Recent findings on the organization of central nervous system structures involved in the innervation of endocrine glands and other organs; observations obtained by the transneuronal viral double-labeling technique.

This review summarizes the data obtained with the aid of the recently introduced dual viral tracing technique, which uses isogenic recombinants of pseudorabies virus that express unique reporter gene. This approach made possible to explore simultaneously neural circuits of two organs. The results of these studies indicate: (1) there are neurons innervating exclusively a given organ; (2) left-sided predominance in the supraspinal innervation of the endocrine glands (adrenal, ovary) studied, so far; (3) viral co-infection of neurons, i.e., special neuronal populations coexist in different brain areas that are transsynaptically connected with both paired endocrine and non-endocrine organs, endocrine glands and non-endocrine organs, and organs of bodily systems other than the endocrine one. The number of common neurons seems to be related to the need of coordinating action of different systems. The data on co-infection of neurons suggest that the central nervous system has the capacity to coordinate different organ functions via common brain neurons providing supraspinal innervation of the organs.

Cerebral neurons involved in the innervation of both the adrenal gland and the ovary: a double viral tracing study.

Previous studies using the viral transneuronal tracing technique demonstrated central autonomic circuits involved in the innervation of the adrenal gland and the ovary. Since the pattern of infection of central nervous system structures is similar after virus inoculation of the adrenal gland and the ovary, and, on the other hand, it is well documented that the activity of the hypothalamo-pituitary-adrenal axis exerts an inhibitory effect on the reproductive system, we investigated whether there are neurons that are transneuronally connected both with the adrenal gland and the ovary. The central circuitry involved in the innervation of the left adrenal and the left ovary was studied in individual rats by dual transneuronal tracing using isogenic recombinant strains (BDG and DS-RED) of Bartha strain of pseudorabies virus. Dual-infected neurons were detected in the ventrolateral medulla, nucleus of the solitary tract, caudal raphe nuclei, A5 cell group, and hypothalamic paraventricular nucleus. The results indicate that there are neurons in the central nervous system that contribute to the transneuronal innervation of both the adrenal gland and the ovary. The data suggest a new type of interaction, i.e. interaction at cellular level that might be involved in regulatory processes integrating the functional activity of the two organs.

Location of parotid preganglionic neurons in the inferior salivatory nucleus and their relation to the superior salivatory nucleus of rat.

In major brain maps the location of the salivatory nuclei of the rat is depicted from the level of the root of the facial nerve to the level of the rostral tip of the nucleus of the solitary tract. Most published data deal with the superior salivatory nucleus (SSN). In the present study the topography of the parasympathetic preganglionic neurons of the inferior salivatory nucleus (ISN) that innervate the parotid gland through the otic ganglion was determined by means of a retrograde transneuronal labeling technique. Parasympathetic, sympathetic and sensory neurons were labeled following injection of the virus into the parotid gland. The majority of the ISN neurons were found dorsal to the facial motor nucleus, embedded in the parvocellular reticular formation. In addition to the ISN neurons, virus-labelled cells were present in the intermediolateral (IML) cell column of the thoracic spinal cord, in the brainstem catecholamine groups, and in medullary raphe neurons. The removal of the ipsilateral superior cervical ganglion prior to the virus injection into the parotid gland did not influence the labeling of the ISN neurons but labeled neurons were not observed in the IML and A5 catecholamine cell group. In our previous study we had defined the relationship between the lacrimal and submandibular subdivison of the SSN, while in the present study we defined the relationship between the ISN and the lacrimal subdivision of SSN: the later located ventrolaterally to the caudal portion of the ISN. On the basis of these data a three-dimensional topography is given suggesting the relationship between the ISN and SSN.

Transneuronal retrograde viral labeling in the brain stem and hypothalamus is more intense from the left than from the right adrenal gland.

