COVID-19 and Neurological Impairment: Hypothalamic Circuits and Beyond.

In December 2019, a novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of Hubei, China. The virus infection, coronavirus disease 2019 (COVID-19), represents a global concern, as almost all countries around the world are affected. Clinical reports have confirmed several neurological manifestations in COVID-19 patients such as headaches, vomiting, and nausea, indicating the involvement of the central nervous system (CNS) and peripheral nervous system (PNS). Neuroinvasion of coronaviruses is not a new phenomenon, as it has been demonstrated by previous autopsies of severe acute respiratory syndrome coronavirus (SARS-CoV) patients who experienced similar neurologic symptoms. The hypothalamus is a complex structure that is composed of many nuclei and diverse neuronal cell groups. It is characterized by intricate intrahypothalamic circuits that orchestrate a finely tuned communication within the CNS and with the PNS. Hypothalamic circuits are critical for maintaining homeostatic challenges including immune responses to viral infections. The present article reviews the possible routes and mechanisms of neuroinvasion of SARS-CoV-2, with a specific focus on the role of the hypothalamic circuits in mediating the neurological symptoms noted during COVID-19 infection.

Testicular Atrophy and Hypothalamic Pathology in COVID-19: Possibility of the Incidence of Male Infertility and HPG Axis Abnormalities.

Coronavirus disease 2019 (COVID-19), which resulted from the pandemic outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes a massive inflammatory cytokine storm leading to multi-organ damage including that of the brain and testes. While the lungs, heart, and brain are identified as the main targets of SARS-CoV-2-mediated pathogenesis, reports on its testicular infections have been a subject of debate. The brain and testes are physiologically synchronized by the action of gonadotropins and sex steroid hormones. Though the evidence for the presence of the viral particles in the testicular biopsies and semen samples from COVID-19 patients are highly limited, the occurrence of testicular pathology due to abrupt inflammatory responses and hyperthermia has incresingly been evident. The reduced level of testosterone production in COVID-19 is associated with altered secretion of gonadotropins. Moreover, hypothalamic pathology which results from SARS-CoV-2 infection of the brain is also evident in COVID-19 cases. This article revisits and supports the key reports on testicular abnormalities and pathological signatures in the hypothalamus of COVID-19 patients and emphasizes that testicular pathology resulting from inflammation and oxidative stress might lead to infertility in a significant portion of COVID-19 survivors. Further investigations are required to monitor the reproductive health parameters and HPG axis abnormalities related to secondary pathological complications in COVID-19 patients and survivors.

Obesity induced by Borna disease virus in rats: key roles of hypothalamic fast-acting neurotransmitters and inflammatory infiltrates.

Human obesity epidemic is increasing worldwide with major adverse consequences on health. Among other possible causes, the hypothesis of an infectious contribution is worth it to be considered. Here, we report on an animal model of virus-induced obesity which might help to better understand underlying processes in human obesity. Eighty Wistar rats, between 30 and 60 days of age, were intracerebrally inoculated with Borna disease virus (BDV-1), a neurotropic negative-strand RNA virus infecting an unusually broad host spectrum including humans. Half of the rats developed fatal encephalitis, while the other half, after 3-4 months, continuously gained weight. At tripled weights, rats were sacrificed by trans-cardial fixative perfusion. Neuropathology revealed prevailing inflammatory infiltrates in the median eminence (ME), progressive degeneration of neurons of the paraventricular nucleus, the entorhinal cortex and the amygdala, and a strikingly high-grade involution of the hippocampus with hydrocephalus. Immune histology revealed that major BDV-1 antigens were preferentially present at glutamatergic receptor sites, while GABAergic areas remained free from BDV-1. Virus-induced suppression of the glutamatergic system caused GABAergic predominance. In the hypothalamus, this shifted the energy balance to the anabolic appetite-stimulating side governed by GABA, allowing for excessive fat accumulation in obese rats. Furthermore, inflammatory infiltrates in the ME and ventro-medial arcuate nucleus hindered free access of appetite-suppressing hormones leptin and insulin. The hormone transport system in hypothalamic areas outside the ME became blocked by excessively produced leptin, leading to leptin resistance. The resulting hyperleptinemic milieu combined with suppressed glutamatergic mechanisms was a characteristic feature of the found metabolic pathology. In conclusion, the study provided clear evidence that BDV-1 induced obesity in the rat model is the result of interdependent structural and functional metabolic changes. They can be explained by an immunologically induced hypothalamic microcirculation-defect, combined with a disturbance of neurotransmitter regulatory systems. The proposed mechanism may also have implications for human health. BDV-1 infection has been frequently found in depressive patients. Independently, comorbidity between depression and obesity has been reported, either. Future studies should address the exciting question of whether BDV-1 infection could be a link, whatsoever, between these two conditions.

