Injection Of Pseudorabies Virus Research Articles

Central Autonomic Network Underlying Cardiometabolic Coupling: Role of the Leptin Receptor Neurons

The autonomic network of the central nervous system (CNS) play an important role in the regulation of many physiological functions. Obesity is often associated with hypertension which is a serious medical condition that increase mortality. Therefore, obesity-induced hypertension may be the consequence of a cardiovascular-metabolic coupling in the CNS autonomic network. Leptin, a hormone secreted by adipose tissue, acts in the brain to regulate energy homeostasis, cardiovascular function, and several other physiological processes. We and others have previously shown the existence of shared nuclei in the autonomic network innervating organs such as the kidney, liver, and brown adipose tissue (BAT). However, whether there are shared neurons within these nuclei associated with these three organs and the anatomical substrates underlying cardiovascular and metabolic control by the leptin receptor (LepR)-containing neurons are unknown. To address this, two groups of wildtype mice received injections of pseudorabies virus (PRV) expressing a green fluorescent protein (GFP) into both kidneys and PRV expressing a red fluorescent protein (RFP) in either the interscapular BAT (iBAT) or the left lobe of the liver. Additionally, mice bearing Cre-mediated expression of a fluorescent protein, td-Tomato, in LepR neurons were injected with PRV in either the kidneys, liver, or iBAT. The animals were sacrificed 5-7 days post-injection and perfused. The brains were extracted, sectioned at 50 μm thickness, stained, and imaged with confocal microscopy. Sections were matched to the mouse brain atlas (Franklin & Paxinos) and neurons co-expressing GFP and RFP throughout the brain were identified. For the double-organ injections, soma morphology was measured using ImageJ software. We found several nuclei in which neurons were co-labeled with GFP (kidney) and RFP (BAT or liver). Co-expressing neurons were observed in areas such as the cortical regions (motor cortex and amygdala), hypothalamus (paraventricular nucleus (PVN), lateral hypothalamus (LH), and dorsomedial hypothalamus (DMH)), midbrain regions such as the periaqueductal gray, and brainstem nuclei such as the locus coeruleus (LC). In general, there appeared to be two scenarios for shared nuclei: 1) in regions such as LH, nucleus of the solitary tract, and dorsal motor nucleus of vagus there was little to very little co-expression, and 2) in regions such as PVN, DMH, LC, and motor cortex there was moderate to high levels of co-expression. Using the Kolmogorov-Smirnov test the kidney and iBAT soma sizes for the PVN, motor cortex, and LC were statistically different (p<0.05), but not for the LH (p>0.1). Approximately 5% of LepR neurons in the lateral hypothalamic area, septal nucleus, ventral tegmental area, and nucleus tractus solitarius (nTS) were linked to kidney or iBAT. Many other common nuclei between the organ associated regions and LepR neurons such as the amygdala and dorsal-medial hypothalamus did not appear to demonstrate any co-localization. This is the first study to provide anatomical evidence linking the autonomic networks regulating cardiovascular and metabolic functions. Moreover, our data show that although there is overlap of neurons in diverse nuclei there may also be a difference in morphological phenotype in the nuclei associated with cardiovascular vs metabolic functions. Our results suggest that leptin may couple cardiovascular and metabolic regulation through a small number of neurons in the CNS autonomic network. Renal & Hypertension T32 (DK007690), American Heart Association (834962), and Diabetes T32 (DK112751). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Parallel Pathways Provide Hippocampal Spatial Information to Prefrontal Cortex.

Long-range synaptic connections define how information flows through neuronal networks. Here, we combined retrograde and anterograde trans-synaptic viruses to delineate areas that exert direct and indirect influence over the dorsal and ventral prefrontal cortex (PFC) of the rat (both sexes). Notably, retrograde tracing using pseudorabies virus (PRV) revealed that both dorsal and ventral areas of the PFC receive prominent disynaptic input from the dorsal CA3 (dCA3) region of the hippocampus. The PRV experiments also identified candidate anatomical relays for this disynaptic pathway, namely, the ventral hippocampus, lateral septum, thalamus, amygdala, and basal forebrain. To determine the viability of each of these relays, we performed three additional experiments. In the first, we injected the retrograde monosynaptic tracer Fluoro-Gold into the PFC and the anterograde monosynaptic tracer Fluoro-Ruby into the dCA3 to confirm the first-order connecting areas and revealed several potential relay regions between the PFC and dCA3. In the second, we combined PRV injection in the PFC with polysynaptic anterograde viral tracer (HSV-1) in the dCA3 to reveal colabeled connecting neurons, which were evident only in the ventral hippocampus. In the third, we combined retrograde adeno-associated virus (AAV) injections in the PFC with an anterograde AAV in the dCA3 to reveal anatomical relay neurons in the ventral hippocampus and dorsal lateral septum. Together, these findings reveal parallel disynaptic pathways from the dCA3 to the PFC, illuminating a new anatomical framework for understanding hippocampal-prefrontal interactions. We suggest that the representation of context and space may be a universal feature of prefrontal function.SIGNIFICANCE STATEMENT The known functions of the prefrontal cortex are shaped by input from multiple brain areas. We used transneuronal viral tracing to discover multiple prominent disynaptic pathways through which the dorsal hippocampus (specifically, the dorsal CA3) has the potential to shape the actions of the prefrontal cortex. The demonstration of neuronal relays in the ventral hippocampus and lateral septum presents a new foundation for understanding long-range influences over prefrontal interactions, including the specific contribution of the dorsal CA3 to prefrontal function.

