distribution and projections of cholecystokinin-immunoreactive neurons in the hypothalamic...

THE JOURNAL OF COMPARATIVE NEUROLOGY 227~173-lSl(l984)

Distribution and Projections of Cholecystokinin-Immunoreactive Neurons

in the Hypothalamic Paraventricular Nucleus of Rat

J.Z. KISS, T.H. WILLIAMS, AND M. PALKOVITS Department of Anatomy, University of Iowa, Iowa City, Iowa 52242 (J.Z.K., T.H.W.) and

Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20205 (M.P.)

ABSTRACT Analysis of coronal sections from colchicine-treated rat brains reveals

that CCK-immunoreactivity (CCK-ir) is present in two distinguishable neuronal systems in the paraventricular nucleus (PVN). More than 60% of these cells were found to be typical parvicellular neurons; the remainder were magnocellular neurons. The magnocellular CCK-ir neurons were concentrated in the medial magnocellular subdivision, while more caudally they formed a ring around a zone of unstained magnocellular neurons. Immuno- stained parvicellular neurons predominate in medial and periventricular parvicellular subdivisions.

The efferent projections of CCK-ir neurons were investigated by looking for retrograde accumulation of CCK-ir in cell bodies after selective knife cuts. A parasagittal cut of the lateral retrochiasmatic area as well as transection of the rostra1 median eminence resulted in an accumulation of CCK- ir material in a large number of both parvi- and magnocellular neurons. After pituitary stalk lesions, however, increased staining was only seen in magnocellular neurons. It is inferred that the magnocellular (presumed oxytocin-CCK) cells send their axons to the pituitary, whereas axons of CCK- ir parvicellular neurons appear to terminate in the median eminence.

After transection of the medial forebrain bundle (MFB), immunostaining increased in a small number of scattered transected fibers proximal to the knife cut and in a few perikarya in the PVN, indicating that very few CCK cells may send descending fibers to the lower brainstem.

Key words: neuropeptides, immunocytochemistry, magnocellular neurons, parvicellular neurons, neuroendocrine regulation

Cholecystokinin (CCK), a peptide hormone, was discov- ered by Ivy and Oldberg in 1928 and shown to cause gall bladder contraction and stimulate pancreatic enzyme secretion. Later, Jorpes and Mutt ('66) isolated from hog intes- tine a molecule of 33 residues (CCK-33) that had the properties of the hormone. Now it is clear that the C terminal octapeptide (CCK-8) of CCK-33 possesses the full biological activity (Mutt, '80). The C terminal pentapeptide is also shared with the antral hormone gastrin (Mutt, '80). Subsequent to the original isolation and characterization from the gastrointestinal tract, CCK-gastrin immunoreac-

tivity has been demonstrated in the brains of several vertebrate species (Vanderhaeghen et al., '75; Dockray et al., '77; Larsson and Rehfeld, '79.) It has been shown that the predominant form of brain CCK immunoreactivity is the octapeptide CCK-8 (Dockray, '76). CCK-8 is a putative synaptic messenger (Dodd et al., '80; Emson et al., '80) that is

Accepted February 29,1984.

Dr. J.Z. Kiss's address is Laboratory of Cell Biology, NIMH, Bldg. 10, Rm. 4N312, Bethesda, MD 20205.

0 1984 ALAN R. LISS, INC.

174

most likely involved in a variety of brain functions since it has been localized in a number of different neuronal systems, including the cortical-limbic areas, brainstem-spinal cord, and the hypothalamus (Innis et al., '79; Vander- haeghen et al.. '80).

