Inhibin β, somatostatin, and enkephalin immunoreactivities coexist in caudal medullary neurons that...

THE JOURNAL OF COMPARATIW NEUROLOGY 291~269-280(1990)

Inhibin p, Somatostatin, and Enkephalin Immunoreactivities Coexist in Caudal Medullary Neurons That Project to the

Paraventricular Nucleus of the Hypothalamus

P.E. SAWCHENKO, C. ARLkS, AND J.C. BITTENCOURT Laboratory of Neuronal Structure and Function, The Salk Institute for Biological Studies, The

Clayton Foundation for Research-California Division, La Jolla? California 92037

ABSTRACT Concurrent and sequential dual immunohistochemical labeling methods

were used in combination, along with retrograde tracing techniques, to determine the extent to which inhibin 0 (ID), somatostatin-28 (SS-28), and enkephalin (ENK) immunoreactivity (IR) might be jointly expressed in neurons centered in the caudal part of the nucleus of the solitary tract (NTS) that project to the paraventricular nucleus of the hypothalamus (PVH). The results indicate that at least 65 7c of 16-stained neurons in the NTS also express SS-28 IR, and at least 33% are ENK-positive. At least 251"~) of the I@ IR population stains positively for all three peptides. A substantial number of cells stained with markers for two, or all three, peptide families, could be retrogradely labeled following tracer deposits centered in the PVH. Prominent 10 and SS- 28 IR projections from the caudal medulla to the hypothalamus have been described and include a preferential input to oxytocinergic (OT) compart- ments of the magnocellular neurosecretory system. The present results suggest that these arise in large measure from a common pool of neurons, a subset of which also shows ENK IR. Implications for the control of OT secretion, and for the processing of sensory information through the NTS, are discussed.

Key words: nucleus of the solitary tract, neurosecretion, oxytocin

The visceral sensory control of several neurosecretory neuron pools localized in the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus is thought to be gated largely through the nucleus of the solitary tract (NTS), the principal terminus of primary vagal and glossopharyngeal afferents. Direct projections from the NTS to components of the parvicellular neurosecretory system have been described, as have projections from discrete regions of the ventrolateral medulla, which appear to receive prominent inputs from the NTS and to project to distinct parvicellular or magnocellular neurosecretory cell groups (Ri- cardo and Koh, '78; Loewy and Burton, '78; McKellar and Loewy, '81, '82; Sawchenko and Swanson, '81b, 82; Ross et al., '85; Cunningham and Sawchenko, '88; Cunningham et al., '89). Both the direct and relayed projection systems arise primarily from catecholamine-containing neurons (Saw-

chenko and Swanson, '81b, '82; Sawchenko et al., '85; Tucker et al., '87; Cunningham et al., '89).

We have recently described novel projections to the neurosecretory hypothalamus from noncatecholaminergic cell clusters centered in the caudal NTS that express either

Accepted September 5,1989.

'Inhibins are a recently characterized family of gonadal glycoprotein hormones, whose best known physiologic action is to suppress the secretion of follicle-stimulating hormone from the anterior pituitary. Recent studies have established that members of this family are expressed in a number of extra- gonadal tissues, including the brain (see Vale et al., '88; for a review).

0 1990 WILEY-LISS, INC.

P.E. SAWCHENKO ET AL.

Fig. 1. 10 IR in the caudal medulla. Brightfield micrographs of avidin-biotin immunoperoxidase-stained sections taken near the rostra1 (A), middle (B), and caudal (C) levels of the Ifl IR cell group. Labeled neurons are concentrated in the ventrolateral aspect of the medial division of the NTS and in the reticular formation at similar rostrocaudal levels. These are the only cell groups in the brain that display perikaryal staining for 10 IR, nuclear staining is seen in many regions, including the spinal trigeminal (STV), the lateral reticular nucleus (LRN), and the area postrema (ap). Other abbreviations: cc, central canal; dc, dorsal col- umns; DMX, dorsal motor nucleus of the vagus; dpy, pyramidal decus- sation; 10, inferior olivary complex; mlf, medial longitudinal fasciculus; py, pyramidal tract; stv, spinal tract of the trigeminal nerve; XII, hypo- glossal nucleus. All micrographs x 15.