Crosstalk in the Central Autonomic Neurocircuitry Underlying Cardiovascular and Metabolic Control

Obesity associated cardiovascular risks such as hypertension are serious medical conditions that increase mortality. The brain autonomic networks play an important role in the regulation of cardiovascular function and metabolism. Therefore, obesity may represent a cardiovascular‐metabolic coupling and possible crosstalk in the central autonomic networks. We and others have previously shown the existence of shared nuclei in the autonomic networks innervating cardiovascular organs such as the kidney and metabolic organs such as the liver and brown adipose tissue (BAT). However, whether there are shared neurons within these nuclei associated with these three organs is unknown. To address this, two groups of mice received injections of pseudorabies virus (PRV) expressing a green fluorescent protein (GFP) into both kidneys and PRV expressing a red fluorescent protein (RFP) in either the interscapular BAT (iBAT) or the left lobe of the liver. The animals were sacrificed 5‐7 days post‐injection and perfused. The brains were extracted, sectioned at 50 µm thickness, stained, and imaged with confocal microscopy. Sections were matched to the mouse atlas (Franklin & Paxinos) and neurons co‐expressing GFP and RFP throughout the brain were identified. We found several nuclei in which neurons were co‐labeled with GFP (kidney) and RFP (BAT or liver). Interestingly, co‐expression does not appear limited to nuclei of specific regions in the brain. Instead, co‐expression was observed in many nuclei from cortical and subcortical regions to hypothalamic areas, midbrain, and brainstem nuclei. Indeed, co‐expressing neurons were observed in areas such as the cortical regions (motor cortex and amygdala), hypothalamic nuclei (paraventricular nucleus (PVN), lateral hypothalamus (LH), and dorsomedial hypothalamus (DMH)), midbrain regions such as the periaqueductal gray, and brainstem nuclei such as the locus coeruleus (LC). In general, there appeared to be two scenarios for shared nuclei: 1) in regions such as LH, nucleus of the solitary tract, and dorsal motor nucleus of vagus there was very little co‐expression, and 2) in regions such as PVN, DMH, LC, and motor cortex there was moderate to high levels of co‐expression. This is the first study to provide anatomical evidence for crosstalk between the autonomic networks regulating cardiovascular and metabolic functions. Moreover, our data demonstrate that crosstalk between the autonomic networks controlling cardiovascular and metabolic functions is distributed throughout the central nervous system.

Propriospinal Neurons of L3-L4 Segments Involved in Control of the Rat External Urethral Sphincter

Coordination of activity of external urethral sphincter (EUS) striated muscle and bladder (BL) smooth muscle is essential for efficient voiding. In this study we examined the morphological and electrophysiological properties of neurons in the L3/L4 spinal cord (SC) that are likely to have an important role in EUS-BL coordination in rats. EUS-related SC neurons were identified by retrograde transsynaptic tracing following injection of pseudorabies virus (PRV) co-expressing fluorescent markers into the EUS of P18-P20 male rats. Tracing revealed not only EUS motoneurons in L6/S1 but also interneurons in lamina X of the L6/S1 and L3/L4 SC. Physiological properties of fluorescently labeled neurons were assessed during whole-cell recordings in SC slices followed by reconstruction of biocytin-filled neurons. Reconstructions of neuronal processes from transverse or longitudinal slices showed that some L3/L4 neurons have axons projecting toward and into the ventro-medial funiculus (VMf) where axons extended caudally. Other neurons had axons projecting within laminae X and VII. Dendrites of L3/L4 neurons were distributed within laminae X and VII. The majority of L3/L4 neurons exhibited tonic firing in response to depolarizing currents. In transverse slices focal electrical stimulation (FES) in the VMf or in laminae X and VII elicited antidromic axonal spikes and/or excitatory synaptic responses in L3/L4 neurons; while in longitudinal slices FES elicited excitatory synaptic inputs from sites up to 400 μm along the central canal. Inhibitory inputs were rarely observed. These data suggest that L3/L4 EUS-related circuitry consists of at least two neuronal populations: segmental interneurons and propriospinal neurons projecting to L6/S1.

