Increased circulating cholecystokinin contributes to anorexia and anxiety behavior in mice overexpressing pancreatic polypeptide.

We have previously reported that pancreatic polypeptide (PP) overexpressing mice display thin phenotype with delayed gastric emptying and decreased food intake. In the present study, we further examined if CCK contributes to anorexia and anxiety behavior in PP overexpressing mice. Plasma CCK levels in PP overexpressing mice and their littermates were determined by radioimmunoassay using antisera specific to sulfated CCK-8 and CCK-33. To elucidate the role of CCK in PP overexpressing mice, CCK-1 receptor antagonist (L-364,718) or saline was administered intraperitoneally and food intake was measured for 2 h. CCK-2 antagonist (L-365,260) or saline was injected intraperitoneally and the elevated plus-maze test was performed to assess anxiety. Plasma CCK levels were significantly increased in PP overexpressing mice. Administration of L-364,718 increased food intake in PP overexpressing mice compared to the saline-injected PP overexpressing group, while L-364,718 did not increase food intake in non-transgenic littermates. PP overexpressing mice exhibited anxiety in the plus-maze test. Administration of CCK-2 receptor antagonist (L-365,260) reversed the decreased percentage of entry into the open arms in PP overexpressing mice. These results indicated that elevated CCK may contribute to anorexic and anxious phenotype of PP overexpressing mice.

Changes of the gastric endocrine cells in the C57BL/6 mouse after implantation of murine lung carcinoma: an immunohistochemical quantitative study.

AIM: The regional distributions and relative frequencies of some gastric endocrine cells of C57BL/6 mice were studied by immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin, somatostatin, gastrin, cholecystokinin (CCK)-8, glucagon and human pancreatic polypeptide (HPP) after subcutaneous implantation of murine lung carcinoma (3LL) cells. METHODS: The experimental animals were divided into two groups, one is non-implanted sham and the other is 3LL-implanted group. Samples were collected from the two regions of stomach (fundus and pylorus) at 28 d after implantation of 3LL cells (1 x 10(5) cell/mouse). RESULTS: In this study, all the seven types of immunoreactive (IR) cells were identified except for HPP. Most of these IR cells in the gastric portion were generally spherical or spindle in shape (open-type cell) while cells showing round in shape (closed-type cell) were found occasionally. The regional distributions of gastric endocrine cells in the 3LL-implanted group were similar to those of non-implanted sham. However, significant decreases of some types of IR cells were detected in 3LL-implanted group compared to those of non-implanted sham. In addition, the IR cells showing degranulation were numerously detected in 3LL-implanted group. CGA-, serotonin- and somatostatin-IR cells in the fundus and pylorus regions, and gastrin-IR cells in the pylorus regions of 3LL-implanted groups significantly decreased compared to those of non-implanted sham. However, no changes on frequencies of CCK-8- and glucagon-IR cells were demonstrated between 3LL-implanted and non-implanted groups. CONCLUSION: Endocrine cells are the anatomical units responsible for the production of gut hormones, and the change in their density would reflect a change in the capacity of producing these hormones. Implantation of tumor cell mass (3LL) induced severe quantitative changes of gastric endocrine cell density, and the abnormality in density of gastric endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.

Terminal field of cholecystokinin-8-like immunoreactive projection neurons of the rat main olfactory bulb.

The terminal field of cholecystokinin-8 (CCK)-like immunoreactive (CCK-IR) tufted cells in the rat main olfactory bulb was examined by means of immunohistochemistry combined with either an anterograde tracer or a degeneration method. CCK immunostaining was carried out in animals in which Phaseolus vulgaris agglutinin (PHA) had been injected into the main olfactory bulb. Pairs of adjacent sections were processed for CCK and PHA immunostaining, respectively. Dense CCK-IR terminallike staining was noted in layer Ia of the anterior olfactory nucleus and lateral part of the olfactory tubercle; weaker staining was also observed in the transitional area between the anterior olfactory nucleus and the piriform cortex, in the medial part of the olfactory tubercle, and in the cortical amygdaloid nucleus. The CCK-IR staining was limited to the area containing PHA-labeled terminals and was diminished in these sites after unilateral olfactory bulbectomy. Immuno-electron microscopic analysis showed that CCK-IR profiles in such regions made asymmetric synaptic contacts, mainly with dendritic spines. These results suggest that CCK-IR tufted cells project mainly to the anterior olfactory nucleus and lateral part of the olfactory tubercle, and act mainly via axospinous synapses.