Using radioimmunoassay (RIA) and immunocytochemistry (IC). CCK has been found in the hypothalamo-neurohypophyseal system of the rat (Rehfeld, '78; Beinfeld et al., '80). Cholecystokiriin-immunoreactive magnocellular neurons were demonstrated in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON), while CCK-ir fibers and terminals have been located in the median eminence (ME) and neurohypophysis (Innis et al., '79; Vander- haeghen et al., '80: Beinfeld et al., '80). Vanderhaeghen et al. ('81) reported that CCK-like peptides coexist with oxytocin in some magnocellular neurons in the hypothalamus. Subsequent studies have shown that much of the CCK in the ME and pituitary disappears following PVN lesions (Beinfeld et al., '80; Palkovits et al., '83), indicating that the major source of this peptide in the ME and neurohypophysis is the PVN. The paraventricular nucleus has been studied in detail by neuroanatomists. Neuroanatomical studies have shown that in addition to the "classical" neurohypophyseal projections, neurons in the PVN also project to the external zone of the ME Wandesande et al., '77; Wiegand and Price, '80; Lechan et al., '80) and to the autonomic centers of lower brainstem-spinal cord (Saper et al., '76; Hosoya and Matsushita, '79; Hosoya, '80; Swanson and Kuypers, '80). Based on this evidence it has been inferred that the PVN has an important role in the integration of autonomic and endocrine functions (Swanson and Sawchenko, '80).

In the rat, the PI 'N consists of a heterogenous population of about 11,000 parvicellular and 6,000 magnocellular neurons (Kiss et al., '83). The nucleus has at least six parvicellular and magnocellular subdivisions (Armstrong et al., '80; Swanson and Kuypers, '80) and more than 15 putative neurotransmitter substances have been found in these cells (Swanson and Sawchenko, '83). While there is no question that a CCK-like peptide is present in some magnocellular neurons, the distribution and morphology of CCK-positive cells have not been mapped. Furthermore, it is not known to what extent these neurons participate in the above-men- tioned divergent efferent projections of the nucleus. To study the distribution and efferent projections of CCK-containing cells in the PVN we have used an antibody against gastrin- cholecystokinin and the unlabeled antibody enzyme method.

MATERIALS AND METHODS Male Sprague-Dawley rats, weights 200-250 gm, were

kept under standard laboratory conditions; 12-12 hours light-dark cycle. standard rat chow pellets and tap water ad libitum. Three experimental groups of rats were exam- ined: 1) Normal, untreated animals. 2) Colchicine-treated animals. In order to increase immunostaining of cell bodies, colchicine (Sigma, 50 pg dissolved in 25 p10.9% NAC1) was injected into the lateral ventricle 48 hours prior to sacrifice. 3) Animals with various experimental lesions. In this experimental group, efferent projections of PVN CCK neurons were studied by transecting presumed CCK fiber pathways followed by immunocytochemical detection of increased staining in neurons that send axons through the lesioned area. This strategy depends on the retrograde accumulation of immunoreactive material in the proximal stump as well as in the perikarya (Barry et al., '73; Makara

J.Z. KISS, T.H. WII,I,IAMS, AND M. F'ALKOVITZ

et al., '83; Antoni et al., '83, '84). All surgery was performed under pentobarbital (Nembu-

tall anesthesia. The animals' heads were fixed in a Kopf stereotaxic instrument (5 mm nose-down position). Transec- tioris were made with a 1-2-mm-wide glass knife cut from a histological cover slip. The following lesions were performed: Group 1 (Fig. 3, cut No. 1): Lesions of the lateral retrochiasmatic area (RCAL). Unilateral, parasagittal cuts, beginning at the caudal edge of the optic chiasm and ex- tending caudally for 2 mm, were made 1 mm from the midline and penetrating to the base of the forebrain. Group 2 (Fig. 3, cut No. 2): Coronal periventricular lesion. In order to destroy CCK-ir fibers of PVN origin in the ME, l-mm- wide coronal knife cuts were made by a vertical penetration near the rostra1 pole of the ME (2.5 mm caudal to the bregma). As a control lesion, the knife was inserted 200- 300 pm short of the basal surface of the brain, thereby leaving the ME intact. Group 3: Pituitary stalk (ST) transection (Fig.3, cut No. 3). To interrupt fibers traveling to the neurohypophysis, a 1-mm-wide coronal cut was made in the midline 4 mm behind the bregma. In controls, the knife was stopped 200-300 pm short of the basal surface. Group 4: Medial forebrain bundle (MFB) lesion (Fig. 3, cut No. 4). To interrupt possible descending fibers from the PVN, 1- mm-wide coronal knife cuts were made 1 mm lateral to the midline, 4 mm caudal to the bregma. To lesion possible ascending CCK-ir fibers from the PVN, the same type of cut was made in the MFB, 0.5 mm caudal to the bregma.