Transsynaptic connections between the hypothalamus and adipose tissue: relationship to reproduction

Neurophysiological mechanisms that control energy balance are reciprocally linked to those that control reproduction. Neuromorphological studies using retrograde tracing methods revealed that nerve cells within the central (CNS) and autonomic (ANS) nervous systems in different species, including the pig, are transsynaptically connected to different fat tissue depots. In the pig, neurons localised in the paraventricular nucleus, supraoptic nucleus and arcuate nucleus were infected with pseudorabies virus (PRV) 9 days after injections into both the perirenal and subcutaneous adipose tissue depots. Infected neurons were in the ventromedial nucleus, dorsomedial nucleus and preoptic area after injection of PRV into perirenal adipose tissue, while infected cells in the lateral hypothalamic area projected only to the subcutaneous adipose tissue depot. Additionally, numerous centres of the ANS innervate adipose tissue depots in the pig. Fast blue stained (FB+) neurons, which projected to the subcutaneous adipose tissue overlaying the thoracolumbar area were located in the thoraco-lumbar region of the sympathetic chain ganglia (SChG). However, neurons supplying perirenal and mesentery adipose tissue depots were found in both the SChG and prevertebral ganglia. The vast majority of labelled neurons, in both the CNS and ANS, which innervated adipose tissue depots, expressed leptin receptor (OBR) immunoreactivity. The purpose of this brief review is to establish evidence for a multisynaptic circuit of neurons, which innervate adipose tissue in the pig and demonstrate that hypothalamic nuclei and sympathetic ganglion neurons involved in reproductive processes are transsynaptically connected to different adipose tissue depots.

Visualization and Quantification of Post-stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain.

Whole-brain volumetric microscopy techniques such as serial two-photon tomography (STPT) can provide detailed information on the roles of neuroinflammation and neuroplasticity throughout the whole brain post-stroke. STPT automatically generates high-resolution images of coronal sections of the entire mouse brain that can be readily visualized in three dimensions. We developed a pipeline for whole brain image analysis that includes supervised machine learning (pixel-wise random forest models via the “ilastik” software package) followed by registration to a standardized 3-D atlas of the adult mouse brain (Common Coordinate Framework v3.0; Allen Institute for Brain Science). These procedures allow the detection of cellular fluorescent signals throughout the brain in an unbiased manner. To illustrate our imaging techniques and automated image quantification, we examined long-term post-stroke motor circuit connectivity in mice that received a motor cortex photothrombotic stroke. Two weeks post-stroke, mice received intramuscular injections of pseudorabies virus (PRV-152), a trans-synaptic retrograde herpes virus driving expression of green fluorescent protein (GFP), into the affected contralesional forelimb to label neurons in descending tracts to the forelimb musculature. Mice were sacrificed 3 weeks post-stroke. We also quantified sub-acute neuroinflammation in the post-stroke brain in a separate cohort of mice following a 60 min transient middle cerebral artery occlusion (tMCAo). Naive e450+-labeled splenic CD8+ cytotoxic T cells were intravenously injected at 7, 24, 48, and 72 h post-tMCAo. Mice were sacrificed 4 days after stroke. Detailed quantification of post-stroke neural connectivity and neuroinflammation indicates a role for remote brain regions in stroke pathology and recovery. The workflow described herein, incorporating STPT and automated quantification of fluorescently labeled features of interest, provides a framework by which one can objectively evaluate labeled neuronal or lymphocyte populations in healthy and injured brains. The results provide region-specific quantification of neural connectivity and neuroinflammation, which could be a critical tool for investigating mechanisms of not only stroke recovery, but also a wide variety of brain injuries or diseases.