inhibin' 0 (Sawchenko e t al., '88c) or somatostatin-28 immunoreactivity (IR) (Sawchenko et al., '88a). These two peptidergic projection systems share basic similarities with re- spect to the distribution of the cells projecting to the paraventricular nucleus of the hypothalamus (PVH) and the distribution of terminals within the magnocellular and parvicellular neurosecretory systems. This includes a terminal distribution that encompasses much of the parvicellular division of the PVH and, in contrast to the catecholaminergic projections (Swanson et al., '81; Sawchenko and Swan- son, '82; Cunningham and Sawchenko, '88), is distributed preferentially to oxytocinergic components of the magnocellular system. Synaptic interactions of terminals immunoreactive for either peptide with OT IR elements have been described (Sawchenko et al., '88b,c), and physiological data supporting roles for both peptides in the control of OT secretion have been provided (Plotsky et al., '88; Raby et al., '88). Despite these commonalities, the extent to which antisera against I@ and SS-28 identify a common population of neurons has not yet been established. The present study was carried out to provide this information. In addition, because it has recently been shown that enkephalin (ENK) IR is commonly expressed jointly with somatostatin in medullary neurons (Millhorn et al., '87a,b), the analysis was broadened to include markers for this peptide family.

MATERIALS AND METHODS Animals and tissue preparation

Thirty adult male Sprague-Dawley albino rats (225-250 g) were used. Ten to thirty days prior to sacrifice, and under sodium pentobarbital anesthesia (45 mg/kg, ip), six animals received stereotaxically guided deposits of a retrogradely transported fluorescent tracer (true blue or fast blue) aimed a t the PVH; these were achieved by ejecting crystalline material from prefilled glass micropipettes with an i.d. of 100-150 Fm equipped with stainless steel stylettes fitted to end flush with the tip (Lind, '86). We have found that this approach allows for generation of circumscribed and highly concentrated tracer deposits and avoids interpretive prob- lems associated with the "trail" of tracer that is inevitably left when conventional injectors filled with aqueous media are removed from the brain. Animals injected with true blue and fast blue were treated similarly, except that postinjec- tion survival periods of 10 and 14 days were used for fast blue-injected rats, whereas longer intervals (21-30 days) were allowed for rats injected with true blue. Eight other animals received no tracer injections and were used only to provide additional material for analysis of colocalization patterns. All animals were treated intracisternally with colchicine (50 pg in 25 p1 saline) 48-72 hours prior to perfusion.

Under chloral hydrate anesthesia (350 mg/kg, ip), all rats were perfused transcardially with saline (50 ml in 1 minute) followed by ice-cold 4 % paraformaldehyde in 0.1 M borate buffer (pH 9.5,350-450 ml in 25 minutes). Brains were post- fixed for 2-4 hours and cryoprotected by overnight incuba- tion in 10% sucrose in 0.1 M phosphate buffer (pH 7.4) at 4°C. Brains were frozen with dry ice and five 1-in-5 series of frozen sections (15 or 20 fim thick) were cut on a sliding microtome and saved. Series of sections from each animal were prepared for localization of three antigens and (where applicable) the transported tracer by using a combination of concurrent and sequential staining methods described elsewhere (Sawchenko et al., '84, '85) and summarized below.