Tissue preparation and immunohistochemistry Postoperative survival times were 3-7 days. Rats were

anesthetized and their brains fixed by transaortic perfusion of ice-cold (4°C) 4% paraformaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.167 M phosphate buffer, pH 7.0. The brains were cut into blocks that were placed in ice- cold fixative for 3 hours and then washed in several changes of potassium phosphate bufrer (pH 7.8). Coronal sections that were 20 pm-50 pm thick were cut with a vibratome (Oxford Co.). The extent and location of surgical cuts were carefully checked on 20-pm-thick vibratome sections. A minimum of three animals (out of a total of 50 operated animals) with verified lesion were used for each surgical group, and brain tissues were processed for immunocytochemistry. Sections were incubated in CCK antiserum (di- luted 1:600 in potassium phosphate buffer, pH 7.8) at 4°C for 24-48 hours, washed in potassium phosphate buffer, incubated in biotinylated antirabbit IgG (1:250) for 1 hour, and placed in Avidin-Biotin-Peroxidase complex (ABC) (90 pl of avidin and 90 pl of biotinylated peroxidase to 10 ml buffer) for 1 hour. The ABC components were obtained in kit form (Vectostain ABC Kits, Vector Laboratories). The chromagen used was 3,3-diaminobenzidine tetrahydro- chloride (DAB) (Hach) Triton-X (Mallinkrodt) was used in all solutions at 0.1% concentration. Sections were mounted on chromium coated slides, dehydrated, and coverslipped.

Antiserum rand controls The antiserum against CCK was produced in rabbits after

conjugation of CCK-8 sulfate to bovine serum albumin with glutaraldehyde. Details of preparation and specificities have been reported previously (Beinfeld et al., '81). The CCK antiserum used in the present study reacts with both CCK and gastrin (Beinfeld et al., '81). However, based on gel filtration and chromatographic characterization, it has been shown that the hypothalamo-hypophyseal system lacks gas-

CHOLECYSTOKININ IN THE PARAVENTRICULAR NUCLEUS 175

trin and the major CCK-like substance here is CCK octapeptide (CCK-8) sulfate (Beinfeld et al., ’80). Absorption of the primary antiserum by 100 pgiml of CCK-8 sulfate elim- inated all immunostaining of sections. Likewise, after omit- ting the primary antiserum or replacing it with normal rat serum, no peroxidase reaction product was seen.

The cross-sectional areas of cells were determined using a method and semiautomated system similar to that described by Cowan and Wann (‘73).

RESULTS The present description of the magnocellular subdivisions

of the PVN is essentially similar to that of Hatton et al. (‘76) and Armstrong et al. (’80). The delineation of the posterior magnocellular subnucleus represents the only dif- ference with previous schemes. This subdivision consists of a mixed population of large to medium-sized, elongated, bipolar neurons (Armstrong et al., ’80). The majority of these cells gives rise to descending projections to the spinal cord and medulla oblongata (Armstrong et al., ’80). The most rostral extension of the subnucleus can be found at the level of 1900 pm caudal to the bregma (Fig. 1D). This portion of the posterior magnocellular subdivision corresponds to the ventral group of spinal projecting neurons of Hosoya and Matsushita (‘79) and was included in the medial parvicellular subdivision by Armstrong et a1.(’80). At more caudal levels (Fig. lE, F), the present delineation of the subnucleus corresponds to the “posterior” subdivision of Armstrong et al. (‘80). Separating parvicellular subdivisions we followed the description and terminology proposed by Swanson and Kuypers (80).

Distribution and morphology of cholecystokinin- immunoreactive neurons

In normal, non-colchicine-treated animals, CCK-ir cell bodies were seen only occasionally, and CCK-ir fibers and varicosities were observed in moderate density throughout the PVN.

