Orexin A (or hypocretin 1)-immunoreactive neurons in the rat lateral hypothalamus project to several areas of the medulla oblongata that are closely associated with cardiovascular regulation. The present study was undertaken to further strengthen the hypothesis that orexin A accelerates cardiovascular response by activating sympathoexcitatory neurons in the rat rostral ventrolateral medulla (RVLM). First, immunohistochemical studies revealed the presence of orexin A-immunoreactive fibers in the RVLM. Double labeling the sections with orexin A- and tyrosine hydroxylase (TH)-antisera further showed that orexin A-immunoreactive fibers are in close proximity with TH-immunoreactive neurons, some of which may be barosensitive, bulbospinal neurons in the RVLM. Second, microinjection of orexin A (6.35, 12.7 and 38.1 μM) into the RVLM, which was verified later by histological examination, caused a significant increase of mean arterial pressure (MAP) and a moderate increase of heart rate (HR) in awake rats. L-glutamate (33.3 mM) injected into the same sites, caused a larger increase in MAP, but a decrease in HR; whereas, saline injection was without significant effect. Results from this study suggest that orexin A, which may be released from the nerve fibers originating from the neurons in the lateral hypothalamus, acting on RVLM neurons in the medulla, increases sympathetic outflow targeted to the heart and blood vessels in awake animals.

Maintenance of a stable arterial blood pressure is a complex physiological phenomenon. In addition to dysfunction of the blood vessels, alterations in homeostasis of circulating signals and humoral factors also contribute significantly to the development of hypertension. Recent evidence indicates that accumulation of the byproducts of cellular respiration, including superoxide anion (O2-) and/or hydrogen peroxide (H2O2), are contributing factors in pathophysiology of hypertension. With respect to the central nervous system, neurons in the rostral ventrolateral medulla (RVLM) play a pivotal role in neural regulation of blood pressure. RVLM neurons not only provide a tonic excitation to maintain the sympathetic vasomotor activity of the blood vessels, they also participate in baroreceptor reflex control of blood pressure. The notion that production of O2- and/or H2O2 in the RVLM participates in central control of blood pressure has recently gained major recognition in the area of hypertension study. Nonetheless, detailed insights into the mechanisms underlying O2- and/or H2O2 promoted hypertension remain to be elucidated. The hypothesis that forms the basis of this study is that enhanced level of O2- and/or H2O2 in the RVLM may be important factors for the manifestation of hypertension in the spontaneously hypertensive rats (SHR), an animal model of human essential hypertension. In comparison to normotensive Wistar-Kyoto (WKY) rats, basal level of O2- in the RVLM region of adult male SHR rats was significantly higher, along with a reduction in the expression of superoxide dismutase 1 (SOD1), SOD2 or catalase. SOD and catalase are enzymes that metabolize cellular O2- or H2O2 respectively. Pharmacologically, microinjection bilaterally into the RVLM of SOD mimetic, Tempol (50 nmol) or a pan SOD/calatase mimetic, FeTMPyP (100 nmol), significantly decreased mean systemic arterial pressure (MSAP) or heart rate (HR) in both SHR and WKY rats. The maximal hypotensive effect produced by Tempol or FeTMPyP was significantly greater in SHR than WKY rats. We also found that in SHR, but not WKY rats, the hypotensive and bradycardiac responses after microinjection bilaterally into the RVLM of FeTMPyP was significantly greater than that by Tempol. In addition, infection of RVLM neurons with adenoviral vector encoding SOD1 (Ad-SOD1), SOD2 (Ad-SOD2) or catalase (Ad-Catalase) gene (5x108 pfu) into the bilateral RVLM resulted in a long-term hypotensive effect in SHR but not WKY rats. The temporal profile of Ad-catalase-promoted hypotension was again longer than that promoted by Ad-SOD1 or Ad-SOD2 alone. At the molecular level, gene transfer of SOD1, SOD2 or catalase into the RVLM region of SHR or WKY rats specifically increased the expression of individual protein, resulting in a reduction in O2- level. Together these results suggest that accumulation of O2- and/or H2O2 in the RVLM is involved in the neural mechanism of hypertension in SHR.

We investigated the role of muscarinic receptor subtypes at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone, in mevinphos (Mev) intoxication. Adult Sprague-Dawley rats anesthetized by pentobarbital (45 mg/kg) and maintained by propofol (30 mg/kg/h) were used. Co-microinjection bilaterally of Mev (10 nmol) and artificial cerebrospinal fluid (aCSF) into the RVLM resulted in an increase (Phase I) followed by a decrease (Phase II) in the power density of the vasomotor components of systemic arterial pressure spectrum, our experimental index for sympathetic vasomotor tone. These changes in sympathetic vasomotor outflow in both phases of Mev intoxication were significantly and dose-dependently reduced on co-microinjection of Mev and the M2 subtype of muscarinic receptor (M2R) antagonist methoctramine (0.5 or 1 nmol) or M4R antagonist tropicamide (0.5 or 1 nmol). On the other hand, the M1R antagonist pirenzepine (0.5 or 1 nmol) or M3R antagonist 4-DAMP (0.5 or 1 nmol) was ineffective. Western blot analysis further revealed that the increase in NOS I protein levels at the RVLM during Phase I Mev intoxication or the augmented level of NOS II during both phases were significantly blunted on co-microinjection bilaterally of Mev and methoctramine (1 nmol) or tropicamide (1 nmol) into the RVLM. Pirenzepine (1 nmol) or 4-DMAP (1 nmol) was again ineffective. We conclude that both M2R and M4R subtypes in the RVLM may be involved in Mev intoxication. Whereas the prevalence of NOS I over NOS II at the RVLM during Phase I results in sympathoexcitation, sympathoinhibition induced by NO from NOS II in the RVLM is primarily involved in Phase II Mev intoxication.

