Bartha Strain Of Pseudorabies Virus Research Articles

Characterization of the neuroinvasive profile of a pseudorabies virus recombinant expressing the mTurquoise2 reporter in single and multiple injection experiments

BackgroundViral transneuronal tracing has become a well established technology used to define the synaptic architecture of polysynaptic neural networks. New MethodIn this report we define the neuroinvasive profile and reporter expression of a new recombinant of the Bartha strain of pseudorabies virus (PRV). The new recombinant, PRV-290, expresses the mTurquoise2 fluorophor and is designed to complement other isogenic recombinants of Bartha that express different reporters of infection.Results & Comparison with Existing Methods: PRV-290 was injected either alone or in combination with isogenic recombinants of PRV that express enhanced green fluorescent protein (EGFP; PRV-152) or monomeric red fluorescent protein (mRFP; PRV-614). Circuits previously defined using PRV-152 and PRV-614 were used for the analysis. The data demonstrate that PRV-290 is a retrograde transneuronal tracer with temporal kinetics similar to those of its isogenic recombinants. Stable expression of the diffusible mTurquoise2 reporter filled infected neurons, with the extent and intensity of labeling increasing with advancing post inoculation survival. In multiple injection experiments, PRV-290 established productive infections in neurons also replicating PRV-152 and/or PRV-614. This novel demonstration of three recombinants infecting individual neurons represents an important advance in the technology. ConclusionCollectively, these data demonstrate that PRV-290 is a valuable addition to the viral tracer toolbox for transneuronal tracing of neural circuitry.

Viral Tracing Identifies Parallel Disynaptic Pathways to the Hippocampus

Electrophysiological and lesion studies in rodents have shown that the dorsal (septal) and ventral (temporal) segments of the hippocampus have functional specializations that can be understood in terms of their anatomical connections with distinct brain areas. Here we explore the circuitry associated with the hippocampus using the pseudorabies virus-Bartha strain (PRV-Bartha) tracer in the rat to examine both direct (first-order) and indirect (second-order) projections to the hippocampus. Based on analysis of PRV-Bartha infection density, we demonstrate two parallel pathways from the limbic cortex to the hippocampus. A dorsal "spatial cognition" pathway provides disynaptic input from the retrosplenial, anterior cingulate, and orbital cortex to the dorsal hippocampus, with potential synaptic relays in the anterior thalamic nuclei and dorsolateral entorhinal cortex. A ventral "executive control" pathway provides disynaptic input from the prelimbic, infralimbic, and orbital cortex to the ventral hippocampus, with potential synaptic relays in the midline thalamic nuclei and the rostral caudomedial entorhinal cortex. These data suggest a new anatomical framework for understanding the functional interactions between the cortex and hippocampus, especially in cognitive disorders that involve both structures, such as frontotemporal dementia.

Gender Specific Pelvic Pain Severity in Neurogenic Cystitis

Interstitial cystitis/painful bladder syndrome is a chronic bladder inflammatory disease of unknown etiology that is often regarded as neurogenic cystitis. The condition is associated with focal inflammation, urothelial lesions, voiding dysfunction and pain in the pelvic/perineal area. Approximately 90% of patients with the condition are women, suggesting the possibility of hormonal involvement in interstitial cystitis/painful bladder syndrome symptoms. We examined the basis of gender specific pelvic pain in a murine model of neurogenic cystitis that recapitulates features of interstitial cystitis/painful bladder syndrome and in which pelvic pain is mediated by mast cell histamine. Murine neurogenic cystitis was induced by tail base inoculation of C57BL/6 or BALB/c mice with the Bartha strain of pseudorabies virus. Pelvic pain behavior was assessed by quantifying tactile allodynia in response to mechanical stimulation with von Frey filaments. Bladder mast cells were quantified by flow cytometry. Female mice of each genetic background showed significantly greater pelvic pain behavior than males, although responses were greater in BALB/c females. Gender specific pelvic pain behavior did not correspond to increased bladder inflammation or barrier dysfunction. Modulating reproductive hormonal status by ovariectomy and subsequent estrogen replacement had no effect on the magnitude of pseudorabies virus induced pain. The number of mast cells was associated with pelvic pain severity in female mice but it did not correlate with gender specific pelvic pain. These data suggest that pelvic pain in mice with murine neurogenic cystitis is mediated by gender specific responsiveness to mast cells while pelvic pain severity is modulated by genetic factors.