Following colchicine treatment, the DAB reaction product was found in large numbers of PVN neurons (Fig. 1). The immunostained perikarya differed in size and shape; and both magno- and parvicellular neurons contained immunoreactive material (Fig. 1). Even so, there were many unstained neuronal cell bodies. Among CCK-ir cell bodies the staining intensity was consistently heavier in magnocellular than parvicellular neurons (Fig. 1). The cross-sectional areas of large and small cells were 172 f 5 and 71 f 3 m2/ mean f SE, n = 82 and n = 120, respectively. A clear topographical segregation of CCK-ir magno- and parvicel- M a r neurons was observed (Fig. 1). The CCK-ir magnocellular neurons were mostly centered in the medial magnocellular subdivision, and more caudally (in the lat-

eral magnocellular subdivision) they surrounded a core of unstained magnocellular cell bodies. Unlike the lateral magnocellular neurons, immunoreactive cells in the medial magnocellular subdivision were evenly distributed. A smaller but significant number of CCK-ir magnocellular neurons were found among the parvicellular cells of PVN. The paucity of CCK-ir cells in the posterior magnocellular subdivision was noteworthy (Fig. 1E,F). While the CCK-ir magnocellular neurons in the medial magnocellular subdivision were both multipolar and bipolar, in the lateral subdivision the majority were bipolar or fusiform in shape.

Most of the stained parvicellular neurons were found in the medial and periventricular parvicellular subdivisions. A substantial number of CCK-ir cells were also found in the anterior, and a few cells were found in the dorsal parvicellular subdivisions. Immunostained parvicellular neurons in the anterior subdivision were mostly multipolar (Fig. 1A). Fusiform, bipolar neurons predominated in the periventricular and lateral regions, respectively, with a vertical orientation in the former and horizontal orientation in the latter. Efferent projections

Considering the specificity of the method of transections and enhanced immunostaining, it is important to empha- size the following points. First, in the control experiments when the primary antiserum was preabsorbed or replaced by normal serum, no DAB reaction product was detected. Second, no apparent signs of cell necrosis were detected in the PVN at the survival times we used after the lesions, although this possibility cannot be excluded entirely. Third, immunoreactive neurons that were found after the lesions were localized only in areas where immunoreactive neurons could be demonstrated following colchicine treatment. Therefore, the possibility that retrograde degenerative changes in neurons may give a false positive peroxidase reaction is unlikely.

Lesions of the lateral retrochiasmatic area (Group 1) resulted in increased CCK-ir of many parvi- and magnocellular neurons (Fig. 2A). Intensely stained fibers could be traced back to the PVN from the lesion site, suggesting that a large number of CCK-ir axons exit the nucleus lat- erally. Immunostained neurons were found in all subdivisions in which CCK-ir cells were identified following colchicine treatment.

After a knife cut at the level of the rostral third of the ME (Group ZA), accumulations of immunoreactive material were present in both magno- and parvicellular neurons. The CCK-ir cells were distributed in identical fashion to the distribution pattern after RCAL cuts (Fig. 2B). When the periventricular knife cut did not reach the ventral surface, i.e., if the ME remained intact (Group 2B), there was no accumulation of the reaction product in PVN neurons,

Abbreviations

F fornix m magnocellular part of the paraventricular

nucleus ME median eminence MFB medial forebrain bundle P

P pituitary S pituitary stalk 3V third ventricle

parvicellular part of the paraventricular nucleus

Subnuclei of the paraventricular nucleus

anterior parvicellular dorsal parvicellular lateral magnocellular medial magnocellular medial parvicellular posterior magnocellular periventricular parvicellular

176 J.Z. KISS, T.H. WILLIAMS, AND M. PaKOVITZ

Fig. 1. Distribution of cholecystokinin-immunoreactive (CCK-ir) cells in the PVN in colchicinc-treated animals (right). Paraventricular nucleus (PVN) subdivisions are riiitlined tin Lux01 fast blue-cresyl violet stained

paraffin sections (left). Serial coronal sections are a t various levels caudally (P, posterior) to the bregma: A,P 1.5 mm; B,1.7 mm; C,1.8 mm; D,1.9 mm: E.2.0 mm; F,2.1 mm. x 124.


