History of the Development of Knowledge about the Neuroendocrine Control of Ovulation-Recent Knowledge on the Molecular Background.

The physiology of reproduction has been of interest to researchers for centuries. The purpose of this work is to review the development of our knowledge on the neuroendocrine background of the regulation of ovulation. We first describe the development of the pituitary gland, the structure of the median eminence (ME), the connection between the hypothalamus and the pituitary gland, the ovarian and pituitary hormones involved in ovulation, and the pituitary cell composition. We recall the pioneer physiological and morphological investigations that drove development forward. The description of the supraoptic-paraventricular magnocellular and tuberoinfundibular parvocellular systems and recognizing the role of the hypophysiotropic area were major milestones in understanding the anatomical and physiological basis of reproduction. The discovery of releasing and inhibiting hormones, the significance of pulse and surge generators, the pulsatile secretion of the gonadotropin-releasing hormone (GnRH), and the subsequent pulsatility of luteinizing (LH) and follicle-stimulating hormones (FSH) in the human reproductive physiology were truly transformative. The roles of three critical neuropeptides, kisspeptin (KP), neurokinin B (NKB), and dynorphin (Dy), were also identified. This review also touches on the endocrine background of human infertility and assisted fertilization.

Fluorescent Molecules That Help Reveal Previously Unidentified Neural Connections in Adult, Neonatal and Peripubertal Mammals.

One hundred and twenty-five years ago there was a lively discussion between Hungarian and Spanish neuroscientists on the nature of neural connections. The question was whether the neurofibrils run from one neuron to the next and connect neurons as a continuous network or the fibrils form an internal skeleton in the neurons and do not leave the cell; however, there is close contact between the neurons. About 50 years later, the invention of the electron microscope solved the problem. Close contacts between individual neurons were identified and named as synapses. In the following years, the need arose to explore distant connections between neuronal structures. Tracing techniques entered neuroscience. There are three major groups of tracers: (A) non-transsynaptic tracers used to find direct connections between two neuronal structures; (B) tracers passing gap junctions; (C) transsynaptic tracers passing synapses that are suitable to explore multineuronal circuits. According to the direction of the transport mechanism, the tracer may be ante- or retrograde. In this review, we focus on the ever-increasing number of fluorescent tracers that we have also used in our studies. The advantage of the use of these molecules is that the fluorescence of the tracer can be seen in histological sections without any other processes. Genes encoding fluorescent molecules can be inserted in various neuropeptide or neurotransmitter expressing transcriptomes. This makes it possible to study the anatomy, development or functional relations of these neuronal networks in transgenic animals.

[Clinical importance of adenylate cyclase-activating polypeptide (PACAP)]. / Az adenilát-cikláz-aktiváló polipeptid (PACAP) klinikai jelentosége.

The pituitary adenylate cyclase-activating polypeptide (PACAP) was isolated and characterized from sheep hypothalami. Its amino acid sequence, gene, receptors and receptor genes and its distribution in the mammalian organism were soon described. PACAP is a member of the secretin peptide family. Its closest relative is the vasoactive intestinal polypeptide (VIP). Its widespread occurrence suggests that it plays a significant role in physiological processes. With the aim of animal models, the role of PACAP was intensively investigated worldwide in a possible treatment of various diseases. The first part of this work contains the most important experimental data regarding the structure, genes and occurrence of the peptide and its receptors in mammalian body. In the second part, we overviewed the ever-increasing data on human material according to organ systems. The review contains the data where there is a chance to use PACAP for therapeutic purposes in the clinical practice. Determining the concentration of PACAP in the blood would help in establishing a clinical and differential diagnosis. In the future, there may be a possibility for non-invasive therapy of tumors expressing PACAP receptors. PACAP regulates the pituitary functions, stimulates vasopressin release, adrenalin secretion, insulin secretion. It is smooth muscle relaxant, immunosuppressant. PACAP is a neurotransmitter, it is neuroprotective in various diseases of the nervous system and cytoprotective in peripheral organs. PACAP inhibits apoptosis and the formation of pro-inflammatory factors and stimulates the anti-inflammatory factors and development of tumor cells. PACAP participates in regulating the daily rhythm of physiological functions. Orv Hetil. 2023; 164(33): 1300-1310.

Sexual dimorphism in the effect of concomitant progesterone administration on changes caused by long-term estrogen treatment in pituitary hormone immunoreactivities of rats.

