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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.

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.

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.