Selective monoaminergic and histaminergic circuit dysregulation following long-term HIV-1 protein exposure.

Between 30 and 60% of HIV-seropositive individuals develop symptoms of clinical depression and/or apathy. Dopamine and serotonin are associated with motivational alterations; however, histamine is less well studied. In the present study, we used fast-scan cyclic voltammetry in HIV-1 transgenic (Tg) rats to simultaneously analyze the kinetics of nucleus accumbens dopamine (DA), prefrontal cortical serotonin (5-HT), and hypothalamic histamine (HA). For voltammetry, subjects were 15 HIV-1 Tg (7 male, 8 female) and 20 F344/N (11 male, 9 female) adult rats. Both serotonergic and dopaminergic release and reuptake kinetics were decreased in HIV-1 Tg animals relative to controls. In contrast, rates of histamine release and reuptake increased in HIV-1 Tg rats. Additionally, we used immunohistochemical (IHC) methods to identify histaminergic neurons in the tuberomammillary nucleus (TMN) of the hypothalamus. For IHC, subjects were 9 HIV-1 Tg (5 male, 4 female) and 9 F344/N (5 male, 4 female) adult rats. Although the total number of TMN histaminergic cells did not differ between HIV-1 Tg rats and F344/N controls, a significant sex effect was found, with females having an increased number of histaminergic neurons, relative to males. Collectively, these findings illustrate neurochemical alterations that potentially underlie or exacerbate the pathogenesis of clinical depression and/or apathy in HIV-1.

Zika Virus Infection in Hypothalamus Causes Hormone Deficiencies and Leads to Irreversible Growth Delay and Memory Impairment in Mice.

Zika virus (ZIKV) can cause microcephaly in the fetus. However, its effects on body growth and the development of children with postnatal ZIKV infection are largely unknown. To examine this, we intraperitoneally challenged mouse pups with ZIKV. Infection causes an irreversible growth delay and deficits in spatial learning and memory, with growth-relevant hormones significantly reduced during infection. These effects are associated with ZIKV RNA expression in the hypothalamus, blood, and brain but not in the pituitary and thyroid. Infection is also associated with hypothalamic inflammation, and ZIKV antigen is detectable in neuroendocrine cells producing thyrotropin-releasing hormone. Moreover, early administration of growth hormone could significantly improve growth delay. Our results demonstrate that ZIKV can infect the hypothalamus, causing multi-hormone deficiencies and delayed growth and development in a mouse model. Therefore, prospective multidisciplinary follow-up of ZIKV-infected children may be necessary to understand potential effects of this virus on childhood development.

Adenovirus-mediated suppression of hypothalamic glucokinase affects feeding behavior.

Glucokinase (GK), the hexokinase involved in glucosensing in pancreatic ß-cells, is also expressed in arcuate nucleus (AN) neurons and hypothalamic tanycytes, the cells that surround the basal third ventricle (3V). Several lines of evidence suggest that tanycytes may be involved in the regulation of energy homeostasis. Tanycytes have extended cell processes that contact the feeding-regulating neurons in the AN, particularly, agouti-related protein (AgRP), neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC) neurons. In this study, we developed an adenovirus expressing GK shRNA to inhibit GK expression in vivo. When injected into the 3V of rats, this adenovirus preferentially transduced tanycytes. qRT-PCR and Western blot assays confirmed GK mRNA and protein levels were lower in GK knockdown animals compared to the controls. In response to an intracerebroventricular glucose injection, the mRNA levels of anorexigenic POMC and CART and orexigenic AgRP and NPY neuropeptides were altered in GK knockdown animals. Similarly, food intake, meal duration, frequency of eating events and the cumulative eating time were increased, whereas the intervals between meals were decreased in GK knockdown rats, suggesting a decrease in satiety. Thus, GK expression in the ventricular cells appears to play an important role in feeding behavior.