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PEPTIDERGIC SOLITARIO-HYPOTHALAMIC PROJECTIONS 271

Fig. 2. Helationship of I@ IH perikarya in the NTS to vagal motor neurons. Three sections through the dorsomedial medulla showing the distribution of cells labeled following injection of true blue into the cervical vagus and cells labeled for Ip IR. IP-stained cells tend to he concentrated a t the dorsolateral margin of the dorsal motor nucleus (DMX), although a few are intermixed within the nucleus. Never were any retro-

gradely laheled cells found to contain 10-IR. The levels illustrated in the top, middle, and bottom rows correspond roughly to those shown in Fig- ure 1 near the rostral, middle, and caudal levels of the IP IR distribution. Other abbreviations not listed in legend to Figure 1: NTS,,m; medial and lateral parts of the NTS. All micrographs x75.

Antisera and controls was abolished by preincubation (overnight at 4°C) with 30 mM SS-28, was reduced slightly by similar treatment with equimolar concentrations of SS-281_,2 and was not affected by SS-14.

I& An affinity-purified polyclonal antiserum (I153 of Vaughan e t al., '89) raised in rabbits against a synthetic peptide fragment of the IiijA chain (81-113) was used (see also Sawchenko e t al., '88a). Staining with this antiserum was

SS-28. A polyclonal antiserum raised in rabbit (S309 of Benoit et al., '82a,b) that preferentially reads SS-28 was used. This choice was based on previous work indicating that this serum stained caudal medullary cells more ro- bustly than sera that preferentially read either SS-14 or SS- 281-12 (Sawchenko et al., '88a). Staining with this serum S309

P.E. SAWCHENKO ET AL. 272


eliminated with 30 WM concentrations of the synthetic immunogen and was reduced following pretreatment with the homologous peptide sequence of the I& chain (IPB [80- 1121). Similar concentrations of the structurally related transforming growth factor P (R&D Systems, Minneapolis, MN; Derynck et al., '85) failed to interfere with staining.

In most experiments, a mouse-derived monoclonal antibody raised against [Leu5]-ENK (Sera Laborato- ries) was used. In some studies, a polyclonal antiserum raised in rabbits against the proenkephalin-derived hepta- peptide [Met5-Arg6-Phe7]-ENK (MERF; Peninsula Labora- tories) was employed. Staining of cells in the caudal medulla with each antiserum was eliminated following preadsorp- tion with 10 mM concentrations of the respective synthetic immunogens but not with similar concentrations of dynor- phin A (1-8).

Cross-reactivity tests were carried out in which each of the primary antisera was preincubated overnight a t 4OC with concentrations of the heterologous peptides at or exceeding that required to eliminate staining in the NTS when applied to the homologous antiserum. No discernible evidence for cross reactivity was found.

Staining procedure and analysis

ENK.

Material obtained from tracer-injected and noninjected animals was treated similarly. Typically, this involved incu- bation of a series of sections in a mixture comprising of the anti-ENK monoclonal antibody and either the anti-10 or SS-28 polyclonal antisera. The primary antisera were localized by exposing the sections to a mixture of affinity-purified fluorescein-conjugated goat antirabbit IgG (Tago Inc., Burlingame, CA) and affinity-purified rhodamine-conjugated goat antimouse IgG (American Qualex Inc., La Mi- rada, CA). To control for the possibility that secondary antisera used in the concurrent dual-labeling phase might cross react with one another, these were applied in a mixture to tissue that had previously been exposed to only one of the pertinent primary antisera. No evidence of such cross reactivity was perceptible. The distribution of the tracer and/or of each of the two antigens in the caudal NTS were photographed a t 50x in each section by using Ilford XP-1 film. The reagents were then eluted from the tissue by means of a minor modification of the method of Tramu et al. ('78) described elsewhere (Sawchenko et al., '84). The material was then reincubated in the mixture of secondary antisera and reexamined. Any signal in cells or fibers was taken as

