 24) would have initiated the cerebral arousal response to nociceptive impulses. Neurons in the POA and hypothalamus fire at a low frequency (11, 21, 26) and do not respond specifically or quickly to somatosensory stimuli which are sufficient for triggering lordosis (5). It is unlikely, therefore, that the CG neurons in this study, which did respond to the same type of stimuli, exert strong, direct effects on these structures. For all these reasons, cells recorded here may be primarily short-axon cells. Successful facilitation of the lordosis reflex from the CG in animals with VMN lesions (38) also rules out ascending projections from CG cells as necessary for the induction of lordosis. Ascending CG projections to the hypothalamus (12, 35) are probably more important for other behavior patterns or neuroendocrine phenomena, such as the control of gonadotropin release (6). Anatomic studies showed that efferent projections from the CG chiefly from a short-axon radial pattern, with a few fibers traced rostrally to the medial thalamic nuclei and hypothalamus, and caudally to the medulla (12, 20, 35). Nauta (27) proposed that the radial projection provides a link between medial hypothalamic output and the diffuse ascending and descending conduction systems in the midbrain reticular formation. The opposing effects of electric stimulation of the VMN and POA on CG cells are of special interest because stimulation of the VMN in free-moving animals facilitated lordosis, whereas POA stimulation inhibited it (3 1, 36). Considering that the CG is a site for facilitation of lordosis (36, 37), the above observations seem to imply that the CG serves partly as a site of interactions between lordosis facilitating and inhibiting outputs from the hypothalamus and forebrain. Anatomic demonstrations of the convergence in the CG of fibers originating from diverse hypothalamic and

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