Alteration of the CNS pathway to the hippocampus in a mouse model of Niemann–Pick, type C disease

Niemann–Pick type C disease (NPC) is an autosomal recessive disorder that results in premature death due to progressive neurodegeneration including dementia. To understand neuronal pathways connecting to the hippocampus, retrograde transneuronal labeling method with Bartha strain of pseudorabies virus (PRV) was employed in 40 NPC+/+, NPC+/− and NPC−/− mice. Immunohistochemistry using polyclonal antibody against PRV and streological counting were used. The number of neurons and synapse in CA2&3 regions of the hippocampus decreased dramatically in the NPC−/− mouse compared to the NPC+/+ or +/− mouse. The number of PRV positive cell was significantly decreased in several regions including the entorhinal and piriform cortex in the NPC−/− mouse. More severely, lateral septal dorsal nucleus, dorsal entorhinal cortex and medial geniculate body showed no positive labeling in the NPC−/− mouse. However, the hippocampus, medial septal and supramammilary nuclei showed increased immunoreactivity in the NPC−/− mouse. Our data suggest that the synaptic loss and discontinuity of the CNS hippocampal pathway may contribute to understanding the mechanism of symptoms and functional disabilities such as memory and learning disturbance in NPC patients.

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.

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.

Phenotypic traits of the hypothalamic PVN cells innervating airway-related vagal preganglionic neurons

The paraventricular nucleus of the hypothalamus (PVN) integrates multiple inputs via projections from arginine vasopressin (AVP)- and oxytocin (OXT)-containing neurons to the brain stem and spinal cord as well as regulates respiratory and cardiovascular stress-related responses, which also affect airway function. In the present study, we used immunocytochemistry and the retrograde transneuronal tracer, Bartha strain of pseudorabies virus expressing green fluorescent protein (PRV-GFP), to localize AVP- and OXT-producing neurons that project to airway-related vagal preganglionic neurons (AVPNs) innervating intrapulmonary airways. PRV-GFP was microinjected into the upper right lung lobe, and after 4 days survival, hypothalamic tissue sections were processed for co-expression of PRV-GFP and AVP or PRV-GFP and OXT. In addition, in a separate group of five rats, Phaseolus vulgaris leucoagglutinin (PHAL), an anterograde tracer, was injected unilaterally into the PVN and cholera toxin beta subunit was microinjected into the tracheal wall. Analysis of five successfully infected animals showed that 14% of PRV-GFP labeled neurons express AVP traits and 18% of transneuronally-labeled neurons contain OXT. Furthermore, the identified AVPNs innervating extrathoracic trachea receive axon terminals of the PVN neurons. The results indicate that AVP- and OXT-producing PVN cells, via direct projections to the AVPNs, could modulate cholinergic outflow to the airways, as a part of overall changes in response to stress.

A spinal cord pathway connecting primary afferents to the segmental sympathetic outflow system

The sympathetic innervation of lumbar dorsal root ganglia (DRGs) and the possible presence of spinal cord circuits connecting primary sensory afferents to the sympathetic outflow to DRGs were investigated. We used simultaneous tracing of the sympathetic input to and sensory output from DRGs. Adult male rats received unilateral microinjections of the Bartha strain of pseudorabies virus into four lumbar DRGs. At 24 h post-inoculation, productive infection was detected in both DRG neurons and sympathetic postganglionic neurons. Infection of spinal cord neurons was first observed in sympathetic preganglionic neurons of the intermediolateral column. Subsequently, the infection spread to the contralateral intermediolateral column, the area around the central canal and the superficial dorsal horn layers. To investigate the relationship between infected spinal cord neurons and primary afferents from the corresponding DRGs, we injected pseudorabies virus for retrograde tracing together with cholera toxin B for anterograde tracing. We found that infected LIV/LV and LX neurons were in close apposition to cholera toxin B labeled afferents. Importantly, immunohistochemical detection of bassoon, a pre-synaptic zone protein, identified such contacts as synapses. Together, this suggests synaptic contacts between primary sensory afferents and neurons regulating sympathetic outflow to corresponding DRGs.

