Zsolt Boldogkoi

Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions

Sympathetic premotor neurons directly control sympathetic preganglionic neurons (SPNs) in the intermediolateral cell column (IML) of the thoracic spinal cord, and many of these premotor neurons are localized in the medulla oblongata. The rostral ventrolateral medulla contains premotor neurons controlling the cardiovascular conditions, whereas rostral medullary raphe regions are a candidate source of sympathetic premotor neurons for thermoregulatory functions. Here, we show that these medullary raphe regions contain putative glutamatergic neurons and that these neurons directly control thermoregulatory SPNs. Neurons expressing vesicular glutamate transporter 3 (VGLUT3) were distributed in the rat medullary raphe regions, including the raphe magnus and rostral raphe pallidus nuclei, and mostly lacked serotonin immunoreactivity. These VGLUT3-positive neurons expressed Fos in response to cold exposure or to central administration of prostaglandin E2, a pyrogenic mediator. Transneuronal retrograde labeling after inoculation of pseudorabies virus into the interscapular brown adipose tissue (BAT) or the tail indicated that those VGLUT3-expressing medullary raphe neurons innervated these thermoregulatory effector organs multisynaptically through SPNs of specific thoracic segments, and microinjection of glutamate into the IML of the BAT-controlling segments produced BAT thermogenesis. An anterograde tracing study further showed a direct projection of those VGLUT3-expressing medullary raphe neurons to the dendrites of SPNs. Furthermore, intra-IML application of glutamate receptor antagonists blocked BAT thermogenesis triggered by disinhibition of the medullary raphe regions. The present results suggest that VGLUT3-expressing neurons in the medullary raphe regions constitute excitatory neurons that could be categorized as a novel group of sympathetic premotor neurons for thermoregulatory functions, including fever.

Local Retinal Circuits of Melanopsin-Containing Ganglion Cells Identified by Transsynaptic Viral Tracing

Intrinsically photosensitive melanopsin-containing retinal ganglion cells (ipRGCs) control important physiological processes, including the circadian rhythm, the pupillary reflex, and the suppression of locomotor behavior (reviewed in [1]). ipRGCs are also activated by classical photoreceptors, the rods and cones, through local retinal circuits [2, 3]. ipRGCs can be transsynaptically labeled through the pupillary-reflex circuit with the derivatives of the Bartha strain of the alphaherpesvirus pseudorabies virus(PRV) [4, 5] that express GFP [6-12]. Bartha-strain derivatives spread only in the retrograde direction [13]. There is evidence that infected cells function normally for a while during GFP expression [7]. Here we combine transsynaptic PRV labeling, two-photon laser microscopy, and electrophysiological techniques to trace the local circuit of different ipRGC subtypes in the mouse retina and record light-evoked activity from the transsynaptically labeled ganglion cells. First, we show that ipRGCs are connected by monostratified amacrine cells that provide strong inhibition from classical-photoreceptor-driven circuits. Second, we show evidence that dopaminergic interplexiform cells are synaptically connected to ipRGCs. The latter finding provides a circuitry link between light-dark adaptation and ipRGC function.

Neuronal Labeling in the Rat Brain and Spinal Cord from the Ovary Using Viral Transneuronal Tracing Technique

In the present investigations the viral transneuronal labeling method, which is able to reveal hierarchial chains of central nervous system (CNS) neurons, was applied to identify sites in the CNS connected with the ovary and presumably involved in the control of ovarian functions. Pseudorabies virus was injected into the ovaries of rats and a few days later (at various times after the injection) the spinal cord and brain were examined for virus-infected neurons from the ovary. The virus-labeled nerve cells were identified by immunocytochemistry using polyclonal antiviral antibody. Virus-labeled neurons were detected both in the spinal cord and the brain. In the spinal cord such elements were observed in the intermediolateral cell column, in the dorsal horn close to the marginal zone and in the central autonomic nucleus. In the medulla oblongata and pons, neurons of several nuclei and cell groups (area postrema, nucleus of the solitary tract, dorsal vagal complex, nucleus ambiguus, paragigantocellular nucleus, parapyramidal nucleus, A1, A5 and A7 cell groups, caudal raphe nuclei, locus ceruleus, subceruleus nucleus, Barrington’s nucleus, Kölliker-Fuse nucleus) were found to be transneuronally labeled. In the mesencephalon, the ventrolateral part of the periaqueductal gray matter contained virus-labeled neurons. In the diencephalon, a very intensive cell body labeling was observed in the hypothalamic paraventricular nucleus and a few virus-infected neurons could be detected in the lateral and dorsal hypothalamus, in the arcuate nucleus, zona incerta, perifornical area and in the anterior hypothalamus. Concerning the telencephalic structures, virus-labeled cells were found in the bed nucleus of the stria terminalis and in the central amygdala nucleus. These findings provide the first neuromorphological evidence for the existence of a multisynaptic neuronal pathway between the ovary and the CNS, and give a detailed account of the structures involved in this pathway.