Pseudo-typed Semliki Forest virus delivers EGFP into neurons.

Semliki Forest virus (SFV), a neurotropic virus, has been used to deliver heterologous genes into cells in vitro and in vivo. In this study, we constructed a reporter SFV4-FL-EGFP and found that it can deliver EGFP into neurons located at the injection site without disseminating throughout the brain. Lacking of the capsid gene of SFV4-FL-EGFP does not block its life cycle, while forming replication-competent virus-like particles (VLPs). These VLPs hold subviral genome by using the packaging sequence (PS) located within the nsP2 gene, and can transfer their genome into cells. In addition, we found that the G protein of vesicular stomatitis virus (VSVG) can package SFV subviral genome, which is consistent with the previous reports. The G protein of rabies virus (RVG) could also package SFV subviral genome. These pseudo-typed SFV can deliver EGFP gene into neurons. Taken together, these findings may be used to construct various SFV-based delivery systems for virological studies, gene therapy, and neural circuit labeling.

H1N1 influenza virus induces narcolepsy-like sleep disruption and targets sleep-wake regulatory neurons in mice.

An increased incidence in the sleep-disorder narcolepsy has been associated with the 2009-2010 pandemic of H1N1 influenza virus in China and with mass vaccination campaigns against influenza during the pandemic in Finland and Sweden. Pathogenetic mechanisms of narcolepsy have so far mainly focused on autoimmunity. We here tested an alternative working hypothesis involving a direct role of influenza virus infection in the pathogenesis of narcolepsy in susceptible subjects. We show that infection with H1N1 influenza virus in mice that lack B and T cells (Recombinant activating gene 1-deficient mice) can lead to narcoleptic-like sleep-wake fragmentation and sleep structure alterations. Interestingly, the infection targeted brainstem and hypothalamic neurons, including orexin/hypocretin-producing neurons that regulate sleep-wake stability and are affected in narcolepsy. Because changes occurred in the absence of adaptive autoimmune responses, the findings show that brain infections with H1N1 virus have the potential to cause per se narcoleptic-like sleep disruption.

Contributions of HIV infection in the hypothalamus and substance abuse/use to HPT dysregulation.

Over the last two decades, consequences of HIV infection of the CNS on disease severity and clinical neuropsychiatric manifestations have changed. These changes are due, in part, to improved control of peripheral infection by new anti-retroviral medications and more efficient CNS penetration of combination anti-retroviral therapies (cART). While the life spans of HIV-infected patients have been prolonged with successful cART, the spectrum of cognitive alterations observed in these patients has broadened. Recent studies report that there does not appear to be a single prototypical pattern of neuropsychological impairment associated with HIV, but includes diverse manifestations. Some co-morbidities, such as substance abuse or depression likely play significant roles in the neuropsychiatric profiles of some HIV-infected patients. Newly recognized factors contributing to neurocognitive impairments include aging and unanticipated side effects from cART. Likewise, disturbances in neuroendocrine functioning are emerging as potentially important contributors to HIV-associated neurocognitive alterations. A retrospective review of clinical data from a small cohort of HIV-infected patients admitted to the psychiatric unit of an inner city hospital indicates that thyroid stimulating hormone levels were abnormal in 27% of the patients. Our data from analyses of post-mortem tissues from HIV patients show for the first time HIV infection of the hypothalamus and altered levels of thyroid hormone processing enzymes. Decreased vasopressin and oxytocin immunoreactivity in hypothalamic neurons was also observed. Thus, HIV infection of the CNS may contribute to changes in hypothalamic thyroid hormone signaling, thereby resulting in abnormal hypothalamic-pituitary-thyroid axis feedback and neuropsychiatric dysfunction.

Hypothalamic abnormalities and Parkinsonism associated with H1N1 influenza infection.