The neural pathway for lacrimal gland tear secretion in New Zealand White rabbits

ObjectiveWe investigated the neural pathway for tear secretion from the lacrimal gland of New Zealand White rabbits. MethodsNine healthy adult New Zealand White rabbits were randomly divided into three experimental groups, namely, an irritant-stimulated group, a non-stimulated group, and a saline-stimulated group. Sanitized dry cotton swabs with menthol were used to wipe both of the rabbits’ eyelids in the irritant-stimulated group, and the non-stimulated group and saline- stimulated group were compared as controls. The animals in the three groups were killed 2h later and the expressions of c-Fos in the frontal cortex, hippocampus, hypothalamus, pons, and medulla oblongata of the rabbits were detected using immunofluorescence labeling. According to the distribution of c-Fos protein expression, 12 healthy adult New Zealand rabbits were similarly divided into three groups for retrograde tract tracing via pseudorabies virus (PRV) injection into the lacrimal gland. Immunofluorescence labeling was used to analyze PRV-infected neurons in the brains of rabbits after survival for 30h, 38h, and 46h. ResultsThe most c-Fos-positive immunolabeled cells were observed in the menthol-stimulated group, whereas fewer c-Fos-positive immunolabeled cells were observed in the saline-stimulated group.The non-treated group showed the least c-Fos-positive immunolabeled cells. At 30h after PRV injection, PRV-positive neurons were found only in the superior salivary nucleus of the pons (SSN). At 38h, PRV-infected neurons were observed in the lateral nucleus of the superior olive (LSO) and the medial nucleus of the superior olive (MSO). At 46h, PRV-infected neurons were found in the nucleus of the trapezoid body (Tz) and the hypothalamic paraventricular nucleus (PVN), and their distributions were dense in the LSO and MSO. ConclusionsMenthol-induced c-Fos protein expression and PRV-mediated tract tracing suggest that in New Zealand White rabbits, the neural pathway that regulates tear secretion from the lacrimal gland proceeds from the PVN to the superior olivary complex of the pons to the SSN and finally to the lacrimal gland.

Bidirectional crosstalk between the sensory and sympathetic motor systems innervating brown and white adipose tissue in male Siberian hamsters.

The brain networks connected to the sympathetic motor and sensory innervations of brown (BAT) and white (WAT) adipose tissues were originally described using two transneuronally transported viruses: the retrogradely transported pseudorabies virus (PRV), and the anterogradely transported H129 strain of herpes simplex virus-1 (HSV-1 H129). Further complexity was added to this network organization when combined injections of PRV and HSV-1 H129 into either BAT or WAT of the same animal generated sets of coinfected neurons in the brain, spinal cord, and sympathetic and dorsal root ganglia. These neurons are well positioned to act as sensorimotor links in the feedback circuits that control each fat pad. We have now determined the extent of sensorimotor crosstalk between interscapular BAT (IBAT) and inguinal WAT (IWAT). PRV152 and HSV-1 H129 were each injected into IBAT or IWAT of the same animal: H129 into IBAT and PRV152 into IWAT. The reverse configuration was applied in a different set of animals. We found single-labeled neurons together with H129+PRV152 coinfected neurons in multiple brain sites, with lesser numbers in the sympathetic and dorsal root ganglia that innervate IBAT and IWAT. We propose that these coinfected neurons mediate sensory-sympathetic motor crosstalk between IBAT and IWAT. Comparing the relative numbers of coinfected neurons between the two injection configurations showed a bias toward IBAT-sensory and IWAT-sympathetic motor feedback loops. These coinfected neurons provide a neuroanatomical framework for functional interactions between IBAT thermogenesis and IWAT lipolysis that occurs with cold exposure, food restriction/deprivation, exercise, and more generally with alterations in adiposity.

Protective effect of resveratrol against pseudorabies virus-induced reproductive failure in a mouse model.

Resveratrol (RES), a natural polyphenol that is abundant in grapes, exerts anti-inflammatory, anti-oxidative, and antiviral bioactive effects. Protective effects of RES against pseudorabies virus (PrV)- induced reproductive failure were investigated in a mice model. Injection of PrV partially induced stillbirth and abortion, and caused poor growth of progeny. Treatment with RES attenuated the reproductive failure induced by the virus with recovery of the serum progesterone level. RES improved the growth performance of newborn mice. RES can attenuate the reproductive failure induced by PrV in mice.

Transneuronal tracing of central autonomic regions involved in cardiac sympathetic afferent reflex in rats

Stimulation of cardiac afferents (CA) increased sympathetic outflow and blood pressure. The goal of the current study is to determine the central autonomic nuclei involved in the regulation of cardiac sympathetic afferent reflex (CSAR) which has been proved in previously functional studies. Neuroanatomical method and pseudorabies virus (PRV) transynaptic retrograde trace technique will be performed to investigate the relationship between kidney and heart and the temporal order of the most PRV-labeled neurons in the central nervous system. Recombinant PRV expressing enhanced green fluorescence protein (EGFP) was injected into the left kidney of rats as a specific trans-synaptic retrograde tracer in neurons. After 2, 3, 4, 5, 6, 7, 8 or 9days, brain, spinal cord and heart were collected for immunofluorescence staining. The temporal order of PRV labeled neurons was found in the ipsilateral intermediolateral nucleus (IML) of T8–T12 spinal segments on day 3; bilateral rostroventrolateral medulla (RVLM), paraventricular nucleus (PVN) and nucleus of the solitary tract (NTS) on day 4; and left and right ventricular walls and ventricular septum of the heart on day 9. In rats with renal denervation, no PRV-infected neurons or cardiomyocytes were found after PRV injection. In conclusion, PRV trans-synaptic retrograde trace confirms that CA, NTS, PVN, RVLM, IML and renal nerves do exist to be involved in the regulation of CSAR and there is a close relationship between heart and kidney. CA is mainly located in the left ventricular wall, right ventricular wall and ventricular septum.