Fig. 3. Ib, SS-28, and ENK IR colocalization patterns in the caudal NTS. Left column: Is, SS-28, and MERF IR demonstrated by means of sequential staining procedures in a section taken at about the level illustrated in Figure 1B. Arrows indicate cells staining for more than a single peptide. Two cells express all three antigens (denoted as ism), one 10 and SS-28 IR (is), and one ID and MERF IR (im). The order of staining was Ip followed by MERF and then SS-28, with interposed elutions. Right column: A section through the NTS a t the level of the area postrema that was first stained concurrently for I@ and ENK IR and then eluted once and stained for SS-28. One cell stains for all three peptides (ise) and three others for ID and SS-28 only (is). Note that the latter approach provides for better preservation of tissue integrity as evi- denced by staining of fibers and varicosities for ENK and SS-28. Note also that I@ and SS-28 IR cells are largely congruent a t these levels of the NTS, whereas more medially situated cells stained only for ENK IR are evident. The central canal is a t the lower left of all panels. All micrographs x150.

evidence that the elution had been ineffective, and the material was either reeluted or discarded. Material from which the immunoreagents were effectively removed was then incubated first in the polyclonal antiserum to which it had not yet been exposed (IB or SS-28) and then in the antirabbit secondary antiserum. The distribution through the NTS was photographed.

In experiments in which the polyclonal anti-MERF was employed, three sequential incubations, with interposed tests for completeness of elution, were used. To control for diminution of antigenicity as a function of subjection to the elution protocol, the sequence with which the antisera were applied to sections was varied systematically.

Colocalization patterns were analyzed photographically. Enlargements (5 x 7 in.) of each frame from good experiments were made, and each cell was scored for its peptide and tracer complement. Estimated counts were extrapo- lated from the number of labeled cells detected on one side of the brain in a 1-in-5 series of 15 or 20 pm thick sections and corrected for double-counting errors (Abercrombie, '46).

Other experiments An additional group of six rats received intracisternal col-

chicine injections as described, and these were prepared for immunoperoxidase localization of the peptidergic antigens of interest. This involved use of the antisera listed above in a conventional avidin-biotin immunoperoxidase protocol described elsewhere (Gerfen and Sawchenko, '84). The principal reagents were obtained in kit form from Vector Labora- tories.

Recause the peptidergic cell group(s) of interest lay in close proximity to the dorsal motor nucleus of the vagus, a final group of 10 rats was prepared to evaluate directly whether any vagal motor neurons might stain for the peptidergic markers of interest. This was accomplished by placing 100-200 nl injections of a aqueous suspension of true blue into the sheath surrounding the vagus nerve a t the level of the nodose ganglion and pretreating the animals with intracisternal colchicine 2-3 days later and waiting an additional 2-3 days before perfusion. The brains were frozen, and 1-in-5 series of 20 pm thick sections through the caudal medulla were prepared and st.ained individually for IP, SS- 28, or MERF IR.

RESULTS The distributions in the caudal medulla of perikarya

stained positively for each of the three antigens of interest were similar to those reported previously (e.g., Millhorn et al., '87a,b; Sawchenko et al., '88a,c). I@ IR cells displayed the most restricted distribution and were seen principally in the caudolateral aspect of the commissural part of the NTS between the levels of the midextent of the area postrema and the spinal medullary transition area (Fig. 1). Fewer (roughly one-fourth as many) were detected in the dorsomedial portion of the medullary reticular formation a t similar rostrocaudal levels, and isolated cells were scattered within the medial longitudinal fasciculus. These were the only loci a t which cells displaying cytoplasmic I@ IR were detected, and this marker thus served as a convenient referent against which to compare the other peptidergic distributions. By contrast, SS-28 and ENK IR cells were widely distributed within the caudal medulla. In line with previous indications (Millhorn et al., '87a; Sawchenko et al., '88a), several areas

274 P.E. SAWCHENKO ET AL.

Fig. 4. I@, SS-28, and MERF IR colocalization patterns in the reticular formation. Arranged and labeled as in Figure 2. The section was stained sequentially for IP, then MERF, then SS-28. Two cells stain for

all three peptides (ism) and one for I@ and MERF IR (im). Note again the presence in the ventrolateral medulla of many enkephalinergic cells that stain for neither I@ nor SS-28. All micrographs x150.