Suprachiasmatic nucleus input to autonomic circuits identified by retrograde transsynaptic transport of pseudorabies virus from the eye.

Intraocular injection of the Bartha strain of pseudorabies virus (PRV Bartha) results in transsynaptic infection of the hypothalamic suprachiasmatic nucleus (SCN), a retinorecipient circadian oscillator. PRV Bartha infection of a limited number of retinorecipient structures, including the SCN, was initially interpreted as the differential infection of a subpopulation of rat retinal ganglion cells, followed by replication and anterograde transport via the optic nerve. A recent report that used a recombinant strain of PRV Bartha (PRV152) expressing enhanced green fluorescent protein demonstrated that SCN infection actually results from retrograde transneuronal transport of the virus via the autonomic innervation of the eye in the golden hamster. In the present study using the rat, the pattern of infection after intravitreal inoculation with PRV152 was examined to determine if infection of the rat SCN is also restricted to retrograde transsynaptic transport. It was observed that infection in preganglionic autonomic nuclei (i.e., Edinger-Westphal nucleus, superior salivatory nucleus, and intermediolateral nucleus) precedes infection in the SCN. Sympathetic superior cervical ganglionectomy did not abolish label in the SCN after intraocular infection, nor did lesions of parasympathetic preganglionic neurons in the Edinger-Westphal nucleus. However, combined Edinger-Westphal nucleus ablation and superior cervical ganglionectomy eliminated infection of the SCN. This observation allowed a detailed examination of the SCN contribution to descending autonomic circuits afferent to the eye. The results indicate that in the rat, as in the hamster, SCN infection after intraocular PRV152 inoculation is by retrograde transsynaptic transport via autonomic pathways to the eye.

Melanopsin and non-melanopsin expressing retinal ganglion cells innervate the hypothalamic suprachiasmatic nucleus.

Retinal input to the hypothalamic suprachiasmatic nucleus (SCN) synchronizes the SCN circadian oscillator to the external day/night cycle. Retinal ganglion cells that innervate the SCN via the retinohypothalamic tract are intrinsically light sensitive and express melanopsin. In this study, we provide data indicating that not all SCN-projecting retinal ganglion cells express melanopsin. To determine the proportion of ganglion cells afferent to the SCN that express melanopsin, ganglion cells were labeled following transsynaptic retrograde transport of a recombinant of the Bartha strain of pseudorabies virus (PRV152) constructed to express the enhanced green fluorescent protein (EGFP). PRV152 injected into the anterior chamber of the eye retrogradely infects four retinorecipient nuclei in the brain via autonomic circuits to the eye, resulting in transneuronally labeled ganglion cells in the contralateral retina 96 h after intraocular infection. In animals with large bilateral lesions of the lateral geniculate body/optic tract, ganglion cells labeled with PRV152 are retrogradely infected from only the SCN. In these animals, most PRV152-infected ganglion cells were immunoreactive for melanopsin. However, a significant percentage (10-20%) of EGFP-labeled ganglion cells did not express melanopsin. These data suggest that in addition to the intrinsically light-sensitive melanopsin-expressing ganglion cells, conventional ganglion cells also innervate the SCN. Thus, it appears that the rod/cone system of photoreceptors may provide signals to the SCN circadian system independent of intrinsically light-sensitive melanopsin ganglion cells.

The attenuated pseudorabies virus strain Bartha fails to package the tegument proteins Us3 and VP22.