MP17-18 BIDIRECTIONAL COMMUNICATION BETWEEN AFFERENT NEURONS AND UROTHELIAL CELLS IN THE MOUSE URINARY BLADDER

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology1 Apr 2014MP17-18 BIDIRECTIONAL COMMUNICATION BETWEEN AFFERENT NEURONS AND UROTHELIAL CELLS IN THE MOUSE URINARY BLADDER Youko Ikeda, Irina Zabbarova, and Anthony Kanai Youko IkedaYouko Ikeda More articles by this author , Irina ZabbarovaIrina Zabbarova More articles by this author , and Anthony KanaiAnthony Kanai More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.551AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It has been widely hypothesized that urothelial cells communicate unidirectionally with afferent terminals to signal changes in the bladder lumen milieu. However, direct interactions between afferents and urothelial cells have not been clearly demonstrated. Our aim was to examine these interactions using pseudorabies virus (PRV) with the genetically-encoded Ca2+ probe, GCaMP to localize its fluorescence in the mouse urinary bladder. METHODS For dorsal root ganglia (DRG)-PRV injections, adult female C57Bl/10 mice were anesthetized and a hemilaminectomy performed at the L5-L6 vertebrae level to expose L6-S1 DRG. 2 μl of PVR468-GCaMP3 (106 pfu/ml) was slowly injected into each DRG using a 1.0 mm glass micropipette with a 10 μm beveled tip. The muscle and skin were sutured, animals allowed to recover and used for experiments after 2-3 days. For sham controls, sterile saline was injected in place of viral vector. In additional experiments, 10 μl of PRV was injected into the wall of the descending colon or tail muscle. Isolated DRG or whole bladder sheets were fixed, sectioned and examined for GCaMP fluorescence. RESULTS Following DRG-PRV injections GCaMP labeling was found in afferent nerves in the detrusor (DM) and within the urothelium (UT), but not the lamina propria (LP) (figure 1A). Labeling in the UT appeared punctate or contained inside vesicles, suggesting there may be urothelial uptake of GCaMP or PRV from afferents. In colon and tail injections, a similar GCaMP expression pattern was found compared to DRG injections (B and C). Colon/tail injected animals also had dense expression in L6/S1 DRG, demonstrating that these structures have shared afferent innervation with the bladder (D and E). Fluorescence was not detected in the DRG or bladders of sham control animals (not shown). CONCLUSIONS The established concept has been that urothelial cells signal to suburothelial afferents to ‘sense’ changes within the bladder lumen milieu. We have demonstrated that communication between afferents and urothelial cells is bidirectional, where afferents can also signal to urothelial cells. Changes in the dynamics of urothelial-afferent communication may underlie sensory disorders of the lower urinary tract. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e142 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Youko Ikeda More articles by this author Irina Zabbarova More articles by this author Anthony Kanai More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

5-HT neurons of the area postrema become c-Fos-activated after increases in plasma sodium levels and transmit interoceptive information to the nucleus accumbens.

Serotonergic (5-hydroxytryptamine, 5-HT) neurons of the area postrema (AP) represent one neuronal phenotype implicated in the regulation of salt appetite. Tryptophan hydroxylase (Tryp-OH, synthetic enzyme-producing 5-HT) immunoreactive neurons in the AP of rats become c-Fos-activated following conditions in which plasma sodium levels are elevated; these include intraperitoneal injections of hypertonic saline and sodium repletion. Non-Tryp-OH neurons also became c-Fos-activated. Sodium depletion, which induced an increase in plasma osmolality but caused no significant change in the plasma sodium concentration, had no effect on the c-Fos activity in the AP. Epithelial sodium channels are expressed in the Tryp-OH-immunoreactive AP neurons, possibly functioning in the detection of changes in plasma sodium levels. Since little is known about the neural circuitry of these neurons, we tested whether the AP contributes to a central pathway that innervates the reward center of the brain. Stereotaxic injections of pseudorabies virus were made in the nucleus accumbens (NAc), and after 4 days, this viral tracer produced retrograde transneuronal labeling in the Tryp-OH and non-Tryp-OH AP neurons. Both sets of neurons innervate the NAc via a multisynaptic pathway. Besides sensory information regarding plasma sodium levels, the AP→NAc pathway may also transmit other types of chemosensory information, such as those related to metabolic functions, food intake, and immune system to the subcortical structures of the reward system. Because these subcortical regions ultimately project to the medial prefrontal cortex, different types of chemical signals from visceral systems may influence affective functions.