Fig. 5. Tracer implantation site in the PVN. Fluorescence and Nissl images of a section through the midextent of a true blue deposit ten- tered in the PVH. The deposit spreads to involve small portions of the medial preoptic area (MPO) and region immediately medial to the for-

nix (fx). Other abbreviations: AHA, anterior hypothalamic area; LHA, lateral hypothalamic area; NC, nucleus circularis; och, optic chiasm; Re, nucleus reuniens; Rt, reticular nucleus (thalamus); SCN, suprachias- matic nucleus; SO, supraoptic nucleus. Both micrographs x 30.

of apparent congruence were evident, and these included the regions in which ID IR cells are concentrated.

Because the peptidergic cell populations in question lie in close proximity to the dorsal motor nucleus of the vagus, experiments were carried out in 10 animals to determine whether peptidergic cells in the dorsomedial medulla could be retrogradely labeled following injections of true blue into the vagus nerve at cervical levels (Fig. 2). Although the peptidergic cells tend to be concentrated a t the lateral margin of the dorsal motor nucleus, and a few lie within the borders of it, we never encountered retrogradely labeled cells that also displayed ID IR. Similarly, neither SS-28 IR nor MERF IR was detected in cells of the caudal dorsal motor nucleus that were labeled by tracer deposits. In addition, none of the retrogradely labeled neurons of the nucleus ambiguus complex displayed I@, SS-28, or MERF IR.

T o obtain estimates of the impact of the elution procedure on subsequent immunostaining, adjoining series of sections from individual animals were prepared in which the order and procedure by which antisera were applied were varied. This involved comparison of counts from two rats in which 1 ) concurrent staining with the I@ antiserum and the leu-ENK monoclonal antibody was followed (after elution) by staining for SS-28; 2) concurrent staining with the SS-28 and the leu-ENK monoclonal was followed by staining for 10, 3) sequential localization of MERF, SS-28, and I@ IR, with interposed elutions; and 4) sequential localization of

10, SS-28, and MERF IR, with interposed elutions. Al- though doubly and triply stained cells were commonly detected with each protocol, procedures 3 and 4 proved least satisfactory, as the number of ID- and MERF-stained cells detected in twice-eluted tissue were, respectively, 19 '2; and 24% lower than values for these antigens obtained in sections before exposure to the elution solutions. By contrast, after a single elution episode (procedures 1 and Z), the estimated number of I0 IR cells was on average 13% lower than control values and the SS-28 IR contingent only 8% below control values (Fig. 3). Colocalization values given below are therefore based on results from four animals, two of which were stained by procedure 1 and two by procedure 2.

Across animals and elution conditions, an average of :386 + 48 cells per side in the NTS stained positively for 10 IR. At the levels of the commissural NTS over which the I@ IH cells are found, SS-28 IR neurons are distributed similarly. Of the I@ 1R neurons in the NTS, 65% (250 * 35) also stained positively for SS-28. Few SS-28 IR cells lacking in I@ IR were found in the caudal NTS. By contrast, ENK- stained perikarya were more numerous and broadly distributed in the caudal NTS, and a majority, tending to lie dorsomedial to the IPISS-28 cell cluster, stained positively only for ENK. Nonetheless, a portion of the ENK IR distribution did overlap with those of the other peptides, and 33% (127 5 53) of all ID-stained cells were also ENK-positive. Triply labeled cells (i.e., those displaying I@, SS-28 and


ENK IR) numbered 98 2 31, and made up 2EiCo of the total I@ TR population (see Fig. 3) . Within the I@ IR population, no obvious topographic organization of cells that expressed I@ and SS-28 and/or ENK TR was apparent.

In the smaller I@ TR cell cluster, localized in the medullary reticular formation, neurons were also visualized that displayed SS-28 and/or ENK immunostaining (Fig. 4). The relative frequency with which the peptides were expressed in dually and triply labeled cells in this region seemed compa- rable to that encountered in the NTS, although this was not evaluated quantitatively.

