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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.

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.

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.

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 β-hydroxylase (DBH), neuropeptide-Y (NPY), vesicular acetylcholine transporter (VAChT), vesicular glutamate transporter (VGluT) and calcitonin gene-related peptide (CGRP) antibodies were able to stain fibers. According to previous data these fibers may be sympathetic (DBH, NPY), parasympathetic (VAChT) or sensory (CGRP). VGluT antibody may stain pinealocytes as well. Some cells were immunoreactive for substance-P, oxytocin, vasopressin, leu-enkefalin and glutamic acid decarboxylase (GAD). These immnoreactivities showed colocalization with neuron-specific nuclear protein immunoreactivity indicating that these cells are 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 the peptidergic neurons may influence the pinealocytes in a paracrine manner.

The major goals of this present study were 1) to further clarify which parasympathetic ganglion sends postganglionic fibers to the lower gingiva and lip that may be involved in the inflammatory processes besides the local factors; 2) to separately examine the central pathways regulating sympathetic and parasympathetic innervation; and 3) to examine the distribution of central premotor neurons on both sides. A retrogradely transported green fluorescent protein conjugated pseudorabies virus was injected into the lower gingiva and lip of intact and sympathectomized adult female rats. Some animals received virus in the adrenal medulla which receive only preganglionic sympathetic fibers to separately clarify the sympathetic nature of premotor neurons. After 72–120 h of survival and perfusion, the corresponding thoracic part of the spinal cord, brainstem, hypothalamus, cervical, otic, submandibular and trigeminal ganglia were harvested. Frozen sections were investigated under a confocal microscope. Green fluorescence indicated the presence of the virus. The postganglionic sympathetic neurons related to both organs are located in the three cervical ganglia, the preganglionic neurons in the lateral horn of the spinal cord on ipsilateral side; premotor neurons were found in the ventrolateral medulla, locus ceruleus, gigantocellular and paraventricular nucleus and perifornical region in nearly the same number on both sides. The parasympathetic postganglionic neurons related to the gingiva are present in the otic and related to the lip are present in the otic and submandibular ganglia and the preganglionic neurons are in the salivatory nuclei. Third order neurons were found in the gigantocellular reticular and hypothalamic paraventricular nuclei and perifornical area.

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.

By means of double-label immunocytochemistry, authors studied the presence of estrogen receptor α (ER-α) protein in vesicular glutamate transporter 2 (VGluT2) protein-immunoreactive neurons in the female rat hypothalamus and amygdala. They examined colocalization of the 2 immunoreactive proteins in structures in which they found a significant overlap in the localization of the distribution of ER-α- and VGluT2-immunopositive nerve cells, namely in the medial preoptic area, the ventral subdivision of the ventromedial hypothalamic nucleus, and the medial amygdaloid nucleus. In the medial preoptic area, only 2.74 % of ER-α-immunoreactive neurons were VGluT2 positive, and conversely, 5 % of VGluT2-immunoreactive neurons contained ER-α immunofluorescent labeling. Highest degree of colocalization was detected in the ventral subdivision of the ventromedial hypothalamic nucleus, where 22.81 % of the ER-α-immunopositive neurons were VGluT2 immunoreactive and 37.14 % of the VGluT2-immunolabeled neurons contained ER-α-positive nucleus. In the medial amygdaloid nucleus, 15.38 % of the ER-α and 18.1 % of the VGluT2-immunoreactive neurons were double labeled. The colocalizations suggest that glutamatergic (VGluT2 protein immunoreactive) neurons are involved in the mediation of the action of estrogen on the rat brain.

The hypothalamic suprachiasmatic nucleus (SCN), which plays a pivotal role in the control of circadian rhythms, consists of several neuronal subpopulations characterized by different neuroactive substances. This prominent cell group has a fairly rich glutamatergic innervation, but the cell types that are targeted by this innervation are unknown. Therefore, the purpose of the present study was to examine the relationship between the afferent glutamatergic axon terminals and the vasoactive intestinal polypeptide (VIP)-, arginine-vasopressin (AVP)- and gamma-aminobutyric acid (GABA)-positive neurons of the SCN. Glutamatergic elements were revealed via immunocytochemical double-labelling for vesicular glutamate transporter type 1 (VGluT1) and type 2 (VGluT2), and brain sections were imaged via confocal laser-scanning microscopy and electron microscopy. Numerous VGluT2-immunoreactive axons were observed to be in synaptic contact with VIP- and GABA-positive neurons, and only a few synapses were detected between VGluT2 boutons and AVP neurons. VGluT1 axon terminals exhibiting very moderate distribution in this cell group were observed to be in synaptic contact with chemically unidentified neurons. The findings provide the first morphological data on the termination of presumed glutamatergic fibres on chemically identified neurons of the rat SCN, and indicate that all three prominent cell types of the cell group receive glutamatergic afferents.