Previous studies using the viral transneuronal tracing technique demonstrated central autonomic circuits involved in the innervation of the adrenal gland. Since increasing number of data indicate laterality in the neuroendocrine system, we aimed to investigate whether the supraspinal innervation of the adrenal gland exhibits asymmetry or not. The central circuitry involved in the innervation of the left and the right adrenal gland was studied in individual rats by dual transneuronal tracing using isogenic recombinant strains (Ba-DupGreen and Ba-Duplac expressing lacZ) of Bartha strain of pseudorabies virus. Viral infection of brain nuclei (dorsal vagal nucleus, nucleus of the solitary tract, caudal raphe nuclei, A5 cell group, hypothalamic paraventricular nucleus) from the left adrenal was more severe than that from the right organ. Dual-infected neurons were present both in the brain stem and in the hypothalamus. The results indicate a predominance in the supraspinal innervation of the left adrenal gland, and that each adrenal gland is innervated both by side-specific neurons and by neurons that project to both organs.

The supraspinal innervation of the left adrenal is more intense than that of the right one.

BACKGROUND AND PURPOSE: Previous studies using the viral transneuronal tracing technique demonstrated that central autonomic circuits are involved in the innervation of the adrenal gland. Since increasing number of data indicate laterality in the neuroendocrine system, we aimed to investigate whether the supraspinal innervation of the adrenal gland exhibits asymmetry or not. METHODS: The central circuitry involved in the innervation of the left and the right adrenal gland was studied in individual rats by dual transneuronal tracing using isogenic recombinant strains (BDG and BDL) of Bartha strain of pseudorabies virus. RESULTS: Viral infection of brain nuclei (dorsal vagal nucleus, nucleus of the solitary tract, caudal raphe nuclei, A5 cell group, hypothalamic paraventricular nucleus) from the left adrenal was more severe than that from the right organ. Dual-infected neurons from the two adrenals were also detected both in the brain stem and in the hypothalamus. CONCLUSION: The results indicate a predominance in the supraspinal innervation of the left adrenal gland. Data further suggest that each adrenal gland is innervated both by side-specific neurons and by neurons which project to both organs.

Predominance of supraspinal innervation of the left ovary.

In our previous studies using the viral transneuronal tracing technique we demonstrated the spinal and supraspinal components of the ovarian innervation. Since increasing number of data indicate the presence of morphological and functional laterality in the control of gonadal functions, we aimed to investigate whether cerebral structures trans-synaptically involved in the innervation of the ovary exhibit asymmetry or not. In one of the studies the left or the right ovary was injected with the red fluorescent protein expressing pseudorabies virus and the number of infected "red" autofluorescent neurons from the right and the left ovary was compared. In another study in order to have distinct labeling of cell groups connected with the right- and left-sided ovary in the same animal, a dual viral labeling was applied. The left- and right-sided ovary were inoculated with genetically engineered pseudorabies virus expressing a red fluorescent protein or a green fluorescent protein gene. Viral infection of brain nuclei including the dorsal vagal nucleus, caudal raphe nuclei, A5 noradrenergic cell group, hypothalamic paraventricular nucleus, from the left ovary in each case was enhanced when compared with labeling from the right gonad. Data suggest a predominance in the supraspinal innervation of the left ovary.

Serotonin-synthesizing neurons in the rostral medullary raphé/parapyramidal region transneuronally labelled after injection of pseudorabies virus into the rat tail.

Serotonin-synthesizing raphé/parapyramidal neurons (5-HT neurons) may function as sympathetic premotor neurons regulating sympathetic outflow to the cutaneous vascular bed. In the present study a genetically engineered pseudorabies virus (PRV) expressing green fluorescent protein (GFP) was injected into the rat tail. After survival for 3-4 days the medulla oblongata was examined using double-label immunohistochemistry, with an antibody against GFP for the virus and an antibody against phenylalanine hydroxylase 8 (PH8) for 5-HT synthesis. Sections were examined using light microscopy, and conventional and confocal fluorescence microscopy. There were two subpopulations of PRV+ve neurons in the raphé/parapyramidal region: a more dorsally and laterally located subgroup of medium-sized and large neurons, mainly non-serotonergic, and a more ventrally located subgroup of small mainly serotonin-synthesizing neurons, including those just dorsal to the pyramids, those in raphé pallidus, and those in close relationship to the ventral surface in the parapyramidal-subependymal zone.