BACKGROUND: Since in clinical practice long-term estrogen (E) treatment is frequently applied, our aim was to study the effect of concomitant progesterone (P) administration on changes caused by long-term estrogen treatment in the secretion of LH, FSH, PRL and GH. MATERIAL/METHODS: Diethylstilbestrol (DES), P or both in silastic capsules were implanted under the skin of prepubertal Sprague-Dawley male and female rats. Animals survived for two or five months. We have also studied whether the changed hormone secretion caused by DES can return to normal level 1 or 2 months after removing DES capsule. RESULTS: 1.) The males more rapidly responded than females with decreasing basal LH release upon treatments. The basal FSH release was decreased only in males. The effect of DES persisted in males; however, in females basal LH and FSH levels were upregulated after removal of DES capsule. 2.) The basal GH levels were low in each group. The body weight and length were depressed by DES in both genders and P little blunted this effect. The body weight and length in males remained low after removal of DES capsule, in females it was nearly similar to intact rats. 3.) There was no sexual dimorphism in the effect of steroids on PRL secretion. In both genders DES extremely enhanced the PRL levels, P prevented the effect of DES. PRL levels returned to intact value after removal of DES influence. 4.) Removal of DES capsule reversed the changes in the immunohistochemical appearance of hormone immunoreactivities. CONCLUSIONS: There was sexual dimorphism in the change of basal gonadotropic hormone and GH secretion but not of PRL upon DES and DES+P treatments. P was basically protective and this role may be mediated by P receptors locally in the pituitary gland.

Chemical characterization of pineal neurons in perinatal rats.

Ample evidence indicates that in several mammalian species the pineal body contains neurons. In adult white albino rats neurons are not present in the pineal body; however, in perinatal rats many neurons were described. It was demonstrated that in adult mammalian species the pineal neurons contained some neuropeptides and neurotransmitters such as leu-enkephalin, met-enkephalin, substance-P, somatostatin and γ-aminobutiric acid. Oxytocin, vasopressin mRNAs and peptides were also demonstrated. No data are available on the chemical nature of the neurons in perinatal rats. In the present experiment we used immunohistochemistry to clarify this issue. After paraformaldehyde fixation frozen sections were prepared and stained for immunoreactivities of several neuropeptides and neurotransmitters. Dopamine ß-hydroxylase, neuropeptide-Y, vesicular acetylcholine transporter, vesicular glutamate transporter and calcitonin gene-related peptide antibodies were able to stain fibers. According to previous data these fibers may be sympathetic, parasympathetic or sensory. Vesicular glutamate transporter antibody may stain pinealocytes as well. Some cells were immunoreactive for substance-P, oxytocin, vasopressin, leu-enkefalin and glutamic acid decarboxylase. These immnoreactivities showed colocalization with neuron-specific nuclear protein immunoreactivity indicating that these cells were neurons. Calbindin was observed in oval and elongated cells resembling pinealocytes. Based on the results obtained in adult mammals, the pineal neurons may be analogue to retinal ganglion cells, or they may function as interneurons in the retino-pinealo-retinal neuronal circuit or peptidergic neurons may influence pinealocytes in a paracrine manner.

Pinealocytes can not transport neurotropic viruses. Pinealo-to-retinal connection in prepubertal rats originates from pineal neurons: Light and electron microscopic immunohistochemical studies.

It is well established that the adult mammalian pineal body (PB), with the exception of rodents, contains nerve cell bodies. Based on our previous results we have proposed that there is a pinealo-to-retinal neuronal connection in adult hamsters and in prebubertal rats. By the time the animals reached puberty, labeled cells in the PB were not observed in rats. In the present experiment, we provide light and electron microscopic immunohistochemical evidence that the labeled cells in the PB of prepubertal rats are neurons. Pinealocytes cannot transport neurotropic viruses. Virus labeled cells do not show S-antigen immunoreactivity typical for pinealocytes of six-day-old rats. Electron microscopic investigation confirmed the neuronal nature of virus labeled cells. These neurons, similarly to that of hamsters, also establish pinealo-to-retinal connections in prepubertal rats.

Current State of Understanding of the Role of PACAP in the Hypothalamo-Hypophyseal Gonadotropin Functions of Mammals.