The hypothalamic suprachiasmatic nucleus is the key structure of the control of circadian rhythms and has a rich glutamatergic innervation. Besides the presence of glutamatergic afferents, several findings also suggest the existence of glutamatergic efferents from the suprachiasmatic nucleus to its target neurons in various prominent hypothalamic cell groups. However, there is no direct neuromorphological evidence for the presence of glutamatergic neurons in the suprachiasmatic nucleus. Therefore, the purpose of the present investigations was to try to clarify this question. Immunocytochemistry was used at the light and electron microscopy level to identify vesicular glutamate transporter type 2 (VGluT2) immunopositive (presumed glutamatergic) neurons in the rat suprachiasmatic nucleus. In addition VGluT2 mRNA expression in neurons of the nucleus was also addressed with radioisotopic in situ hybridization. Both at the light and electron microscopy level we detected VGluT2 positive neurons, which did not contain GABA, vasoactive intestinal polypeptide or vasopressin. Further, we demonstrated the expression of VGluT2 mRNA in a few cells within the suprachiasmatic nucleus; these glutamatergic cells were distinct from somatostatin mRNA expressing neurons. As VGluT2 is a selective marker of glutamatergic neuronal elements, the present observations provide direct neuromorphological evidence for the presence of glutamatergic neurons in the cell group.

In our present work utilizing the retrograde or anterograde transport of tracers (biotinylated dextran amine and Fluorogold, respectively) we have provided direct evidence for the cells of origin of the limboretinal pathway in rats and their termination in the retina using light microscopic approach. Administration of biotinylated dextran amine into the vitreous body resulted in nerve cell body labeling in several structures: the supraoptic and paraventricular nuclei, the hippocampus (CA1, CA3), the dentate gyrus, the indusium griseum, the olfactory tubercle, and the medial habenula, all of them belong to the limbic system. We estimated that the total number of retrogradely labeled cells is 1495±516. We have seen fiber labeling in the retinorecipient suprachiasmatic nucleus and in the primary visual center, the lateral geniculate body, but labeled nerve cell bodies in these structures were never seen. Iontophoretic application of Fluorogold into the hippocampal formation, where the major part of the biotinylated dextran amine-labeled cell bodies was observed, resulted in labeled fibers in the optic nerve and in the retina indicating that the retrogradely labeled cells in the hippocampus and the dentate gyrus among others are the cells of origin of the centrifugal visual fibers. Sections showing biotinylated dextran amine labeling were stained for vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity using immunohistochemistry. Some biotinylated dextran amine-labeled cells also showed vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity. We conclude that the limboretinal pathway exists and that the cells of origin are partially vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactive.

It is well established that the supramammillary nucleus plays a critical role in hippocampal theta rhythm generation/regulation by its direct and indirect (via the septal complex) connections to the hippocampus. Previous morphological and electrophysiological studies indicate that both the supramammillo-hippocampal and supramammillo-septal efferents contain excitatory transmitter. To test the validity of this assumption, transmitter specific retrograde tracer experiments were performed. [3H]D-aspartate was injected into different locations of the hippocampus (granular and supragranular layers of the dentate gyrus and CA2 and CA3a areas of the Ammon's horn) and septal complex (medial septum and the area between the medial and lateral septum) that are known targets of the supramammillary projection. Consecutive vibratome sections prepared from the entire length of the posterior hypothalamus, including the supramammillary area, were immunostained for calretinin, tyrosine hydroxylase, or calbindin, and further processed for autoradiography. Radiolabeled, radiolabeled plus calretinin-containing, and calretinin-immunoreactive neurons were plotted at six different oro-caudal levels of the supramammillary area. The results demonstrated that following both hippocampal and septal injection of the tracer, the majority of the retrogradely radiolabeled (glutamatergic/aspartatergic) cells are immunoreactive for calretinin. However, non-radiolabeled calretinin-containing neurons and radiolabeled calretinin-immunonegative cells were also seen, albeit at a much lower density. These observations clearly indicate the presence of glutamatergic/aspartatergic projections to both the hippocampus and septal complex. It may be assumed that this transmitter could play a role in hippocampal theta rhythm generation/regulation.