Occasional transsynaptic viral labeling in the central nervous system from the polycystic ovary induced by estradiol valerate.

Increased density of catecholaminergic nerves in the human polycystic ovary has been observed. The aim of the present study was to investigate the distribution of transsynaptically virus-labeled neurons in the central nervous system from the rat polycystic ovary to see whether is it different or not from that of cycling control rats. To induce a polycystic ovary, a single injection of estradiol valerate was given to adult female rats and 30 days later a neurotropic virus was injected into the right ovary. Rats were sacrificed 72 or 96 hours after viral infection. Weight of the ovaries of the estradiol valerate-treated rats was significantly lower compared to controls, and the histology of the ovaries of the treated rats displayed severely atretic large antral follicles. There was almost no viral labeling in the central nervous system from the ovaries showing precystic morphology, in spite of the fact that such altered organs are rich in nerve fibres. It is assumed that presently unidentified factors in the precystic ovary, presumably related to the link between the immune and the nervous system, might be involved in the infectivity of the virus, and thus be responsible for the lack of viral labeling from such an ovary.

Comparison of transsynaptic viral labeling of central nervous system structures from the uterine horn in virgin, pregnant, and lactating rats.

Using the transneuronal viral tracing method, the central nervous system (CNS) connections of the uterine horn were studied in virgin, pregnant, and in lactating rats. The frequency of viral labeling in the brain and the distribution of virus-infected neurons from the uterine horn were compared among groups. There was a marked difference in the frequency of viral labeling in the brain stem. In virgin rats more than half of the brain stems (5 out of 9) were labeled. In contrast, in pregnant animals viral-labeled neurons were detected in only a few cases (3 out of 16) and almost each brain stem of the lactating group was labeled (12 out of 13). A similar, less marked difference was observed in the hypothalamus. The pattern of distribution of infected neurons was similar in each group. In the brain stem, the nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, gigantocellular and paragigantocellular nucleus, ventrolateral medulla, A5 cell group, and caudal raphe nuclei were the most frequently labeled structures. In the diencephalon, viral-infected neurons were detected primarily in the hypothalamic paraventricular nucleus. The telencephalon was devoid of infected cells. Data suggest that the CNS control of the uterine horn varies depending on reproductive status. The low frequency of brain labeling in pregnant rats may be related to the almost complete lack of sympathetic fibers in the uterus prior to parturition and the very high frequency of labeling in lactating animals to the postpartum hyperinnervation of the uterus.

Intracochlear injection of pseudorabies virus labels descending auditory and monoaminerg projections to olivocochlear cells in guinea pig.

Pseudorabies virus was used to label transneuronally descending auditory projections following intracochlear injections. At different time points after injection, virus-infected cells were detected immunohistochemically in the central nervous system. Initially (25 h), virus was transported retrogradely to olivocochlear cells in the pons. At 32-72 h after injection, labelling occurred in higher order auditory brainstem nuclei as well as in the locus coeruleus and pontine dorsal raphe. At 90-108 h, virus-infected neurons were found bilaterally in the medial geniculate body and in layer V of the auditory cortex. Viral transneuronal labelling in the auditory cortex after intracochlear application confirms the existence of a continuous descending chain of neurons from the auditory cortex to the cochlea, via the medial and lateral olivocochlear systems. The transneuronal labelling of the locus coeruleus and pontine dorsal raphe suggests that noradrenergic and serotonergic inputs may substantially influence the activity of olivocochlear cells, and thus the cochlea.

Identification of neurones of the brain and spinal cord involved in the innervation of the ductus deferens using the viral tracing method.