PACAP was discovered 30 years ago in Dr. Akira Arimura's laboratory. In the past three decades since then, it has become evident that this peptide plays numerous crucial roles in mammalian organisms. The most important functions of PACAP are the following: 1. neurotransmitter, 2. neuromodulator, 3. hypophysiotropic hormone, 4. neuroprotector. This paper reviews the accumulated data regarding the distribution of PACAP and its receptors in the mammalian hypothalamus and pituitary gland, the role of PACAP in the gonadotropin hormone secretion of females and males. The review also summarizes the interaction between PACAP, GnRH, and sex steroids as well as hypothalamic peptides including kisspeptin. The possible role of PACAP in reproductive functions through the biological clock is also discussed. Finally, the significance of PACAP in the hypothalamo-hypophysial system is considered and the facts missing, that would help better understand the function of PACAP in this system, are also highlighted.

Intranasal application of PACAP and ß-cyclodextrin before the "critical period of proestrous stage" can block ovulation.

INTRODUCTION: It was previously shown that intracerebroventricular administration of pituitary adenylate cyclase-activating polypeptide (PACAP) prior to GnRH mobilization in proestrus prevents ovulation in rats. In this study, we examined whether PACAP given intranasally could influence luteinizing hormone (LH) and prolactin (PRL) surges and ovulation. METHODS: On the day of proestrus PACAP, p-cyclodextrin (modifier of blood-brain barrier) or PACAP + p-cyclodextrin was applied intranasally between 12:30 and 13:00. Blood samples were taken at 16:00, 18:00, and 20:00 for measuring plasma hormone levels. In the next morning, the expelled ova were counted. p-Cyclodextrin was also administered to male and diestrous female rats between 12:30 and 13:00 and blood was taken at 18:00. RESULTS: PACAP prevented LH and PRL surges and ovulation in about half of the rats, p-cyclodextrin alone more effectively prevented ovulation. When PACAP and p-cyclodextrin were administered together, more rats ovulated like when PACAP given alone. p-Cyclodextrin did not influence LH and PRL levels in diestrous females; however, in males, it significantly enhanced PRL level. DISCUSSION: Not only the intracerebroventricular, but the intranasal application of PACAP prevented ovulation. p-Cyclodextrin alone is more effective than PACAP and enhances PRL levels in male rats. PACAP and p-cyclodextrin given together weaken each other's effect. p-Cyclodextrin, as excipient of various drugs, has to be used carefully in human medications.

PACAP is transiently expressed in anterior pituitary gland of rats: in situ hybridization and cell immunoblot assay studies.

In this work the expression of PACAP (pituitary adenylate cyclase activating polypeptide) in rat anterior pituitary was demonstrated for the first time using in situ hybridization. The number of cells showing PACAP signal in intact male rats was negligible similarly to that of diestrous rats. In proestrous rats sacrificed at 10h there was a moderate increase in the expression and after a decrease at 16 h and 18 h, there was a transient peak at 20 h and then the number of labeled cells was declined again (22 h). In the cell immunoblot assay study it was observed that the number of PACAP blot forming (PACAP releasing) cells in an anterior pituitary cell culture changed according to a similar pattern as the number of PACAP expressing cells. The number of blots was also the highest when the animals were sacrificed in the evening of proestrus at 20h. The results obtained by in situ hybridization and cell immunoblot assay well correlate with each other. The above-mentioned results support our hypothesis that the enhanced expression and secretion of PACAP in the pituitary gland may be involved in ceasing the LH surge.

Ontogenesis of the pinealo-retinal neuronal connection in albino rats.

It was accepted for a long time that in mammals there is only retinofugal neuronal connection between the eye and the pineal body (PB). In our previous paper we described that nerve cells were present in hamster PB and these neurons could establish a reverse connection with the retina through a transsynaptic pathway. In adult albino rats neuronal perikarya were not found. In this present experiment it was examined whether the lack of these nerve cells in the PB of adult rats is the result of an apoptotic phenomenon or the lack of migration during the fetal period. Green fluorescence protein expressing pseudorabies virus, spreading only in retrograde direction, was injected into the vitreous body of rats at various postnatal ages. Virus labeled cell bodies were not observed in the PB of adult rats; however, labeling with gradually decreasing number of cells was present in animals aged 3-6, 13-14, 20, 35 and 41 postnatal days. Injection of virus, spreading in anterograde direction (expressing red fluorescence protein), into the PB of young prepubertal animals resulted in labeling in the retina. This observation indicates that the pinealo-retinal connection in prepubertal period is active. Immunostaining revealed that some of the labeled neuronal perikarya showed activated caspase-3 (an apoptotic marker) immunoreactivity. Our results clearly show that the neurons migrate to the PB and later, during the prepubertal period, they disappear. Caspase-3 immnoreactivity indicates that these cells die off by apoptosis.