Laminar organization of peptide-like immunoreactivity in the anuran optic tectum.

Peptide, 5-hydroxytryptamine (5-HT)-, tyrosine hydroxylase (TOH)-, and glial fibrillary acidic protein (GFAP)-like immunoreactivity was studied in the optic tectum of Rana pipiens. Peroxidase-antiperoxidase and indirect immunofluorescence single- and double-labeling methods were used to compare differential laminar distribution of each of these substances. Substance P (SP), leucine-enkephalin (LENK), cholecystokinin octapeptide (CCK8), bombesin (BOM), avian pancreatic polypeptide (APP), and possibly neurotensin display unique individual patterns of laminar distribution of processes and cell bodies throughout the tectum. A correlative analysis of the topographical distribution of SP, LENK, BOM, and APP on the basis of double-labeled sections shows a precise laminar segregation of these substances. Vasoactive intestinal peptide-, beta-endorphin-, and ranatensinlike immunoreactivity is consistently absent from our material. 5HT- and TOH-like immunoreactivity discloses a reticular array of fibers without clear evidence of laminar organization. This peptide-like laminar organization is particularly elaborate throughout the superficial neuropil of the optic tectum, the major retinorecipient zone. The pattern of lamination demonstrated in the present study differs in several important features from that previously described on the basis of several histological methods. The cells of origin of processes (axons and/or dendrites) in the superficial tectal neuropil may be either intrinsic or extrinsic to the tectum. Special reference is made to conflicting evidence regarding the possibility of a retinal contribution to peptide-like tectal lamination.

Fine structural studies of cholecystokinin-8-like immunoreactive neurons and axon terminals in the nucleus of tractus solitarius of the rat.

Cholecystokinin (CCK)-8-like immunoreactive structures in the nucleus of tractus solitarius (NTS) were studied by using the peroxidase-antiperoxidase (PAP) immunohistochemical method. Immunoreactivity was localized in cell bodies and nerve fibers. The perikarya were oval or fusiform (average length 13 micron) and were mostly located in the dorsal half of the medial subnucleus of the NTS at the level of the area postrema (AP). One to three straight immunoreactive dendritelike processes emerged from the perikarya. Neurons that had first been identified under light microscopy were also studied by electron microscopy. Each neuron had a moderate amount of cytoplasm and an oval or elongated nucleus that was eccentrically located in the soma. A few synaptic inputs were found on the CCK immunoreactive perikarya, while a moderate number were seen on both proximal and distal dendrites. These neurons received both asymmetrical and symmetrical synaptic inputs. The immunoreactive dendrites were most frequently in asymmetrical synaptic contact with nonreactive boutons (max. 2.7 micron in diameter) containing fairly densely packed, small round vesicles. CCK immunoreactive boutons located in the NTS at the level of the AP were analyzed using electron microscopy; these boutons formed asymmetrical synaptic contact with other neuronal elements. Their postsynaptic targets were immunoreactive and nonreactive perikarya and dendrites. These data suggest that CCK-containing afferents might affect the neurotransmission of heterogenous types of solitary neurons.

GABAergic neurons containing CCK-8-like and/or VIP-like immunoreactivities in the rat hippocampus and dentate gyrus.

The coexistence of cholecystokinin-octapeptide-like (CCK-L) and/or vasoactive-intestinal-polypeptide-like immunoreactive (VIP-LI) materials and glutamate decarboxylase (GAD) was studied in the rat hippocampus and dentate gyrus by means of immunohistochemistry. Consecutive 40-micron-thick sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for these peptides and GAD in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell, incubated in two different antisera. Almost all of the CCK-LI neurons were also GAD immunoreactive, whereas only about 10% of the GAD-immunoreactive neurons were CCK-LI. The percentages of GAD-immunoreactive neurons which were also immunoreactive for CCK were dependent on the laminar area in which they were found: i.e., 15-20% in the stratum radiatum of the hippocampus, about 10% in the stratum pyramidale, and about 6% in the stratum oriens. In contrast to the CCK-LI neurons, only about 40% of the VIP-LI neurons were identified to be also GAD immunoreactive, which might correspond to only part of the GAD-immunoreactive neurons. Furthermore the coexistence of VIP-LI and CCK-LI materials was recognized in about 10% of the CCK-LI neurons or about 35% of the VIP-LI neurons, indicating that some GABAergic neurons (presumably about 1%) in the rat hippocampus and dentate gyrus may contain both CCK-LI and VIP-LI materials.

Radioimmunoassay of cholecystokinin: comparison of different tracers.