Using the viral transneuronal tracing technique cell groups of the spinal cord and brain transsynaptically connected with the ductus deferens were identified. Neurotropic (pseudorabies) virus was injected into the muscular coat of the ductus deferens and after survival times of 3, 4 and 5 days the spinal cord and brain were processed immunocytochemically. Virus-labelled neurones could be detected in the preganglionic sympathetic neurones and the dorsal commissural nucleus (upper lumbar segments) and in the sacral parasympathetic nucleus (L6-S1). Virus-infected perikarya were present in several brain stem nuclei including the gigantocellular and paragigantocellular nucleus, the lateral reticular nucleus, the nucleus of the solitary tract, the caudal raphe nuclei, the A1/C1, A2, A5 and A7 noradrenergic cell groups and the locus coeruleus. In the hypothalamus significant numbers of virus-infected neurones could be detected in the paraventricular nucleus. In most cases moderate numbers of virus-labelled cells were present in the lateral hypothalamic area, in the retrochiasmatic area, in the periventricular region and in the median preoptic area. Double-labelling immunofluorescence detection of virus-infected neurones and thyrosine hydroxylase (TH) showed colocalization of virus protein and TH in portion of neurones of the A1/C1, A2, A5 and A7 noradrenergic cell groups, in the locus coeruleus and in the hypothalamic paraventricular nucleus. The present results provide the first morphological data on the multisynaptic circuit of neurones innervating the ductus deferens.

Mechanisms of pain-induced local cerebral blood flow changes in the rat sensory cortex and thalamus.

It is a well-known phenomenon that cerebral blood flow is coupled to neural activation induced by non-noxious somatosensory stimulation. However, basic questions related to pain-induced cerebral blood flow changes remain unanswered. In the present study, the sciatic nerve of anesthetized rats was subjected to electric stimulation with noxious and non-noxious parameters. Changes in local cerebral blood flow and neuronal activity were determined simultaneously in the sensory cortex and in the thalamus by laser-Doppler flowmetry and c-fos immunohistochemistry, respectively. The role of different vasoregulatory mechanisms and the pain-induced increase in mean arterial blood pressure (MABP) were examined with specific blocking agents and by means of rapid intra-arterial transfusion. Noxious stimulation resulted in significant enhancement of neuronal activity both in the thalamus and in the somatosensory cortex indicated by marked c-fos expression in these areas. Cortical and thalamic blood flow (cBF and tBF) increased by 47+/-4 and 44+/-3% during the stimulation while the MABP elevated by 35+/-2%. Similar changes in MABP induced by intra-arterial transfusion had no effect on tBF, while cBF increased only by 18+/-5%. Blockade of ATP sensitive potassium channels (K(+)(ATP)) and sympathetic beta-receptors significantly attenuated the pain-induced blood flow increases in both investigated areas, while inhibition of nitric oxide synthase was effective only in the thalamus. The blockade of the sympathetic alpha-receptors, opiate receptors, and the cyclooxygenase enzyme had no effect on the pain-induced cerebral blood flow elevations. These findings demonstrate that during noxious stimulation, cerebral blood flow is adjusted to the increased neural activity by the interaction of vasoconstrictor autoregulatory and specific vasodilator mechanisms, involving the activation of sympathetic beta-receptors, K(+)(ATP)-channels and the release of nitric oxide.

Construction of recombinant pseudorabies viruses optimized for labeling and neurochemical characterization of neural circuitry.

In this study we have modified the neuroinvasiveness of pseudorabies virus strain Bartha, a commonly utilized trans-synaptic tract-tracer. In addition, we sought to facilitate detection of cellular mRNAs in neurons infected with the virus. In order to modify spreading characteristics, we inserted the lacZ or the GFP (green fluorescent protein) genes into the genomic loci containing the putative latency-associated transcript promoter (P(LAT2)), resulting in the disruption of the promoter function. Following rat kidney injection, mutant viruses labeled central autonomic neurons in a slower and much more restricted manner than the parent Bartha strain. Since both reporter genes were controlled by the human cytomegalovirus immediate early (IE) 1 promoter, they exhibited IE expression kinetics. This property proved to be important for the co-detection of reporter proteins with neuronal mRNAs, readily detected at early but not at late stage of infection, as shown in tyrosine-hydroxylase expressing A5 catecholaminergic neurons and in serotonin transporter expressing raphe magnus neurons.