OBJECTIVE: To describe a case of a young adult with severe H1N1 influenza illness associated with hypothalamic abnormalities and post-influenza parkinsonism. DESIGN: Case report. PATIENT: A 22-year-old woman with H1N1 influenza infection developed encephalopathy followed by diverse hypothalamic dysfunction manifestations, sleeplessness, and persistent parkinsonian features. RESULTS: CSF analysis, brain imaging and EEG ruled out hypoxic brain injury or other illnesses. CONCLUSIONS: A number of viruses have been associated with both acute and chronic parkinsonism. A link between parkinsonism and influenza viruses is somewhat controversial. This is the first reported case of parkinsonism following an H1N1 influenza infection.

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 trajectory of sensory pathways from the lamina terminalis to the insular and cingulate cortex: a neuroanatomical framework for the generation of thirst.

The pathways involved in the emotional aspects of thirst, the arousal and affect associated with the generation of thirst and the motivation to obtain satiation, have been studied but remain poorly understood. Rats were therefore injected with the neurotropic virus pseudorabies in either the insular or cingulate cortex. After 2 days of infection, pseudorabies-positive neurons were identified within the thalamus and lamina terminalis. In a separate group of rats, the retrograde tracer cholera toxin subunit b (CTb) was used in combination with either isotonic (0.15 M NaCl) or hypertonic (0.8 M NaCl) saline (1 ml/100 g body wt ip). Rats injected with CTb in the insular cortex and stimulated with hypertonic saline had increased numbers of Fos/CTb double-positive neurons in the paraventricular, rhomboid, and reuniens thalamic nuclei, whereas those rats injected with CTb in the cingulate cortex and challenged with hypertonic saline had increased numbers of Fos/CTb double-positive neurons in the medial part of the mediodorsal, interanteromedial, anteromedial, and ventrolateral part of the laterodorsal thalamic nuclei. Rats injected with CTb in the dorsal midline of the thalamus and challenged with hypertonic saline had increased numbers of Fos/CTb double-positive neurons within the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and insular cortex but not the subfornical organ. A small proportion of the CTb-positive neurons in the OVLT were immunopositive for transient receptor potential vanilloid 1, a putative osmoresponsive membrane protein. These results identify functional thalamocortical pathways involved in relaying osmotic signals to the insular and cingulate cortex and may provide a neuroanatomical framework for the emotional aspects of thirst.

Differential effects of recombinant adeno-associated virus-mediated neuropeptide Y overexpression in the hypothalamic paraventricular nucleus and lateral hypothalamus on feeding behavior.

It is well known that neuropeptide Y (NPY) increases food intake. The hypothalamic paraventricular nucleus (PVN) and the lateral hypothalamus (LH) are both involved in the acute, hyperphagic effects of NPY. Although it is obvious that increased energy intake may lead to obesity, it is less understood which aspects of feeding behavior are affected and whether one or multiple neural sites mediate the effects of long-term increased NPY signaling. By long-term overexpressing NPY in either the PVN or the LH, we uncovered brain site-specific effects of NPY on meal frequency, meal size, and diurnal feeding patterns. In rats injected with adeno-associated virus-NPY in the PVN, increased food intake resulted from an increase in the amount of meals consumed, whereas in rats injected in the LH, increased food intake was attributable to increased meal size. Interestingly, food intake and body weight gain were only temporarily increased in PVN-injected rats, whereas in LH-injected rats hyperphagia and body weight gain remained for the entire 50 d. Moreover, in LH-NPY rats, but not in PVN-NPY rats, diurnal rhythmicity with regard to food intake and body core temperature was lost. These data clearly show that the NPY system differentially regulates energy intake and energy expenditure in the PVN and LH, which together adjust energy balance.

Lentivirus-mediated downregulation of hypothalamic insulin receptor expression.