We have compared the binding of cholecystokinin (CCK) antibodies with different sequence-specificities to Bolton-Hunter labeled CCK-33 (125I-BH-CCK-33), CCK-8 (125I-BH-CCK-8) and chloramine-T iodinated gastrin-17 (125I-gastrin-17). The antibody binding was expressed as the final antiserum dilution ('titer') and the effective equilibrium constant of the binding (Ko eff). Antibodies specific for the C- or the N-terminal sequence of CCK-8 all bound well to 125I-BH-CCK-8. In contrast, some of the antibodies directed against the common C-terminus of CCK and gastrin displayed remarkably low binding of 125I-gastrin-17 or 125I-BH-CCK-33, whereas all antisera specific for the N-terminal or midsequence of CCK-33 bound 125I-BH-CCK-33 well. The lower binding of 125I-BH-CCK-33 to some C-terminal antibodies raised against gastrin may be due to a C-terminal conformation of CCK-33 different from that of gastrin. In accord with the high specific radioactivity of 125I-BH-CCK-8, the best sensitivity of CCK radioimmunoassays was obtained with the CCK-8 tracer.

Reversal of tolerance to morphine but no potentiation of morphine-induced analgesia by antiserum against cholecystokinin octapeptide.

Tolerance to morphine analgesia was induced in rats by chronic treatment with morphine (5-30 mg/kg, t.i.d. for 6 days). Intracerebroventricular (i.c.v.) injection of antiserum against cholecystokinin octapeptide (CCK-8) reversed tolerance to morphine by 50% (P less than 0.001). Intrathecal (ith) injection of the CCK-8 antiserum produced a similar, although less marked, reversal of tolerance to morphine. Rats made tolerant to analgesia induced by morphine developed a cross tolerance to electroacupuncture-induced analgesia. This cross tolerance was also reversed by the CCK-8 antiserum by more than 50% (P less than 0.001). Intracerebroventricular or intrathecal injection of the CCK-8 antiserum per se produced no significant changes in the basal level of the latency of the tail flick response, nor did it affect the analgesia induced by morphine in naive rats. The results suggest that prolonged activation of opioid receptors may trigger the CCK-8 system in the central nervous system to exert a negative feedback control, which may constitute one of the mechanisms for the development of tolerance to opioids.

Regulation of human T lymphoblast growth by sensory neuropeptides: augmentation of cholecystokinin-induced inhibition of Molt-4 proliferation by somatostatin and vasoactive intestinal peptide in vitro.

The effects on proliferation of Molt-4 lymphoblasts of cholecystokinin (CCK-8), somatostatin-14 (SS), vasoactive intestinal peptide (VIP) and substance P (SP) were investigated using different combinations of the peptides, peptide analogs and their antagonists. In vitro proliferation of the cells was measured by a colorimetric assay for cell growth and survival. Results indicate that SP and SP (3-11) stimulated, whereas CCK-8, VIP and SS inhibited, proliferation in a dose-dependent manner (P < 0.05). Unsulfated CCK-8 had no effect on growth of Molt-4 lymphoblasts, and a specific antagonist of CCK, at a concentration 10(-6) M, diminished the inhibitory effect of CCK-8 on Molt lymphoblasts (P < 0.05). This suggests that the inhibitory action of CCK-8 was mediated by peripheral-type CCK receptors. SS and VIP, at equimolar concentrations of 10(-6) M, significantly augmented the CCK-8-induced inhibition of Molt-4 lymphoblast proliferation. However, none of the inhibiting neuropeptides suppressed stimulation of Molt-4 lymphoblast proliferation in response to SP. These data suggest a role of sensory neuropeptides including CCK in modulating human T lymphoblast proliferation during neuroendocrine interactions with the immune system.

Neurotrophic effects of testosterone on the medial nucleus of the amygdala in adult male rats.