Four of the six animals that received injections of tracer (one with fast blue, three with true blue) bore deposits that were centered in the PVH, with minor involvement of the overlying nucleus reuniens of the thalamus and/or the un- derlying anterior hypothalamic area (Fig. 5). Neither of these structures is known to receive prominent inputs from the NTS. Deposits of true blue appeared as consisting of a core of crystalline tracer (approximately 100-150 pm in diameter) surrounded by a zone (200-400 pm diameter) in which the neuropil was clearly labeled. Fast blue deposits were similar, except that the halo surrounding the crystalline rore was larger (300-500 pm diameter). The distribution of retrogradely labeled neurons seen in the medulla in animals with well placed deposits was similar to that described previously (e.g., Sawchenko and Swanson, '82; Sawchenko et al., '85,88a.c) and included cells in the regions of the medullary adrenergic and noradrenergic cell groups and in the regions of the two prominent I,B/SS-ZB/ENK cell clusters as well. No pronounced differences in the number or distribution of cells retrogradely labeled with true blue vs. fast blue were apparent. Retrogradely labeled cells in the NTS that stained for I@ and SS-28 and/or ENK were encountered in each experiment (Fig. 6). Among the subsets of I@ IR cells in the NTS defined by colocalization patterns, retrograde labeling was most commonly seen in cells stained for I@ alone, I@ and SS-28, or all three peptides. Counts made in one particularly good experiment, showed that of 64 retrogradely labeled, ID IR cells detected in the NTS, 12 stained positively only for 10, four for TB and SS-28, eight for I@ and ENK, and 40 for all three peptides. Retrogradely labeled cells stained for one or more of the peptides of interest were also seen in the reticular formation (Fig. 7). Although these were not counted, they were consistently fewer than those seen in the NTS.

DISCUSSION The results indicate that a substantial fraction of the I@

IR neuron population in the caudal medulla also expresses SS-28 and, to a lesser extent, ENK IR. These include cells that project to the region of the PVH.

Because of the uncertain relative sensitivities of the dif- ferent antisera, and the observed decrement in sensitivity imposed by the sequential staining (elution) procedure, it is likely that we have underestimated the extent to which the three peptides are colocalized. Although on average 66 % of all IB IR neurons were also SS-28-positive, this value reached as high as 8670 in individual experiments, and staining for these peptides was always carried out sequentially. As a practical matter, it would seem reasonable to consider the populations identified by the two antisera to be largely, if not completely, congruent. Sequential and concurrent methods were used with antisera against proen-

kephalin-derived peptides, and the results were consistent in identifying the enkephalinergic contingent as a subset (3350 on average and ranging as high as 60%) of the ID IR population. This result is in line with those of Millhorn et al. ('87a), who found. over the entire NTS, that on the order of 15'( of all SS-IR neurons are also ENK-positive.

The cell group in question will likely prove even more bio- chemically heterogeneous than the present results would indicate. As this study was in progress, a report appeared indicating that proglucagon-derived peptides display a distribution in cells and fibers (Jin et al., '88) that is seemingly identical to those stained for ID. We have confirmed this (unpublished observations) with antisera raised against either glucagon or glucagon-like peptide 1 (GLP-1). That neither peptide effectively competes for ID immunostaining would suggest that at least one additional class of peptides may be expressed by the caudal medullary IP/SS-28/ENK cell groups.

Findings obtained in material in which retrograde tracer deposits had been placed in the PVH confirm the results of previous studies treating the 16 and SS-28 systems individually (Sawchenko et al., '88a,c). In addition, the results estab- lish that caudal medullary cells projecting to the PVH commonly express both peptides. The present results also provide the first indication that medullary ENK neurons, and specifically those in which I@ and/or SS-28 IR are also expressed, contribute to the innervation of the PVH. It must be noted, however, that a thorough survey of the ori- gins of enkephalinergic inputs to the PVH is yet to be undertaken, and additional contributing sources seem likely (see, e.g., Cobbett et al., '83).