Figure 1 (continued)

178 J.Z. KISS, T.H. WILLIAMS, AND M. PALKOVITZ


suggesting that CCK-ir PVN neurons may not project via the periventricular fiber system. This indicated also that increased staining in PVN neurons following this cut was not caused by interruption of inhibitory input reaching the nucleus from a more caudal site.

Lesions of the pituitary stalk (Group 3A) enhanced the immunostaining of magnocellular neurons (Fig. 2C). Fol- lowing this type of lesion, immunoreactive neurons were distributed as magnocellular CCK-ir neurons after colchicine treatment. No immunostained cells were found after the control lesion (Group 3B).

Increased CCK-immunoreactivity was noted in nerve fibers after the caudal MFB lesion (Group 4A) and was observed on both aspects of the cut. A few PVN cells in the lateral parvi- and magnocellular subdivisions showed increased immunostaining ipsilateral to the lesion (Fig. 2C). This did not occur after the rostra1 MFB lesion (Group 4B).

DISCUSSION The finding that a very few CCK-positive cell bodies can

be visualized in the PVN of normal, non-colchicine-treated rats is consistent with previous reports (Vanderhaeghen et al., '80). Likewise, the presence of CCK-ir in magnocellular neurons following colchicine treatment is in agreement with earlier studies (Innis et al., '73; Vanderhaeghen et al., '80). However, the present study extends these reports as follows: 1) CCK-ir neurons form a morphologically heterogenous population in the PVN with CCK-ir present in both magno- and parvicellular cells; 2) parvicellular neurons outnumbered their magnocellular counterparts by 2:l; 3) magnocellular CCK-ir neurons send their axons to the posterior lobe of the pituitary, whereas parvicellular cells project to the neurohemal (external) zone of the median eminence; and 4) it appears that CCK neurons give only a minor proportion of descending projections of the PVN.

Distribution of cells Recent neuroanatomical studies have clearly demon-

strated that the PVN neurons are segregated into at least eight parvi- and magnocellular subdivisions (Armstrong et al., '80; Swanson and Kuypers, '80). The subnuclei are characterized not only on the basis of cytoarchitectonic criteria but also because subdivisions project differentially to the neurohypophysis, the external zone of the ME, and the lower brainstem (Swanson and Kuypers, '80; Swanson et al., '80). Furthermore, different, immunocytochemically characterized neuronal subtypes were shown to be un- evenly distributed among the various subdivisions (see re- view of Swanson and Sawchenko, '83). Both magno- and parvicellular CCK-immunostained neurons also have a characteristic distribution pattern in the PVN. The magnocellular CCK-ir neurons predominate in the medial and lateral subdivisions, from where neurohypophyseal-neurosecretory pathways arise (Armstrong et al., '80), while the small CCK neurons were concentrated in those parts of

Fig. 2. Topography of CCK-ir cells in the PVN after various hypothalamic transections. A. Magno- and parvicellular CCK-ir neurons and immunostained processes (arrowheads) following retrochiasmatic area (RCAL). transection (arrows). No. 1 cut is shown on the insert B. Median eminence transection (No. 2 cut), magno- and parvicellular (arrows) CCK-ir neuron in the PVN. C. Pituitary stalk-transection (No. 3 cut); only magnocellular CCK cells are immunostained. D. Medial forebrain bundle (MFB) transection at a posterior hypothalamic level (No. 4 cut); only scattered CCK-ir cells in the PVN (arrows). x135.