Cushing's syndrome in a child with pancreatic acinar cell carcinoma.

A case of pancreatic acinar cell tumor (ACC) is presented in a 10-year-old boy. The tumor manifested clinically with Cushing's syndrome, high serum adrenocorticotropic hormone (ACTH) and cortisol concentrations. In addition, excessive serum levels of alpha-fetoprotein (AFP) were detected. Surgical resection was not possible due to retroperitoneal invasion. Biopsy of the mass showed a solid, poorly differentiated ACC of the pancreas. Periodic acid Schiff positive cytoplasmic granules, trypsinogen, keratins, alpha-1-antitrypsin, and AFP were identified in the tumor cells. Electron microscopy demonstrated zymogen granules as well as isolated dense core granules. Using immunochemiluminometric assay, a high quantity of ACTH was found in the fresh frozen tumor extract. ACTH, chromogranin A, and corticotropin-releasing factor were identified only in a few cells by immunohistochemistry. Combined radiochemotherapy was temporarily effective in reducing the tumor mass and serum AFP. Serum ACTH and cortisol levels dropped progressively and definitively to normal values after chemotherapy, and the Cushing's syndrome subsided. Two years later, the patient died with metastatic disease. The presented case of ACC is interesting due to high serum AFP values and ectopic ACTH secretion resulting in Cushing's syndrome.

Is a neuronal chain between the pineal body and the retina in rats and hamsters? Transneural tracing studies.

Neuronal chains between the retina and the pineal body were investigated. Transneuronal tracers, retrograde spreading pseudorabies virus (labeled with green fluorescent protein, memGreen-RV) and virus spreading in both ante- and retrograde directions (labeled with red fluorescent protein, Ka-VHS-mCherry-A-RV) were injected into the right eye of vitreous body of intact or bilaterally sympathectomized Wistar male rats. Intact golden hamsters also received memGreen-RV into the eye and Ka-VHS-mCherry-A-RV into the pineal body. Four-five days later the animals were sacrificed. Frozen sections were prepared from the removed structures. In intact rats memGreen-RV resulted in green fluorescent labeling in the trigeminal and the superior cervical ganglia, the lateral horn of the spinal cord, the paraventricular and the suprachiasmatic nuclei, the perifornical region, the ventrolateral medulla, the locus ceruleus, and the raphe nuclei. In sympathectomized rats the labeling was missing from the brainstem but further existed in the hypothalamus. This observation indicates that the hypothalamic labeling is not mediated by the sympathetic system. One labeled neuron in the pineal body was only observed in 2/13 rats. It was independent from the sympathectomy. When the animals received Ka-VHS-mCherry-A-RV the distribution of the labeling was very similar to that of the intact group receiving retrograde virus. In golden hamsters the memGreen-RV labeled structures were seen in similar places as in rats, but virus labeled nerve cell bodies were always seen in the pineal body. Injection of Ka-VHS-mCherry-A-RV into the pineal body of hamsters resulted in labeling of the retina at both sides. It was concluded that the retinopetal neuronal chain in golden hamsters is always present but in rats it is stochastic.

The role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels.

The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its mRNAin the three levels of the hypothalamo-hypophyseal-ovarian axis was previously demonstrated using immunohistochemistry, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). In the hypothalamus, PACAP is present in neuroendocrine effector cells and in the median eminence. In the anterior pituitary and ovary, PACAP is transiently present during the proestrous stage of the estrous cycle. In the pituitary, PACAP was observed in gonadotropes. In the ovary, PACAP was demonstrated in the granulosa cells of the preovulatory ovarian follicles. The effect of PACAP on luteinizing hormone (LH) secretion was demonstrated in in vivo and in vitro models. In our work we have studied the role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels. At the hypothalamic level, PACAP, administered intracerebroventricularly to female rats before the critical period of the proestrus stage, can inhibit LH release and ovulation. Its inhibiting effect is mediated through corticotropin-releasing factor (CRF) and endogenous opioids. PACAP administered to neonatal female rats delayed the onset of puberty by influencing the luteinizing hormone-releasing hormone (LHRH) neuronal system. In the pituitary gland, the release of PACAP depended on the stage of the estrous cycle and on the time of day the animals were sacrificed. On the day of proestrus, the number of PACAP-releasing cells showed a diurnal change with two peaks (in the morning and in the evening). The peak was much higher in the evening at the end of the LH surge than in the morning.