The Bartha strain of pseudorabies virus has several recognized mutations, including a deletion in the unique short region encompassing the glycoprotein I (gI), gE, Us9, and Us2 genes and point mutations in the gC, gM, and UL21 genes. We have determined that Bartha has mutations in the serine/threonine kinase encoded by the Us3 gene relative to the wild-type Becker strain. Our analysis revealed that Becker virions contain the Us3 protein, whereas Bartha virions do not. To test whether the mutations in the Bartha Us3 protein were responsible for this observation, we constructed a recombinant Bartha strain, PRV632, which expresses the Becker Us3 protein. PRV632 failed to package Us3 into the tegument, indicating that mutations other than those in the Us3 primary amino acid sequence were responsible for the failure of Bartha to package its Us3 protein. A recombinant Becker strain, PRV634, which expresses the Bartha Us3 protein, was constructed to test whether it was capable of being packaged into virions. The Bartha Us3 protein was not incorporated into PRV634 virions efficiently, suggesting that the primary sequence of the Bartha Us3 protein affects packaging into the tegument. To determine whether the packaging of other tegument proteins was affected in the Bartha strain, we examined VP22. Whereas Becker packaged VP22 into virions, Bartha had a severe deficiency in VP22 incorporation. Analysis of VP22 expression in Bartha-infected cells revealed that Bartha VP22 had a slower mobility on sodium dodecyl sulfate-polyacrylamide gels, indicating either primary sequence differences and/or different posttranslational modifications relative to Becker VP22. Taken together, these data indicate that, while the primary sequence of the Us3 protein does affect its incorporation into the tegument, other factors are involved. Furthermore, our data suggest that one or more of the gI, gE, Us9, or Us2 genes influences the localization of the Us3 protein in infected cells, and this effect may be important for the proper incorporation of Us3 into virions.

Neural circuitry of the adult rat central nervous system after spinal cord injury: a study using fast blue and the Bartha strain of pseudorabies virus.

The distribution of retrogradely and transneuronally labeled neurons in the adult rat brain and spinal cord after contusive mid-thoracic spinal cord injury (SCI) was studied using Fast Blue (FB) and the Bartha strain of pseudorabies virus (PRV), respectively. When FB was injected into the distal spinal cord at 2 days after graded SCI at the 10th vertebral level, labeled neurons were consistently found 7 days later in supraspinal areas that normally project to the spinal cord. The number of FB-labeled neurons decreased as the injury severity increased. An inverse correlation between the number of FB-labeled neurons and injury severity was seen in most investigated brain nuclei with coefficient of correlations (r) ranging from -0.84 in the red nucleus to -0.92 in the raphe nuclei. The coefficient of correlation was relatively poor in the motor cortex (r = -0.63), where a mild injury (6.25 g.cm) resulted in a 99% damage of the corticospinal tract. Such a prominent difference between the corticospinal tract and other descending pathways can be related to the difference in location of these pathways within the adult rat spinal cord. When PRV was injected into the right sciatic nerve one month after the injury, labeled cells were consistently identified 5 days later in the spinal cord rostral to the injury and in certain supraspinal regions that regulate autonomic outflow. In these nuclei, the distribution and number of PRV-labeled neurons markedly decreased after SCI as compared to the control group. In contrast, PRV-labeled neurons were inconsistently found in the supraspinal nuclei that contribute to somatic motor outflow in normal controls and no labeling was observed in these nuclei after injury. These results demonstrate that (1) a proportion of neural network across the injured spinal cord has been spared after acute contusive SCI, (2) the proportion of spared axons of a particular pathway is closely correlated to the injury severity and the position of that pathway, and (3) the transneuronal labeling method using PRV may provide a unique approach to investigate multi-synaptic neural circuitry of the central autonomic control after SCI, but its application to the somatic motor system is limited.

Topographic organization of suprachiasmatic nucleus projection neurons.