Our previous reports of major sex differences in the substance P-immunoreactive (SPir) innervation of the medial posterior divisions of the bed nucleus of the stria terminalis (BST) and medial nucleus of the amygdala in rats raised the question of the hormonal regulation of this innervation. We now report the results of two experiments which examined the effects of castration of adult males on the SPir innervation of these regions. In experiment 2 we asked whether castration might also alter the cytoarchitecture of these regions. In experiment 1 three groups; sham operated (Sham), castrated (C) and castrated plus testosterone (C+T) were examined at each of the three survival periods (2, 4 and 8 weeks) post castration. Animals of the C+T groups each received a 45 mm silastic implant of testosterone sc at the time of castration to maintain testosterone levels postoperatively. Castration produced a consistent and highly significant decrease in the area of dense SPir fiber staining in the posterior medial amygdala which became greater with increasing survival. By 8 weeks the area of staining was 42% smaller in group C as compared to the matched sham-operated group. Smaller decreases were seen in the size of the dense field of SPir fibers in the posterior part of the dorsomedial BST. Testosterone implants maintained the size of the SPir fields of fibers in both the medial amygdala and BST, as the areas of staining in the C+T groups were not significantly different from those in the Sham groups at any of the 3 survival times. In experiment 2 we measured the area and optical density of SPir fiber staining in the medial amygdala and medial BST at 8 weeks post-castration. In addition, we measured the size of the cell groups within these regions using cresyl-stained sections. As in experiment 1, at 8 wks following castration there was a marked decrease in the area of dense SPir staining in both the BST and medial amygdala. The sizes of the dense fields of fibers were reduced by approximately 23% in the BST and by 40% in the posterior medial amygdala. Castration also significantly reduced the optical density of staining within the medial amygdala. The major finding of experiment 2 is that castration affects the cytoarchitecture as well as the SPir staining in these areas. In the BST, the cell group BSTMPM receives most of the dense SPir innervation.(ABSTRACT TRUNCATED AT 400 WORDS)

Principles of rat subcortical forebrain organization: a study using histological techniques and multiple fluorescence labeling.

In the present study, we introduce new views on neuro- and chemoarchitectonics of the rat forebrain subcortex deduced from traditional and current concepts of anatomical organization and from our own results. It is based on double and triple immunofluorescence of markers for transmitter-related enzymes, calcium-binding proteins, receptor proteins, myelin basic protein (MBP) and neuropeptides, and on histological cell/myelin stains. The main findings can be summarized as follows: (i) the dorsal striatum of rat and other myomorph rodents reveals a small caudate equivalent homotopic to the caudate nucleus (C) of other mammals, and a large putamen (Pu). (ii) Shell and core can be distinguished also in the 'rostral pole' of nucleus accumbens (ACC) with the calretinin/calbindin and neuropeptide Y (NPY) immunostaining. The shell reveals characteristics of a genuine striatal but not of an extended amygdala (EA) subunit. (iii) EA and lateral septum show striking similarities in structure and fiber connections and may therefore represent a separate parastriatal complex. (iv) The meandering dense layer (DL) of olfactory tubercle (OT) forms longitudinal gyrus- and sulcus-like structures converging in its rostral pole. (v) The core regions of the islands of Calleja that border the ventral pallidum (VP) sharing some of its features are invaded by myelinated fibers of the medial forebrain bundle (MFB). The island of Calleja magna is also apposed to an inconspicuous, slender dorsal appendage of VP. (vi) The VP is composed of a large dorsal reticulated part traversed by the myelinated GABAergic parvalbumin-immunoreactive axons of the MFB and a slender ventral non-reticulate part close to the islands of Calleja. (vii) Considering their close association to the limbic system, ventral striatum (VS) and VP may represent the oldest part of basal ganglia, whereas dorsal striatopallidal subunits were progressively developed in parallel to the growing neocortical influence on motor behavior.

Measurement of cholecystokinin octapeptide using a new specific radioimmunoassay.

A peptide analogue of CCK-8 (Tyroc) which has a tyrosine in place of the amide group in the C-terminal end, has been used both for raising antisera and for iodination. The antisera produced by immunisation with Tyroc are directed towards the N-terminal end of the CCK-8 molecule. The assay system appears totally specific for the CCK-8 sulphated molecule and shows no significant cross-reaction with other molecular forms of CCK, or with the gastrins. The assay can detect changes between adjacent tubes of 0.25 fmol/tube CCK-8 with 95% confidence. The assay is robust, reliable and reproducible and can be used to measure tissue and plasma levels of CCK-8.

Cholecystokinin octapeptide immunoreactivity in the nucleus tractus solitarius of the cat.

The nucleus tractus solitarius possessed distinct patterns of cholecystokinin immunoreactive fibers and cell bodies within its various subdivisions. The commissural, medial, intermediate, parvocellular, dorsolateral and interstitial subdivisions contained relatively dense amounts of CCK immunolabelled fibers. In contrast, CCK immunoreactivity within the ventrolateral subdivision consisted of a few scattered fibers and small neurons. The commissural, intermediate, medial, dorsolateral and parvocellular subdivisions contained CCK immunoreactive neurons following colchicine treatment. The presence of CCK in the NTS suggest that it may be involved as a neuromodulator and/or neurotransmitter in circuitry that mediate cardiovascular, respiratory, gastrointestinal and taste functions.