Secretin and autism: a basic morphological study about the distribution of secretin in the nervous system.

For the first time, the relationship between secretin and autism has been demonstrated by one of us. Intravenous administration of secretin in autistic children caused a fivefold higher pancreaticobiliary fluid secretion than in healthy ones and, at least in some of the patients, better mental functions were reported after the secretin test. Because the precise localization of secretin in the brain is still not completely known, the abovementioned observation led us to map secretin immunoreactivity in the nervous system of several mammalian species. In the present work, the distribution of secretin immunoreactivity in cat and human nervous systems was compared with that of rats using an immunohistochemical approach. Secretin immunoreactivity was observed in the following brain structures of both humans and in colchicine-treated rats: (1) Purkinje cells in the cerebellar cortex; (2) central cerebellar nuclei; (3) pyramidal cells in the motor cortex; and (4) primary sensory neurons. Additionally, secretin immnoreactive cells were observed in the human hippocampus and amygdala and in third-order sensory neurons of the rat auditory system. In cats, secretin was only observed in the spinal ganglia. Our findings support the view that secretin is not only a gastrointestinal peptide but that it is also a neuropeptide. Its presence or the lack of its presence may have a role in the development of behavioral disorders.

Distribution of PACAP and its mRNA in several nonneural tissues of rats demonstrated by sandwich enzyme immunoassay and RT-PCR technique.

The presence of PACAP in various organs was previously demonstrated using immunohistochemistry and radioimmunoassay. The aim of our work was to get information whether the presence of immunoreactive PACAP in various organs, mainly in the gastric mucosa, also indicates the place of its synthesis. The immunoreactive PACAP and its mRNA were measured in parallel assays using sandwich enzyme immunoassay (S-EIA) and RT-PCR technique. PACAP and its mRNA were demonstrated in the pancreas, testes, adrenal glands, ovaries, and in the oxyntic mucosa of the stomach. These results support our previous observation that PACAP is present not only in the nervous system and endocrine glands, but might be synthetized in the oxyntic mucosa of the stomach as well.

Advent and recent advances in research on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of gonadotropic hormone secretion of female rats.

PACAP (ADCYAP1) was isolated from ovine hypothalami. PACAP activates three distinct receptor types: G-protein coupled PAC1, VPAC1, and VPAC2 with seven transmembrane domains. Eight splice variants of PAC1 receptor are described. A part of the hypothalamic PACAP is released into the hypophyseal portal circulation. Both hypothalamic and pituitary PACAP are involved in the dynamic control of gonadotropic hormone secretion. In female rats, PACAP in the paraventricular nucleus is upregulated in the morning and pituitary PACAP is upregulated in the late evening of the proestrus stage of the reproductive cycle. PACAP mRNA peak in the hypothalamic PVN precedes the LHRH release into the portal circulation. It is supposed that PACAP peak is evoked by the elevated estrogen on proestrous morning. At the beginning of the so-called critical period of the same day, PACAP level starts to decline allowing LHRH release into the portal circulation, resulting in the LH surge that evokes ovulation. Just before the critical period, icv-administered exogenous PACAP blocks the LH surge and ovulation. The blocking effect of PACAP is mediated through CRF and endogenous opioids. The effect of the pituitary-born PACAP depends on the intracellular cross-talk between PACAP and LHRH.

Distribution of secretin receptors in the rat central nervous system: an in situ hybridization study.

Secretin shows a wide distribution in the brain. Functional significance of central secretin is stressed since it has been associated with autism and schizophrenia. The presence of the secretin receptor was previously demonstrated in the brain by different methods. Neurons in the cerebellum, hypothalamic paraventricular and supraoptic nuclei, and in the vascular organ of lamina terminalis were shown to express secretin receptor mRNA by using in situ hybridization with digoxigenin-labeled probe. In this work, we used a very sensitive radioactive in situ hybridization technique and systematically mapped the expression of secretin receptor mRNA in the brain. The densest labeling was observed in the nucleus of solitary tract and in the laterodorsal thalamic nucleus, where decreasing number of receptors was seen in the vascular organ of lamina terminalis, and the lateral habenular complex, and then in the supraoptic nucleus. Only a few scattered labeled cells were observed in the median frontal gyrus, entorhinal cortex, hypothalamic paraventricular nucleus, perifornical region, lateral hypothalamic area, head of the caudate nucleus, spinal trigeminal nucleus, and cerebellum. Secretin receptor mRNA showed a far wider distribution than was known before, suggesting a more significant functional relevance than thought earlier.