The cell groups under scrutiny here hold both organiza- tional and physiological interest. Organizationally, the results help to define and characterize an NTS population whose cells of origin and terminal distributions are distinct from those of closely juxtaposed groups of catecholamine- containing neurons and support the view that the sensory control of various neurosecretory neuron pools in the PVH is achieved by cell groups that are anatomically and bio- chemically differentiated. From a functional perspective, although the I@ and SS-28 IR projections touch several classes of PVH output neurons, the prominent and preferential inputs to OT-TR components of the magnocellular neurosecretory system are most distinctive and intriguing.

OT secretion can be elicited following intra-PVH injection of activin A, an TPA homodimer, a t femtomolar doses, or following electrical stimulation of the caudal NTS (Plotsky et al., '88). Of the three agents in question, only SS-28 and ENK examined for their effects on identified OT neurons, and both appear to exert predominantly hyperpolarizing effects when delivered iontophoretically (Arnauld et al., '83; Raby et al., '88). Paradoxically, intracerebroventricular in- fusion of 58-28 is a strong stimulus for OT (and vasopressin) secretion (Brown et al., '88a,b). Overall, the available ana- tomic, physiologic, and pharmacologic data converge to support roles for the three peptide systems in the control of OT secretion. The nature of their interactions, the conditions under which each may be released, and the kind(s) of information conveyed by this multiply determined pathway re- main to be clarified.

In the latter regard, the region of the caudal NTS in which the IBISS-28IENK cell group resides, falls within a zone that may receive primary sensory inputs from the vagus and glossopharyngeal nerves (see, e.g., Contreras et al., '82; Kalia and Sullivan, '82; Norgren and Smith, '88). Thus these pep-

PEPTLDERGIC SOLITARIO-HYPOTHALAMIC PROJECTIONS 277


tidergic neurons could play a role in relaying hemodynamic, particularly blood volume-related, information, which is known to influence OT secretion powerfully (see Poulain and Wakerley, '82). In addition, influences of vagally me- diated stimuli related to ingestion or nausea on OT secretion and on the activity of identified OT neurons have been described (Verbalis et al., '86; Renaud et al., '87) and provide a second potential context for this pathway. Alter- natively, the termination of spinal afferents, including some from the dorsal horn, in the caudal NTS (Menetrey and Basbaum, '87), raises the possibility that the caudal medullary peptidergic cell group could constitute the long-elusive central relay for the classic (somatic) stimuli for OT secretion, such as suckling or vaginal distension. The recently demonstrated attenuation of suckling-induced OT secretion following administration of anti-IP sera to the F'VH (Plotsky et al., '88) supports, but by no means estab- lishes, such a role. In this context, it is worthy of mention that, although the present study was carried out in male rats, we have shown previously that the organization of I@ IR pathways to the hypothalamus is similar in males and females (Sawchenko et al., '88b). If this circuitry does indeed play a role in gender-specific (i.e., suckling- and/or parturition-related) controls of OT secretion, it would seem likely that the pertinent dimorphisms would be exhibited distal to the NTS a t the level of the spinal cord and/or the primary somatosensory afferents.

ACKNOWLEDGMENTS This work was supported by NIH grant HL-35137 and

was conducted in part by the Clayton Foundation for Research-California Division. P.E.S. is a Clayton Founda- tion Investigators. J.C.B. was supported by a fellowship from the Brazilian Research Council, CNPQ. We thank Dr. W. Vale and <J. Rivier for inhibin antisera and synthetic peptides, Dr. R. Benoit for somatostatin antisera and for thoughtfully reviewing a draft of the manuscript, and K. Trulvck for assistance with photography.

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Inhibin β, somatostatin, and enkephalin immunoreactivities coexist in caudal medullary neurons that...

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