Regulation of feeding behavior and energy balance are among the central effects of insulin. For example, intracerebroventricular administration of insulin decreases food intake and body weight, whereas antisense oligodeoxynucleotide downregulation of insulin receptors (IRs) produces hyperphagia. To further examine the role of IRs in the central actions of insulin, we designed an IR antisense lentiviral vector (LV-IRAS) and injected this vector into the third ventricle to selectively decrease IR expression in the rat hypothalamus. Three weeks after LV-IRAS administration, the expression of IRs in the hypothalamus was significantly decreased, whereas no changes were observed in hippocampal IR levels. LV-IRAS administration decreased insulin-stimulated phosphorylation of hypothalamic IRs and translocation of the insulin-sensitive glucose transporter GLUT4 in the hypothalamus; no changes in IR signaling were observed in the hippocampus of LV-IRAS-treated rats. Lentivirus-mediated downregulation of IR expression and signaling produced significant increases in body weight, as well as increases in fat mass that were selective for the subcutaneous compartment. Conversely, lean muscle mass and water mass were not affected in LV-IRAS-treated rats compared to rats treated with control virus. Changes in peripheral adiposity were associated with increases in basal hypothalamic leptin signaling in the absence of changes in leptin receptor expression in LV-IRAS rats. Collectively, these data illustrate the important functional relationships between hypothalamic insulin and leptin signaling in the regulation of body composition and provide insight into the mechanisms through which decreases in IR expression and signaling dysregulates leptin activity, thereby promoting increases in peripheral adiposity.

Human herpesvirus 6 meningoencephalitis in allogeneic hematopoietic stem cell transplant recipients.

We retrospectively investigated the clinical characteristics of human herpesvirus 6 (HHV-6) meningoencephalitis within 100 days after allogeneic hematopoietic stem cell transplantation (HSCT). Of 1148 patients who received transplants between January 1999 and December 2003, 11 patients (0.96%) with HHV-6 meningoencephalitis were identified. Ten of 11 recipients received hematopoietic stem cells from donors other than HLA-identical siblings. Confusion was the most frequent central nervous system (CNS) symptom, and a skin rash with high-grade fever preceded the CNS symptoms in 9 patients. Magnetic resonance imaging of the brain showed an abnormal increased T2 signal in the hypothalamus of 5 patients. Eight patients were treated with ganciclovir, and an improvement of CNS symptoms was obtained in 3 patients; 3 patients treated with acyclovir showed no improvement. Improvement in the meningoencephalitis seemed less frequent in patients with abnormal findings in the hypothalamus than in those without such findings. Because the symptoms of HHV-6 meningoencephalitis mimicked those of cyclosporine- or tacrolimus-induced encephalopathy, the drugs were withdrawn at the onset of CNS symptoms in 10 patients, resulting in the development of grade IV graft-versus-host disease (GVHD) in 5 patients. Three patients died of HHV-6 meningoencephalitis, and 6 died of other causes, including GVHD. In conclusion, HHV-6 meningoencephalitis is a rare but potentially life-threatening complication in patients who undergo allogeneic HSCT. Careful assessment of the clinical findings and the brain may allow early and precise diagnosis of HHV-6 meningoencephalitis and contribute to improving its prognosis.

Olfactory inputs to hypothalamic neurons controlling reproduction and fertility.

In order to gain insight into sensory processing modulating reproductive behavioral and endocrine changes, we have aimed at identifying afferent pathways to neurons synthesizing luteinizing hormone-releasing hormone (LHRH, also known as gonadotropin-releasing hormone [GnRH]), a key neurohormone of reproduction. Injection of conditional pseudorabies virus into the brain of an LHRH::CRE mouse line led to the identification of neuronal networks connected to LHRH neurons. Remarkably, and in contrast to established notions on the nature of LHRH neuronal inputs, our data identify major olfactory projection pathways originating from a discrete population of olfactory sensory neurons but fail to document any synaptic connectivity with the vomeronasal system. Accordingly, chemosensory modulation of LHRH neuronal activity and mating behavior are dramatically impaired in absence of olfactory function, while they appear unaffected in mouse mutants lacking vomeronasal signaling. Further visualization of afferents to LHRH neurons across the brain offers a unique opportunity to uncover complex polysynaptic circuits modulating reproduction and fertility.

Structural and functional evidence supporting a role for leptin in central neural pathways influencing blood pressure in rats.