CENTRAL AUTONOMIC CONTROL OF THE BONE MARROW: MULTISYNAPTIC TRACT TRACING BY RECOMBINANT PSEUDORABIES VIRUS

Bone marrow is the primary place of hematopoiesis, where the development, survival and release of multipotent stem cells, progenitors, precursors and mature cells are under continuous humoral and neural control. Dense network of nerve fibers, containing various neurotransmitters is found in the bone marrow, however, the central neuronal circuit that regulates the activities of the bone marrow through these fibers remained unexplored. Transsynaptically connected neurons were mapped by virus-based transneuronal tracing technique using two isogenic, genetically engineered pseudorabies viruses, Bartha-DupGreen and Ba-DupLac expressing green fluorescent protein and beta-galactosidase, respectively. Bartha-DupGreen was injected into the femoral bone marrow of male rats and the progression of infection was followed 4-7 days post-inoculation. Virus-labeled cells were revealed in ganglia of the paravertebral chain and in the intermediolateral cell column of the lower thoracic spinal cord. Neurons were retrogradely labeled in the C1, A5, A7 catecholaminergic cell groups and several other nuclei of the ventrolateral and ventromedial medulla, the periaqueductal gray matter, the paraventricular and other hypothalamic nuclei, and in the insular and piriform cortex. Nerve transections and double-virus tracing from the bone marrow and the surrounding muscles were used to confirm the specific spreading of the virus. These results provide anatomical evidence for the CNS control of the bone marrow and identify putative brain areas, which are involved in autonomic regulation of the hematopoiesis, the release of progenitor cells, the blood supply and the immune cell function in the bone marrow.

Neurons of the rat preoptic area and the raphe pallidus nucleus innervating the brown adipose tissue express the prostaglandin E receptor subtype EP3

The major effector organ for thermogenesis during inflammation or experimental pyrogen‐induced fever in rodents is the brown adipose tissue (BAT). Prostaglandin E2 (PGE2) microinjection into the medial preoptic area (POA) of rats leads to hyperthermia through an increase in BAT thermogenesis and induces pyrogenic signal transmission towards the raphe pallidus nucleus (RPa), a brainstem nucleus known to contain sympathetic premotor neurons for BAT control. The medial POA has a high expression of prostaglandin E receptor subtype EP3 (EP3R) on POA neurons, suggesting that these EP3R are main central targets of PGE2 to mediate BAT thermogenesis. To reveal central command neurons that contain EP3R and polysynaptically project to the BAT, we combined EP3R immunohistochemistry with the detection of transneuronally labelled neurons that were infected after injection of pseudorabies virus into the BAT. Neurons double‐labelled with EP3R and viral surface antigens were particularly numerous in two brain regions, the medial POA and the RPa. Of all medial POA neurons that became virally infected 71 h after BAT inoculation, about 40% expressed the EP3R. This subpopulation of POA neurons is the origin of a complete neuronal chain that connects potential PGE2‐sensitive POA neurons with the BAT. As for the efferent pathway of pyrogenic signal transmission, we hypothesize that neurons of this subpopulation of EP3R expressing POA neurons convey their pyrogenic signals towards the BAT via the RPa. We additionally observed that two‐thirds of those RPa neurons that polysynaptically project to the interscapular BAT also expressed the EP3R, suggesting that RPa neurons themselves might possess prostaglandin sensitivity that is able to modulate BAT thermogenesis under febrile conditions.