Differential Joint-Specific Corticospinal Tract Projections within the Cervical Enlargement

The motor cortex represents muscle and joint control and projects to spinal cord interneurons and–in many primates, including humans–motoneurons, via the corticospinal tract (CST). To examine these spinal CST anatomical mechanisms, we determined if motor cortex sites controlling individual forelimb joints project differentially to distinct cervical spinal cord territories, defined regionally and by the locations of putative last-order interneurons that were transneuronally labeled by intramuscular injection of pseudorabies virus. Motor cortex joint-specific sites were identified using intracortical-microstimulation. CST segmental termination fields from joint-specific sites, determined using anterograde tracers, comprised a high density core of terminations that was consistent between animals and a surrounding lower density projection that was more variable. Core terminations from shoulder, elbow, and wrist control sites overlapped in the medial dorsal horn and intermediate zone at C5/C6 but were separated at C7/C8. Shoulder sites preferentially terminated dorsally, in the dorsal horn; wrist/digit sites, more ventrally in the intermediate zone; and elbow sites, medially in the dorsal horn and intermediate zone. Pseudorabies virus injected in shoulder, elbow, or wrist muscles labeled overlapping populations of predominantly muscle-specific putative premotor interneurons, at a survival time for disynaptic transfer from muscle. At C5/C6, CST core projections from all joint zones were located medial to regions of densely labeled last-order interneurons, irrespective of injected muscle. At C7/C8 wrist CST core projections overlapped the densest interneuron territory, which was located in the lateral intermediate zone. In contrast, elbow CST core projections were located medial to the densest interneuron territories, and shoulder CST core projections were located dorsally and only partially overlapped the densest interneuron territory. Our findings show a surprising fractionation of CST terminations in the caudal cervical enlargement that may be organized to engage different spinal premotor circuits for distal and proximal joint control.

A subset of presympathetic-premotor neurons within the centrally projecting Edinger–Westphal nucleus expresses urocortin-1

Numerous motivated behaviors require simultaneous activation of somatomotor and autonomic functions. We have previously characterized the organization of brain circuits that may mediate this integration. Presympathetic premotor neurons (PSPMNs) that are part of such circuits are distributed across multiple brain regions, which mediate stress-elicited behavioral and physiological responses, including the Edinger–Westphal nucleus (EW). Based on its connectivity and function, EW has recently been re-classified into a preganglionic (EWpg) and a centrally projecting (EWcp) population. Neurons within EWcp are the major source of urocortin 1 (Ucn-1), an analog of the corticotropin-releasing factor that binds the CRFR1 and CRFR2 receptors and has been implicated in mediating homeostatic responses to stress. We hypothesized that a subset of EWcp PSPMNs expresses Ucn-1. Utilizing dual-label immunofluorescence, we initially mapped the distribution of Ucn-1 and cholinergic neurons within EW in colchicine pre-treated rats. Based on this labeling we divided EWcp into three neuroanatomical levels. To examine connections of EWcp neurons to the gastrocnemius muscle and the adrenal gland, we next employed trans-synaptic tract-tracing in a second group of rats, utilizing two pseudorabies virus (PRV) recombinants that express unique reporter proteins. Using multi-label immunofluorescent staining, we identified the presence of Ucn-1-positive PSPMNs, dually labeled with PRV and present throughout the entire extent of EWcp and intermingled with Ucn-1 neurons infected with one or neither of the viral recombinants. Compared to rats pretreated with colchicine, we observed significantly fewer Ucn-1 neurons in animals that received PRV injections. Post hoc analyses revealed significantly fewer Ucn-1 neurons at the rostral level as compared to the caudal and middle levels. These data suggest functional and anatomic heterogeneity within EWcp; this organization may coordinate various aspects of stress-elicited and emotionally salient behaviors.