The mammalian circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), has two subdivisions. The core is located above the optic chiasm, receives primary and secondary visual afferents, and contains neurons producing vasoactive intestinal polypeptide and gastrin-releasing peptide. The shell largely surrounds the core, receives input from nonvisual sources and contains neurons producing arginine vasopressin and calretinin. In this study, we tested the hypothesis that SCN efferent projections are topographically organized with respect to the subdivision of origin. Injections of retrograde tracers were placed in major sites of efferent termination, described from prior studies that used anterograde tracers (Watts and Swanson, [1987] J. Comp. Neurol. 258:230-252; Watts et al. [1987] J. Comp. Neurol. 258:204-229). After retrograde tracer injections in the medial preoptic area, dorsomedial and paraventricular hypothalamic nuclei, bed nucleus of stria terminalis, paraventricular thalamic nucleus, zona incerta, and medial subparaventricular zone, retrogradely labeled SCN cells are clustered in the shell with few labeled neurons in the core. After injections centered in the lateral subparaventricular zone, peri-suprachiasmatic region, lateral septum, or ventral tuberal area, the majority of neuronal label is in the core with moderate to sparse neuronal label in the shell. Both subdivisions are labeled after injections in the paratenial thalamic nucleus. The same pattern of retrograde labeling is found with four tracers, cholera toxin-beta subunit, Fluoro-Gold, the Bartha strain of pseudorabies virus, and biotinylated dextran amine. These data extend our understanding of the significance of the division of the SCN into shell and core by demonstrating that the subdivisions differ in the pattern of projections. Together with prior observations that the subdivisions differ with respect to afferents, local connections, and neuroactive substances, the present study provides an anatomic basis for discrete control of circadian function by the SCN core and shell. In this novel view, the nature of the signal conveyed to areas receiving core or shell projections varies as a function of the subdivision from which innervation is derived.

Activity of Cardiorespiratory Networks Revealed by Transsynaptic Virus Expressing GFP

A fluorescent transneuronal marker capable of labeling individual neurons in a central network while maintaining their normal physiology would permit functional studies of neurons within entire networks responsible for complex behaviors such as cardiorespiratory reflexes. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alpha herpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and then travels transsynaptically to continue labeling the second- and higher-order neurons in a time-dependent manner. A Bartha PRV mutant that expresses green fluorescent protein (GFP) was used to visualize and record from neurons that determine the vagal motor outflow to the heart. Here we show that Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties and that the labeled baroreflex pathways that control heart rate are unaltered by the virus. This novel transynaptic virus permits in vitro studies of identified neurons within functionally defined neuronal systems including networks that mediate cardiovascular and respiratory function and interactions. We also demonstrate superior laryngeal motorneurons fire spontaneously and synapse on cardiac vagal neurons in the nucleus ambiguus. This cardiorespiratory pathway provides a neural basis of respiratory sinus arrhythmias.

CNS cholinergic innervation to the hippocampus in the rat using pseudorabies virus as a neurotracer.

The hippocampus is a central area of the memory-related neural system. Combined immunohistochemistry against choline acetyl transferase and retrograde transneuronal labelling of the pseudorabies virus were used to identify cholinergic neurons in the central nervous system projecting to the hippocampal formation of the rat. Five to ten microL of Bartha strain of pseudorabies virus were injected into the dentate gyrus, CA1 and CA3 of the hippocampus of 20 Sprague Dawley rats using stereotaxic instrument. Forty eight to 96 hr after the injection, the brains were removed and the tissue sections were processed for double immunofluorescence procedure using polyclonal antibodies against pseudorabies virus or choline acetyl transferase. The double labelled neurons were distributed at several different nuclei and the labelling patterns of three different areas of the hippocampus were similar. These data suggests that the cholinergic innervation to the hippocampus were distributed in a transsynaptic manner throughout the whole brain area.

Spatial and temporal patterns of transneuronal labeling in CNS neurons after injection of pseudorabies virus into the sciatic nerve of adult rats