The subcellular distribution of peptides immunoreactive for the amino-terminal of pro-cholecystokinin in rat brain.

Antiserum 1942 raised against the synthetic peptide V-9-M is specific for the amino-terminus of pro-cholecystokinin (pro-CCK). It detects three major peptides in whole rat brain extracts with molecular weights of about 13 000 (peak 1), 8000 (peak 2) and 2700 (peak 3), of which the major one is peak 3. Rat brain was found to contain large quantities of these V-9-M-like peptides. Subcellular fractionation of whole rat brain was performed to determine what cellular component was enriched in these peptides. The molecular weight of the V-9-M-like and CCK-8-like peptides enriched in various subcellular fractions has been determined by Sephadex G-50 chromatography. Primary subcellular fractionation experiments indicated a significant enrichment of V-9-M-like peptides in the mitochondrial pellet (P2), a lesser amount in the microsomal pellet (P3), and a slight enrichment in the soluble fraction (S3). Further purification of the P2 fraction demonstrated an increase of V-9-M-like immunoreactivity in purified synaptosomes. With the exception of the enrichment in the soluble fraction, V-9-M-like peptides follow a similar distribution to that of CCK-8-like peptides. Sephadex chromatography of P2 and P3 fractions indicates that the major form of V-9-M present is the peak 3 (2700) form. This V-9-M-like peptide may represent an intermediate in the processing of CCK, and its presence in synaptosomes may indicate that the proteolytic cleavage of pro-CCK into CCK 58 and peak 3 takes place in synaptic vesicles.

Apomorphine affects cholecystokinin content via preferentially D1 or D2 dopamine receptor according to the regions of the rat brain.

Cholecystokinin octapeptide-like immunoreactivity (CCK-8IR) was measured in several regions of the rat brain after the intraperitoneal administration of apomorphine, SKF-38393 (D1 agonist), LY-171555 (D2 agonist). In the medial prefrontal cortex and striatum, apomorphine and SKF 3839 decreased CCK-8IR. In the anterior and posterior nucleus accumbens, on the other hand, the inhibitory effect of apomorphine was mimicked by LY-171555. These results suggest that apomorphine affects CCK-8IR via either the D1 dopamine (DA)-receptor or D2 DA-receptor according to the brain region.

Dopaminergic agents affected neuronal transmission of cholecystokinin in the rat brain.

In order to study the interaction between cholecystokinin (CCK) and dopamine (DA), we prepared an anti-CCK-8 antibody with low cross reactivity, and observed effects of administered various dopaminergic agents on CCK-immunoreactivity (CCK-IR) in discrete brain regions of rats. CCK-8 IR (boiling water extraction) and CCK-33 IR (acetic acid extraction) were also measured in the same sample. A single administration of haloperidol decreased the CCK-8 IR in the corpus striatum and that of racemic sulpiride significantly decreased the CCK-8 IR in the frontal cortex and the limbic system. In contrast, a single administration of apomorphine or methamphetamine increased the CCK-8 IR in the same regions. These findings suggest that an acute response of the CCK system to administered dopaminergic agents may be due to a change in the rate of release of CCK-8, but not to a change in its synthesis in areas in which DA neurons originating in the midbrain innervate. After chronic administration of racemic sulpiride or methaphetamine, CCK-8 IR in various brain regions exhibited a tendency close to that of control.

Distribution of calcitonin-gene-related peptide, neuropeptide-Y, vasoactive intestinal polypeptide, cholecystokinin-8, substance P and islet peptides in the pancreas of normal and diabetic rats.