Role of Spinal Cord in Regulating Mouse Kidney: A Virally Mediated Trans-synaptic Tracing Study

To determine the spinal innervation and neuronal connections is important for studying renal metabolic responses. In this study, the spinal cords of 10 adult male C57BL/6J strain mice were mapped retrograde using injections of pseudorabies virus (PRV)-614. The virus, injected into the kidney, was specifically transported to the spinal cord. At 5 days after injection of the PRV-614, PRV-614-positive cells were found in the intermediolateral cell column, the intercalates nucleus, or the central autonomic nucleus of spinal cord segments T4-L1, and most PRV-614-labeled cells were found in the T9 segment. Our results revealed neuroanatomical circuits between kidney and the spinal intermediolateral cell column neurons.

Projections from the hypothalamic paraventricular nucleus and the nucleus of the solitary tract to prechoroidal neurons in the superior salivatory nucleus: Pathways controlling rodent choroidal blood flow

Using intrachoroidal injection of the transneuronal retrograde tracer pseudorabies virus (PRV) in rats, we previously localized preganglionic neurons in the superior salivatory nucleus (SSN) that regulate choroidal blood flow (ChBF) via projections to the pterygopalatine ganglion (PPG). In the present study, we used higher-order transneuronal retrograde labeling following intrachoroidal PRV injection to identify central neuronal cell groups involved in parasympathetic regulation of ChBF via input to the SSN. These prominently included the hypothalamic paraventricular nucleus (PVN) and the nucleus of the solitary tract (NTS), both of which are responsive to systemic BP and are involved in systemic sympathetic vasoconstriction. Conventional pathway tracing methods were then used to determine if the PVN and/or NTS project directly to the choroidal subdivision of the SSN. Following retrograde tracer injection into SSN (biotinylated dextran amine 3K or Fluorogold), labeled perikarya were found in PVN and NTS. Injection of the anterograde tracer, biotinylated dextran amine 10K (BDA10K), into PVN or NTS resulted in densely packed BDA10K+terminals in prechoroidal SSN (as defined by its enrichment in nitric oxide synthase-containing perikarya). Double-label studies showed these inputs ended directly on prechoroidal nitric oxide synthase-containing neurons of SSN. Our study thus establishes that PVN and NTS project directly to the part of SSN involved in parasympathetic vasodilatory control of the choroid via the PPG. These results suggest that control of ChBF may be linked to systemic blood pressure and central control of the systemic vasculature.

Noxious Colorectal Distention in Spinalized Rats Reduces Pseudorabies Virus Labeling of Sympathetic Neurons

The retrograde transsynaptic tracer pseudorabies virus (PRV) has been widely used as a marker for synaptic connectivity in the spinal cord. Notably, the PRV-152 construct expresses enhanced green fluorescent protein (EGFP). We recently reported a significant attenuation of PRV-152 labeling of the intermediolateral cell column (IML) and celiac ganglia after complete T4 spinal cord transection versus sham injury in rats at 96 h after PRV-152 inoculation of the left kidney. Here we found a significant increase in noxious colorectal distention (CRD)-evoked c-Fos expression in spinal cords of injured versus sham rats without PRV infection. In order to assess whether enhancing neuronal activity in spinalized rats might increase PRV-152 labeling, we subjected awake spinalized rats to 1.5 h of intermittent noxious CRD either: (1) just prior to inoculation, or (2) 96 h after inoculation (n = 3/group). Equal numbers of spinalized rats in both groups received PRV-152 inoculations without CRD (non-stimulated; n = 3/group). At 96 h post-inoculation fixed spinal cords and left celiac ganglionic tissues were assessed for the distribution and quantification of EGFP-labeled cells. The injured cohort that received CRD just prior to PRV injection showed a significant reduction in EGFP-labeled cells in both the IML and left celiac ganglion compared to non-stimulated injured rats. In contrast, the injured cohort that received CRD 96 h after PRV-152 inoculation showed no differences in EGFP-labeled cell numbers in the IML or celiac ganglia versus non-stimulated injured rats. Interestingly, microglia near c-Fos-positive cells after acute CRD appeared more reactive compared to non-stimulated spinalized rats, and activated microglial cells markedly reduce viral transduction and progression following PRV inoculation of the CNS. Hence our results imply that increased CRD-induced c-Fos expression in the injured paradigm, prior to but not after PRV injection, further attenuates PRV-152 uptake, perhaps through changes in neuronal activity and/or innate neuro-immune responses.