the PVN (anterior parvicellular subdivision and the ventral and periventricular part of the medial subnucleus) that are known to project to the neurohemal (external) zone of the ME (Lechan et al., '80). Thus CCK neurons were mainly in subdivisions that are known to project to the ME and neurohypophysis. The topographical segregation of large CCK cells resembles the previously reported distribution of oxytocin cells in the PVN (Rhodes et al., '81; Sawchenko and Swanson, '82), thereby supporting the view that CCK probably coexists with oxytocin in magnocellular neurons (Van- derhaeghen et al., '81); but it should be recognized that CCK may not be a constituent of all oxytocin-containing cells. In line with this caveat, CCK-ir cell bodies were rarely found in the posterior magnocellular subnucleus, where substantial numbers of oxytocin cells have been previously described (Rhodes et al., '81; Sawchenko and Swan- son, '82). These oxytocin cells give rise to long descending projections to the lower brainstem (Sawchenko and Swan- son, '82); and these oxytocinergic neurons that project to autonomic centers of the lower brainstem most probably lack CCK. On the other hand, CCK likely exists in most of the oxytocin neurons that project to the neurohypophysis since 1) large numbers of CCK-ir neurons were found in all subdivisions where oxytocin neurons that project to the neurohypophysis from the PVN, SON, and magnocellular accessory neurons and 2) all of the oxytocin nerve terminals in the neuronal lobe of the pituitary are reportedly immunoreactive with CCK antiserum (Martin et al., '83). It follows from the above that oxytocin neurons that are known to have different efferent projections might differ their ca- pability for generating CCK-like peptide.

Nearly ten different putative neurotransmitter substances have been demonstrated so far in small cells of the PVN. Does CCK-ir exist in a separate neuron population or does it coexist in parvicellular neurons of the PVN with other putative neurotransmitter substances such as other peptides or biogenic amines? The distribution patterns of oxytocin (Rhodes et al., '81; Sawchenko and Swanson, '821, somatostatin (Dierickx and Vandersande, '79), dopamine (Swanson et al., '81), enkephalin (Sawchenko and Swanson, '821, corticotropin-releasing factor (CRF) (Swanson et al., '82; Antoni et al., '83a,b), and thyrotropin-releasing factor (TRH) (Lechan et al., '82) have already been described, and none is identical to the distribution of CCK parvicellular neurons, although there may be some overlap. In case of CRF, for example, there is a distribution pattern similar to that of CCK-positive neurons in the anterior subnucleus and in the more caudal parts of the medial parvicellular subdivisions (Antoni et al., '83a,b), raising the possibility of a partial coexistence of these substances. This hypothesis deserves further testing.

Efferent projections of cholecystokinin- immunoreactive neurons

The distribution pattern of CCK-ir cells suggests that these neurons project to the ME and neurohypophysis. To confirm this supposition, however, we sought a more direct approach. The approach applied was identical to that once widely used "classical" strategy of analyzing retrograde cell reactions following transection of fibers to identify the cells of origin (Cowan, '70). Recent immunocytochemical reports indicate that a dramatic increase in staining intensity can occur following axotomy in the proximal stump of axon as well as in the neuronal perikarya (Barry et al., '73;

180

Antoni et al., '83, '84; Makara et al., '83). Similarly, some of the transections used in the present study induced an enhanced immunoreactivity in certain neurons of PVN.

A number of earlier studies demonstrated that the lesions of the pituitary stalk results in cell loss (20-80%, depending on the site of the lesions) in hypothalamic magnocellular nuclei (Rasmussen, '40; Frykman, '42; Bodian and Maren, '51; Olivecrona, '57). The same cell loss may not have occurred in the present series of experiments because the survival times differed from those in the earlier studies. It is generally believed that retrograde, reactive neuronal changes occur in the acute stage after axotomy, and cell death, if it happens, occurs later (Cowan, '70; Grant '75). The postoperative survival times of 1-2 months used in the studies cited above contrast with the 3-7 days survival intervals used in the present study, which may correspond to the stage of "cell struggle" (for recovery). Some authors, e.g., Grant ( 'TO), have distinguished between "retrograde cellular reaction" and "retrograde degeneration"; the former leaves open the possibility for recovery.

Previous studies have shown that most PVN efferents leave the nucleus in a lateral direction and loop into the ME through the lateral retrochiasmatic area (Palkovits, '82). Cholecystokinin-immunoreactive axons follow this route since the lateral knife cut in the RCAL (Group 1) generated a marked accumulation in fibers on the PVN side. This accumulation in a large number of parvi- and magnocellular neurons indicated that many magno- and parvicellular CCK-ir neurons are projection neurons (Fig. 3).