Neuropeptides and peptides are particularly important in the co-ordination of pancreatic exocrine and endocrine secretions. In diabetes mellitus, pancreatic endocrine secretion is particularly impaired. This study investigates whether there is a change in the pattern of distribution of neuropeptides including calcitonin-gene-related peptide (CGRP), neuropeptide-Y (NPY), vasoactive intestinal polypeptide (VIP), cholecystokinin-octapeptide (CCK-8), substance P (SP), and islet peptides including insulin (INS), glucagon (GLU), somatostatin (SOM) and pancreatic polypeptide (PP) in the pancreas of streptozotocin (STZ)-diabetic rats. After the onset of diabetes, the pattern of distribution of INS, GLU, SOM and PP cells was deranged. CGRP was demonstrated in ganglion cells of both normal and diabetic pancreas. CGRP was also localized in nerve fibres innervating the blood vessels of both normal and diabetic pancreas. The pancreata of both normal and diabetic rats contained numerous NPY-immunopositive varicose nerve fibres in the wall of blood vessels. In normal pancreatic tissue, VIP-immunopositive nerve fibres were observed in all areas of the pancreas. After the onset of diabetes, VIP-positive nerve fibres were still discernible in the interacinar regions of the pancreas. CCK-8 was identified in nerve fibres innervating both the normal and diabetic rat pancreata. These CCK-8-immunopositive nerves were varicose in nature and distributed in the wall of blood vessels. SP was demonstrated in neurons located in the interlobular areas of normal tissue and in fine varicose nerve fibres of the interacinar region of STZ-induced diabetic pancreas. In conclusion, CGRP, NPY, VIP, CCK-8 and SP are well distributed in both normal and diabetic pancreas.

Cholecystokinin innervation of rat thalamus, including fibers to ventroposterolateral nucleus from dorsal column nuclei.

The distribution of cholecystokinin octapeptide immunoreactive fibers and puncta in the adult rat thalamus was studied using immunocytochemical methods. Small to moderate numbers of immunoreactive fibers were present in the lateral habenular nucleus, ventral lateral geniculate nucleus, zona incerta, parataenial, mediodorsal, medioventral, and submedial nuclei, the rhomboid, paracentral, central lateral and parafascicular nuclei, and in the medial geniculate and dorsal lateral geniculate nuclei. Moderate to large numbers of cholecystokinin (CCK)-positive fibers were present in the paraventricular nuclei, the reticular nucleus, the anteroventral, anteromedial, and central medial nuclei, and in the rostral extension of the internal medullary lamina between the parataenial and anteroventral nuclei. Dense concentrations of immunoreactive fibers were also found in a principal sensory relay nucleus, the ventroposterolateral nucleus (VPL), of the ventrobasal complex. The number of CCK-positive fibers in VPL showed a marked unilateral decrease in rats which had received lesions of the contralateral gracile and cuneate nuclei. The results of this study demonstrate that CCK-immunoreactive fibers and puncta are widely distributed in the rat thalamus, and that the source of these fibers in VPL is probably the dorsal column nuclei.

Cholecystokinin octapeptide-like immunoreactive material in neurons of the intralaminar nuclei of the cat's thalamus.

Cholecystokinin-like immunoreactive material (CCK-IR) was revealed in the cat's thalamus by using the peroxidase-antiperoxidase method. The most dense collection of perikarya containing CCK-IR was seen in the rostral group of the intralaminar nuclei, in rostral parts of the rhomboid nucleus and the anterodorsal nucleus. Cells with CCK-IR were also found in the caudal group of the intralaminar nuclei, in the paraventricular nucleus and the parataenial nucleus. The remaining thalamic nuclei were void of CCK-IR. By combining immunohistochemistry with retrograde transport of horseradish peroxidase, CCK-IR was shown to be present in neurons of the intralaminar nuclei projecting to the neocortex. Our findings suggest that CCK might act as a transmitter in the efferent projections of the intralaminar and midline nuclei of the cat's thalamus.

Peripheral territory and neuropeptides of the trigeminal ganglion neurons centrally projecting through the oculomotor nerve demonstrated by fluorescent retrograde double-labeling combined with immunocytochemistry.

The peripheral territories of sheep trigeminal neurons which send their central process to the brainstem through the oculomotor nerve were investigated by the use of fluorescent tracers in double-labeling experiments. For this purpose Diamidino yellow (DY) injection into the oculomotor nerve was combined with Fast blue (FB) injection either into the extraocular muscles (EOMs), or the cornea, or the superior eyelid. Double-labeled DY + FB cells were found in the ophthalmic region of the trigeminal ganglion in addition to single-labeled DY or FB cells. The DY and DY + FB-labeled trigeminal cells were analysed immunocytochemically for their content of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, and cholecystokinin-8 (CCK-8)-like. All single-labeled DY cells showed SP-, CGRP- or CCK-8-like immunoreactivity. Double-labeled DY + FB neurons innervating the EOMs were immunoreactive for each of the three peptides, whereas double-labeled neurons supplying the cornea were only CGRP-like positive. The findings suggest that, in the sheep, trigeminal neurons which send their process centrally through the oculomotor nerve supply the EOMs, the cornea, and the superior eyelid and contain neuropeptides which are usually associated with pain sensation.