Innervation of skeletal muscle by leptin receptor-containing neurons

In addition to suppressing food intake, leptin reduces body adiposity by altering metabolism within peripheral tissues such as adipose tissue and muscle. Recent work indicates that leptin action within the brain is sufficient to promote glucose uptake and increase fat oxidation within skeletal muscle, and that these effects are dependent on the sympathetic nervous system. To identify neuronal circuits through which leptin impacts skeletal muscle metabolism, we used LepRb-GFP reporter mice in combination with muscle-specific injection of an mRFP-expressing pseudorabies virus (PRV), which acts as a transsynaptic retrograde tracer. Consistent with previous observations in the rat, muscle-specific PRV injection lead to labeling within multiple areas of the hypothalamus and brainstem. However, the only areas in which PRV and LepRb colocalization was detected were within the brainstem nucleus of the solitary tract (NTS) and the hypothalamic retrochiasmatic area. Within the NTS 28.5 ± 9.4% of PRV-positive neurons contained LepRb-GFP, while in the RCH 37 ± 1.7% of PRV neurons also contained LepRb. In summary, these data clearly implicate the NTS and RCH as key sites through which brain leptin impacts skeletal muscle, and as such provide an anatomical framework within which to interpret physiological data indicating that leptin acts in the brain to influence metabolism within skeletal muscle.

Endogenous activation of spinal 5-hydroxytryptamine (5-HT) receptors contributes to the thermoregulatory activation of brown adipose tissue

Neurons in the rostral raphe pallidus (RPa) play an essential role in the regulation of sympathetically mediated metabolism and thermogenesis in brown adipose tissue (BAT). The presence of serotonergic neurons in the RPa that are retrogradely labeled following pseudorabies virus injections into BAT suggests that these neurons play a role in the regulation of BAT. In urethane/chloralose-anesthetized rats, whole body cooling decreased skin (-5.7 +/- 2.3 degrees C) and core (-1.3 +/- 0.2 degrees C) temperatures and resulted in an increase in BAT sympathetic nerve activity (SNA; +1,026 +/- 344% of baseline activity). Serial microinjections of the 5-hydroxytryptamine (5-HT) receptor antagonist, methysergide (1.2 nmol/site), but not saline vehicle, into the intermediolateral cell column (IML) in spinal segments T2-T5 markedly attenuated the cooling-evoked increase in BAT SNA (remaining area under the curve, AUC: 36 +/- 9% of naive cooling response). Microinjections of the 5-HT(1A) receptor antagonist, WAY-100635 (1.2 nmol/site), or the 5-HT(7) receptor antagonist, SB-269970 (1.2 nmol/site), into the T2-T5 IML also attenuated the cold-evoked increase in BAT SNA (remaining activity at peak inhibition: 47 +/- 8% and 39 +/- 12% of the initial cold-evoked response, respectively). The increases in BAT SNA evoked by microinjection of N-methyl-d-aspartate (NMDA) (12 pmol) or bicuculline (30 pmol) into the RPa were attenuated following microinjections of methysergide, but not saline vehicle, into the T2-T5 IML (NMDA remaining AUC, 64 +/- 13% of naive response; bicuculline remaining AUC, 52 +/- 5% of naive response). These results are consistent with our earlier demonstration of a potentiating effect of 5-HT within the IML on BAT SNA and indicate that activation of 5-HT(1A) and 5-HT(7) receptors in the spinal cord contributes to increases in BAT SNA and thermogenesis.

Functional recovery of stepping in rats after a complete neonatal spinal cord transection is not due to regrowth across the lesion site

Rats receiving a complete spinal cord transection (ST) at a neonatal stage spontaneously can recover significant stepping ability, whereas minimal recovery is attained in rats transected as adults. In addition, neonatally spinal cord transected rats trained to step more readily improve their locomotor ability. We hypothesized that recovery of stepping in rats receiving a complete spinal cord transection at postnatal day 5 (P5) is attributable to changes in the lumbosacral neural circuitry and not to regeneration of axons across the lesion. As expected, stepping performance measured by several kinematics parameters was significantly better in ST (at P5) trained (treadmill stepping for 8 weeks) than age-matched non-trained spinal rats. Anterograde tracing with biotinylated dextran amine showed an absence of labeling of corticospinal or rubrospinal tract axons below the transection. Retrograde tracing with Fast Blue from the spinal cord below the transection showed no labeled neurons in the somatosensory motor cortex of the hindlimb area, red nucleus, spinal vestibular nucleus, and medullary reticular nucleus. Retrograde labeling transsynaptically via injection of pseudorabies virus (Bartha) into the soleus and tibialis anterior muscles showed no labeling in the same brain nuclei. Furthermore, re-transection of the spinal cord at or rostral to the original transection did not affect stepping ability. Combined, these results clearly indicate that there was no regeneration across the lesion after a complete spinal cord transection in neonatal rats and suggest that this is an important model to understand the higher level of locomotor recovery in rats attributable to lumbosacral mechanisms after receiving a complete ST at a neonatal compared to an adult stage.