Severing the neurosecretory-neurohypophyseal pathways (Group 3A) induced accumulation only in magnocellular neurons, indicating that these magnocellular (presumed oxytocin-CCK) cells send their axons to the pituitary. This

J.Z. KISS, T.H. WILLIAMS, AND M. PALKOVITZ

Cut No. 3

Fig. 3. Three~dirnensional schematic drawing ofthe projections of CCK- ir cells to the median cminence (ME). MFB, and posterior pituitary through the lateral KCAL. Topography of surgical transections are also schemati- cally indicated: No. 1, KCAI, transection; No. 2, ME transection; No. 3, pituitary stalk transectuin; No. 4 , MFB transection. For details see "Mate- rials and Methods."

supports previous indicatio'ns that the PVN is the major source of neurohypophyseal CCK (Beinfeld et al., '80; Pal- kovits et al., '83).

In addition to a pathway from the PVN to the neurohypophysis via the internal zone of ME, CCK immunostaining was observed in PVN neurons that innervate the neurohemal (external) layer of the ME. The notable accumulation occurred in parvicellular neurons following the RCAL and ME cut, but not after the MFB and stalk transection, indicating that parvicellular CCK-ir neurons in the PVN give rise to this projection. These results are consistent with those studies that showed a dramatic depletion of CCK content (6040%) in the ME after PVN lesion (Bein- feld et al., '80, Palkovits et al., '83). The report of Palkovits et al. '(83) also suggests tha.t the PVN is a major source of CCK in the ME since the RCAL cut, which eliminates all afferent input to the ME, results in a comparable depletion.

A substantial number of PVN neurons send axons to the lower brainstem and to the spinal cord (Hosoya and Mat- sushita, '79; Hosoya, '80; Swanson and Kuypers, '801, and several attempts have been made to characterize this pathway immunocytochernically. So far only a small percentage of these projecting neurons have been identified. Thus, 10% of the neurons that give rise to long descending projections are oxytocinergic, and a few enkephalin-, dopamine-, and somatostatin-containing cells were also shown to project to the lower brainstem (Sawchenko and Swanson, '82). Since a very few immunostained cells were found after transection of the caudal MFB, it follows that CCK neurons con- tribute few fibers to the descending system.

Our basic conclusion is that a CCK-like substance has been identified in two distinct neuronal systems of the PVN, and both of these systems have been linked with neuroendocrine regulation (Fig. 3). First, CCK-immunoreactivity exists in parvicellular neurons that are poten- tially involved in regulation of adenohypophyseal hormone secretion through projections to the external zone of the ME. Second, CCK-ir was identified in the magnocellular- neurohypophyseal system. .Although we found CCK-ir in these two, separate outputs of the PVN, we are not able to grasp the precise physiological significance of these outputs. Based on some previous evidence, one can speculate that CCK coexists with other putative transmitter substances in both magno- and parvicellular neurons and thereby helps to regulate (or modulate) the release or action of these other substances in both systems. The coexistence of CCK with oxytocin in magnocellular neurons has been established (Vanderhaeghen et al., '81). Furthermore, it has been reported that the CCK content of the pituitary CCK is decreased dramatically by physiological manipulations that stimulate the release of vasopressin or oxytocin (Bein- feld et al., '80). Thus, CCK may be involved in regulation of vasopressin and oxytocin neurosecretion. Similar indi- rect evidence suggests that CCK may be involved in central mechanisms activating the pituitary-adrenal axis. For example, the CCK content of the medial basal hypothalamus is reduced substantially after bilateral adrenalectomy (An- hut et al., '83). Interestingly, as pointed out earlier, there appears to be a re1ationshi.p between CCK- and CRF-ir neurons, in regard to their distribution in parts of the PVN. This favors the possibility that in some neurons there may be coexistence and interaction between these two peptides. These are phenomena that we need to understand more thoroughly.


ACKNOWLEDGMENTS The authors are grateful to Margery Beinfeld for supply-

ing the antibody and to Patricia Thurston for her excellent secretarial help.

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distribution and projections of cholecystokinin-immunoreactive neurons in the hypothalamic...

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