Characterization of the autonomic innervation of mammary gland in lactating rats studied by retrograde transynaptic virus labeling and immunohistochemistry.

The aim of experiments was to characterize the neurons of the autonomic chain that innervates the nipple and the mammary gland of lactating rats using retrograde transynaptic virus labeling and neurotransmitter and neuropeptide immunohistochemistry. Two days after injection of green fluorescence protein labeled virus in two nipples and underlying mammary glands, labeling was observed in the ipsilateral paravertebral sympathetic trunk and the lateral horn. Three days after inoculation the labeling appeared in the brain stem and the hypothalamic paraventricular nucleus. Above the spinal cord the labeling was bilateral. A subpopulation of virus labeled cells in the paraventricular nuclei synthesized oxytocin. Labeled neurons in the lateral horn showed cholinergic immunoreactivity. These cholinergic neurons innervated the paravertebral ganglia where the virus labeled neurons were partially noradrenergic. The noradrenergic fibers in the mammary gland innervate the smooth muscle wall of vessels, but not the mammary gland in rats. The neurons in the lateral horn receive afferents from the brain stem, and paraventricular nucleus and these afferents are noradrenergic and oxytocinergic. New findings in our work: Some oxytocinergic fibers may descend to the neurons of the lateral horn which innervate noradrenergic neurons in the paravertebral sympathetic trunk, and in turn these noradrenergic neurons reach the vessels of the mammary gland.

Intranasal application of secretin, similarly to intracerebroventricular administration, influences the motor behavior of mice probably through specific receptors.

Secretin and its receptors show wide distribution in the central nervous system. It was demonstrated previously that intravenous (i.v.) and intracerebroventricular (i.c.v.) application of secretin influenced the behavior of rat, mouse, and human. In our previous experiment, we used a special animal model, Japanese waltzing mice (JWM). These animals run around without stopping (the ambulation distance is very limited) and they do not bother with their environment. The i.c.v. secretin attenuated this hyperactive repetitive movement. In the present work, the effect of i.c.v. and intranasal (i.n.) application of secretin was compared. We have also looked for the presence of secretin receptors in the brain structures related to motor functions. Two micrograms of i.c.v. secretin improved the horizontal movement of JWM, enhancing the ambulation distance. It was nearly threefold higher in treated than in control animals. The i.n. application of secretin to the left nostril once or twice a day or once for 3 days more effectively enhanced the ambulation distance than i.c.v. administration. When secretin was given twice a day for 3 days it had no effect. Secretin did not improve the explorative behavior (the rearing), of JWM. With the use of in situ hybridization, we have found very dense secretin receptor labeling in the cerebellum. In the primary motor cortex and in the striatum, only a few labeled cells were seen. It was supposed that secretin exerted its effect through specific receptors, mainly present in the cerebellum.

Secretin mRNA in the subdivision of primary sensory neurons in the trigeminal ganglion of rats.

The primary sensory neurons use glutamate as a major neurotransmitter. Several neuropeptides are also found in these neurons. In our laboratory we demonstrated secretin-like immunoreactivity in primary sensory neurons of several species including human, rat and cat. In the present experiment utilizing in situ hybridization, we have demonstrated for the first time that secretin is not only immunostained but is also expressed in the primary sensory neurons of the trigeminal ganglion of male rats. In intact rats, secretin mRNA was not observed; we had to use intracerebroventricular colchicine administration to induce the expression of secretin. Secretin was expressed in about 5% of the cells in all the three subdivisions of the trigeminal ganglion. The secretin-synthetizing cells were large and medium sized, and their mean diameter was about 50 µm. When we compared the percentage and the size of secretin to that of calcitonin gene-related peptide (CGRP), substance-P (SP) and vasoactive intestinal polypeptide (VIP) cells, it was found that CGRP, SP and VIP are present in about 15-20% of the cells and their mean diameter is about 20-25 µm. The morphometric data indicate that secretin is present in a subdivision of neurons that is different from the subdivision of the CGRP, SP and VIP cells. It is suggested that secretin may modulate the function of the primary neurotransmitter.