Leptin, a peptide hormone normally associated with body weight homeostasis, is implicated in the generation of obesity-induced hypertension. Administration of leptin increases sympathetic nerve activity and blood pressure; however, the neural circuity involved in this pressor effect is not clearly defined. In this review we describe experiments in which pseudorabies virus was injected into the heart, kidney and the vasculature within skeletal muscle to reveal the distribution of neurones in the hypothalamus that project to these cardiovascular tissues. This distribution is compared to the well-documented distribution of leptin receptors. Finally we discuss microinjection studies designed to examine the effect of leptin, in these regions, on sympathetic nerve discharge and arterial blood pressure. Leptin injected directly into the ventromedial hypothalamus, arcuate nucleus and lateral hypothalamic area (particularly the perifornical area) increased lumbar sympathetic nerve activity. In addition, microinjection into the ventromedial hypothalamus and parvocellular paraventricular nucleus increased blood pressure. Our results demonstrate a discrete set of hypothalamic pathways that may underlie the involvement of leptin in obesity-induced hypertension.

Specific alteration of the expression of selected hypothalamic neuropeptides during acute and late mouse brain infection using a morbillivirus: relevance to the late-onset obesity?

Neurotropic viruses are involved in pathologies of the central nervous system, triggering transient or irreversible disorders, such as neurological diseases or homeostasis imbalance. In experimental animals, viruses have been shown to cause obesity, a complex disease depending on multiple factors, including genetic susceptibility and environmental components. Using a mouse model of virally induced obesity following brain infection by the Canine Distemper Virus (CDV), a morbillivirus closely related to the human measles virus, we investigated the modulation of expression of several hypothalamic neuropeptides known to intervene in the regulation of body weight and energy expenditure, both during the acute and late stages of infection. During the acute stage, while viral replication occurs, we found a dramatic decrease of expressions of neuropeptides, in particular neuropeptide Y, melanin-concentrating hormone (MCH), hypocretin, vasopressin and tachykinins, the magnitude of which seemed to be linked to the viral burden and the individual susceptibility. The effect of the virus, however, varied with the hypothalamic nucleus and neuropeptide involved, suggesting that certain circuits were affected while others remained intact. During the late stage of infection, marked recovery to the initial hypothalamic levels of peptide expression was seen in a number of lean animals, suggesting recovery of homeostasis equilibrium. Interestingly, some neuropeptidergic systems remained disturbed in mice exhibiting obese phenotype, arguing for their involvement in triggering/maintaining obesity. Even though our data could not fully explain the viral-induced obesity, they may be helpful in understanding the molecular events associated with obesity and in investigating therapeutic alternatives.

Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons.

Analysis of gene regulatory sequences in primary cultures of neurons has been hampered by inefficient transfection of post-mitotic neurons with reporter plasmids. We describe detailed conditions that allowed a significant improvement of transfection efficiency in primary cultures of serum-supplemented rat fetal hypothalamic cells. Transfected cells expressed the green fluorescent protein (GFP) under the control of the strong but non-cell-specific cytomegalo virus (CMV) promoter or under the thyrotropin-releasing hormone (TRH) promoter, to direct expression only in TRH neurons. Using the CMV promoter-GFP plasmid, we tested several commercially available transfection reagents; the best results were obtained with polyethylenimine (PEI) and Lipofectamine 2000. We optimized the transfection procedure with PEI because it rendered more reproducible results. Transfection with PEI was optimal when cells were transfected at a cellular density of 2.9 x 10(6) cells in 35-mm dishes, with 10 microg of DNA, a PEI/DNA ratio of 8.8 and PEI pH of 6.9. Using these conditions, we were able to detect GFP positive neurons after transfecting the TRH promoter-GFP plasmid. GFP positive cells were successfully purified by FACS. This opens the possibility to use transfection of mammalian CNS post-mitotic neurons for new applications including the purification of specific neuronal subtypes.

Hypothalamic co-localization of substance P receptor and transneuronal tracer from the lordosis-relevant lumbar epaxial muscles in the female rat.

Previous studies indicated that within the ventromedial hypothalamus (VMH), the estrogen receptor alpha (ERalpha)-containing neurons express substance P (SP), but do not comprise the majority of projection neurons. The present study tested the hypothesis that projection neurons within the VMH express SP receptors (NK1), allowing responsiveness to signals from ERalpha-containing neurons. Pseudorabies virus was transneuronally transported from the lordosis-relevant lumbar epaxial muscles to the VMH, labeling 28% of the NK1-containing neurons in the VMH and surrounding area. Thus, SP may influence sexual behavior through its release from the ERalpha-containing neurons, perhaps synaptically affecting NK1 receptor-labeled lordosis-relevant projection neurons within the VMH.