The distribution of labeled neurons in the brain and spinal cord was studied after injecting the Bartha strain of pseudorabies virus (PRV) into the sciatic nerve to provide a baseline for studying neural circuitry after spinal cord injury (SCI) and regeneration. Following a single injection of viral particles into the left sciatic nerve, PRV labeling was found in the spinal cord at 2 days post-injection (p.i.). Increasing complexity in viral labeling from the spinal cord to supraspinal regions became apparent with increasing survival time. In brain regions, several neuronal groups that regulate sympathetic outflow, such as the rostroventrolateral medulla, the lateral paragigantocellular nuclei, and the A5 cells, were densely labeled. However, relatively sparse labeling was noticed in the lateral vestibular nuclei, the red nucleus and the motor cortex whose spinal projections regulate somatic motor function, although those areas were abundantly labeled with Fast blue (FB) in a double-labeling experiment in which FB was co-injected into the lumbar cord. The pattern of viral labeling became more complex beyond 5 days p.i. when increased numbers of cell groups were labeled with PRV but not FB. In addition, some infected neurons started to lyse, as evidenced by a decrease in viral labeling at 7 days p.i. Thus, the 5th day post-viral injection would appear to be an appropriate survival time to obtain maximal labeling with acceptable specificity. We suggest that transneuronal labeling using PRV should be appropriate for studying multi-neural circuitry after SCI and regeneration.

Identification of neural projections from the forebrain to the kidney, using the virus pseudorabies

These data demonstrate a chain of synaptically connected neurons extending to the rat kidney through several levels of the neuraxis from the forebrain, and the lamina terminalis — an area known to be involved in the regulation of body fluid homeostasis. The Bartha strain of pseudorabies virus was injected into the kidney of male Sprague–Dawley rats, resulting in retrograde infections in spinal cord segments (T 1–T 8), and successive infection in five autonomic `premotor' areas of the brain, the rostroventrolateral medulla, rostroventromedial medulla, raphe nuclei, A5 region of the pons, and the paraventricular nucleus of the hypothalamus, as well as the nucleus of the solitary tract, locus coeruleus, and subcoeruleus nuclei. Higher order labelling was found in regions of the forebrain, including the organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, medial preoptic area, bed nucleus of the stria terminalis, anteroventral periventricular nucleus, lateral preoptic area, suprachiasmatic nucleus, retrochiasmatic nucleus, primary motor cortex, and visceral cortex. This polysynaptic pathway to the kidney may form the substrate underlying the impact of forebrain structures on renal function.

Tracing autonomic innervation of the rat pineal gland using viral transneuronal tracing.

We have used the neurotropic Bartha strain of pseudorabies virus (PRV) to characterise the pathway linking the endogenous circadian pacemaker of the suprachiasmatic nucleus (SCN) to the pineal gland. This low virulent strain of virus replicates within synaptically linked neurones and is ideally suited to visualise the multisynaptic pathways through which the SCN modulates the activity of the rat pineal gland. Using specific antibodies against PRV, we could follow the immunohistochemical pattern of the spatiotemporal passage of virus through the sympathetic trunk and the neuraxis. The time course of virus infection indicated that the most prominent pathway from the SCN to the pineal gland is via a final sympathetic innervation from the superior cervical ganglion (SCG). The pathway arises in the dorsomedial portion of the SCN from where neurones project to the dorsal parvicellular subdivision of the hypothalamic paraventricular nucleus (PVN) to form synaptic contact with neurones descending to the intermediolateral nucleus (IML) of the upper thoracic spinal cord. The neurones of the IML constitute the presynaptic sympathetic input synaptically connected to postsynaptic sympathetic neurones in the SCG which constitute the final input to the pineal gland. Removal of the superior cervical ganglion (SCGX) prior to viral infection completely abolished infection of neurones in this circuit. However, an additional parasympathetic projection from the superior salivatory nucleus via the sphenopalatine ganglion to the pineal gland was observed in SCGX animals.

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.

The nuclei innervating digastric muscle do not project to the hypoglossal nucleus in the rat.

We employed the retrograde neural tracing method using the pseudorabies virus bartha strain (PRV-Ba) to investigate the projection from accessory trigeminal nucleus (Acc5) or accessary facial nucleus (Acc7) to hypoglossal nucleus (HP). After injection of PRV-Ba into the bellies of the digastric muscle, the PRV-Ba containing neurones were observed in Acc5 of the cranial belly injected rats and Acc7 of caudal belly injected rats ipsilaterally, but not in HP. These results suggest that Acc5 and Acc7 may not project to HP and that movements of the digastric muscle are not related to cooperation with tongue movements during mastication.