CNS structures presumably involved in vagal control of ovarian function

The contribution of the vagus nerve to viral transneuronal labeling of brain structures from the ovaries demonstrated recently by us was investigated. Unilateral vagotomy was performed prior to ipsilateral intraovarian virus injection. Virus-infected neurons were visualized by immunostaining. In vagotomized rats such neurons were detected only in certain cell groups of the brain (parapyramidal nucleus, A(1), A(5) cell group, caudal raphe nuclei, hypothalamic paraventricular nucleus, lateral hypothalamus). Vagotomy interfered with labeling of several structures that were labeled in controls, including area postrema, nucleus of the solitary tract, dorsal vagal complex, nucleus ambiguus, A(7) cell group, Barringtons nucleus, locus coeruleus, periaqueductal gray, dorsal hypothalamus. Findings provide a morphological basis to study the functional significance of brain structures presumably involved in the control of ovarian function and acting via the vagus or the sympathetic nerves.

Lacrimal preganglionic neurons form a subdivision of the superior salivatory nucleus of rat: transneuronal labelling by pseudorabies virus

Transneuronal viral tracing was applied to localize preganglionic parasympathetic neurons in the brainstem which innervate the extraorbital lacrimal gland in the rat. The Bartha strain of pseudorabies virus was injected into the lacrimal gland, and after different survival times, the superior cervical and Gasserian ganglia, the upper thoracic spinal cords and the brainstems were immunostained by antiviral antiserum. Virus-labelled neurons appeared in the ganglia and in the ventrolateral part of the ipsilateral brainstem at the pontomedullary junction 45 h after inoculation. The virus-labelled brainstem neurons comprised a subgroup of the superior salivatory nucleus (SSN) located between the root fibers of the facial nerve and the nuclei of the superior olive, and were clearly distinguished from the tyrosine hydroxylase (TH)-immunopositive, A5 catecholaminergic neurons by double immunostaining. The number of infected cells in the ipsilateral SSN was increased by 72 h, and labelled neurons appeared in the intermediolateral cell column (IML) of the ipsilateral thoracic spinal cord. In rats with cervical ganglionectomy prior to the virus injection in the lacrimal gland, virus-infected cells appeared in the SSN, but not in the thoracic spinal cord, indicating that preganglionic SSN cells were infected via parasympathetic axons of the facial nerve. A double-virus tracer labelling technique was applied to determine the topographical relationship between the preganglionic parasympathetic neurons of the lacrimal gland and those of the submandibular gland within the SSN. Simultaneous injection of Bartha strain of pseudorabies virus into the submandibular gland, and a lacZ gene-containing Bartha-derived virus strain into the lacrimal gland (and vice versa) demarcated a ventral lacrimal and a dorsal submandibular subgroup in the SSN.

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.

Identification of CNS neurons involved in the innervation of the epididymis: A viral transneuronal tracing study

Cell groups of the spinal cord and the brain transsynaptically connected with the epididymis (caput, cauda) were identified by means of the viral transneuronal tracing technique. Pseudorabies virus was injected into the caput or the cauda epididymidis, and after survival times 4 and 5 days, the spinal cord and brain were processed immunocytochemically. Virus-labeled neurons could be detected in the preganglionic sympathetic neurons (lower thoracic and upper lumbar segments) and following virus injection into the cauda epididymidis, also in the sacral parasympathetic nucleus (L6-S1). Virus-infected perikarya were present in several brain stem nuclei (lateral reticular nucleus, gigantocellular and paragigantocellular nucleus, A5 noradrenergic cell group, caudal raphe nuclei, locus coeruleus, Barringtons nucleus, nucleus of the solitary tract, periaqueductal gray) and in the diencephalon (hypothalamic paraventricular nucleus, lateral hypothalamus). At the longer survival time, some telencephalic structures also exhibited virus-labeled neurons. The distribution of infected neurons in the brain was similar after virus injection into the caput or cauda epididymidis; however, earlier onset of infection was observed after inoculation into the cauda. The present findings provide the first morphological data on a multisynaptic circuit of neurons innervating the epididymis and presumably involved in the control of epididymal functions. reserved.

Gene and Cancer Therapy - Pseudorabies Virus: A Novel Research and Therapeutic Tool?

The past decade has been marked by significant advances in the application of gene transfer into living cells of animals and humans, with the resulting catapulting of preclinical and basic scientific concepts into therapeutic trials. A variety of virus-mediated gene delivery techniques have proved to be superior to other methodologies. This article concisely reviews the current status of gene and tumor therapy, focusing on virus-based technologies, describes the molecular biology of neurotropic herpesviruses and the application of herpes simplex virus, a relative of pseudorabies virus (PRV) in gene transfer and cancer therapy protocols. Finally, it addresses the issue of whether PRV, a nonhuman pathogen, could serve as a suitable research and therapeutic tool as concerns genetic and tumor therapy.