The neuropeptides dynorphin and cholecystokinin (CCK), and their associated pronociceptive effects were investigated in the RVM. Utilizing a nerve-injury model (SNL), RT-PCR analysis revealed increases (p<0.05) of prodynorphin mRNA, and bradyinkin, B1- and B2-receptor mRNA, post-SNL, 14-days, 2-days, and 14-days, respectively. Administration of dynorphin into the RVM produced both acute and long-lasting (>30-days) tactile hypersensitivity. Administration of the B1-antagonist, DALBK and the B2-antagonist, Hoe-140, into the RVM significantly attenuated dynorphin-induced tactile hypersensitivity. Nerve-injury induced tactile hypersensitivity was significantly reversed by RVM administration of dynorphin antiserum or the B2-antagonist, Hoe-140. These data suggest that dynorphin is up-regulated in the RVM in nerve-injury, and via the activation of bradykinin receptors in the RVM, produces abnormal pain. Like dynorphin, CCK is up-regulated in the RVM in nerve-injury, with studies suggesting that elevated levels of CCK in the RVM mediate pronociceptive activity through CCK2 receptor activation, resulting in enhanced spinal nociceptive transmission. At present, it is unknown what key neurotransmitters are mediating this RVM CCK-driven effect at the level of the spinal cord. Here, spinal cerebrospinal fluid (CSF) levels of serotonin (5-HT) and prostaglandin E2 (PGE2) were measured in the lumbar spinal cord in naïve rats following CCK administration into the RVM. Following RVM CCK microinjection, an approximate 5-fold increase in spinal (CSF) PGE2 levels was observed, as compared to baseline controls. PGE2 levels showed a progressive increase with peak levels observed at the 80-minute post-CCK injection timepoint, whereas 5-HT levels in the spinal CSF remained unchanged following CCK administration into the RVM. This release of PGE2 coincided with the timecourse for CCK-induced mechanical hypersensitivity. Administration of the CCK2-antagonist YM022 prior to CCK into the RVM, significantly attenuated (>50%) the release of PGE2 in the spinal cord. The non-selective COX-inhibitor naproxen and the 5-HT3 antagonist ondansetron, both administered intrathecally, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. In summary, these data suggest a bradykinin- or CCK2-receptor antagonist could be used alone or in conjunction with current therapies in the treatment of chronic pain.

Sustained administration of morphine in humans and in animals induces a state of abnormal pain (i.e., hyperalgesia) which may be associated with the development of reduced analgesic efficacy (i.e., tolerance). Evidence suggests that opiate treatment may upregulate cholecystokinin (CCK), a pronociceptive peptide, in the brain and spinal cord. Therefore, we hypothesized that CCK may be upregulated by opiate treatment in the rostral ventromedial medulla (RVM) and to subsequently drive descending facilitation mechanisms to elicit hyperalgesia and antinociceptive tolerance in rats.CCK administered into the RVM of naive rats elicited hyperalgesia which was blocked by either RVM CCK2 receptor antagonist L365,260; or by bilateral lesion of dorsolateral funiculus, a major bulbospinal descending pain modulation pathway from the RVM to spinal cord.Sustained subcutaneous morphine induced hyperalgesia and spinal antinociceptive tolerance. Both effects were reversed by RVM CCK2 antagonist, suggesting that the up-regulation of the endogenous RVM CCK system played a critical role in the expression of these phenomena.Lesion of cells in the RVM which selectively express CCK2 receptors with a saporin construct (CCK-SAP) to inhibit ribosome activity, prevented morphine-induced hyperalgesia and spinal antinociceptive tolerance. These findings suggest that the integrity of the RVM CCK system is required for the development of hyperalgesia and antinociceptive tolerance induced by sustained morphine.The CCK system does not seem to play a role in setting the baseline sensory thresholds in normal rats because neither RVM L365,260 nor CCK-SAP treatment altered baseline sensory thresholds in naive rats.CCK appears to be present exclusively in nerve terminals of RVM neurons in naive rats. There was no obvious change in the levels of CCK-LI, CCK2 receptor, or CCK2 receptor mRNA in the RVM after sustained morphine treatment. However, microdialysis studies showed an approximately 5-fold increase in basal CCK levels in the RVM after sustained morphine treatment.Taken together, our results support the hypothesis that increased release of CCK in the RVM is induced by sustained morphine and drives descending facilitation to mediate morphine-induced paradoxical "pain" and spinal antinociceptive tolerance.

Huntington’s disease is an inherited neurodegenerative disease characterized by the degeneration of the cerebral cortex, thalamus, and striatum. The loss of neurons in the cerebral cortex and the thalamus may affect the synaptic circuitry in the striatum as these regions send glutamatergic projections (corticospinal & thalamostriatal) to neurons in the striatum. Prior studies have suggested the detrimental impact that the mutant Huntingtin protein (mHTT) may have on corticostriatal afferents, but less is known thalamic inputs to the dorsal striatum. In this study, we report a 50% reduction in thalamostriatal axospinous synapse density and significant reductions in dendritic spine volume at the ultrastructural level using electron microscopy. Additionally, dystrophic alterations to mitochondria size and morphology were also found. At the microcircuit level, we report a reduction in the spatial abundance of thalamostriatal axon terminals at the rostral, middle, and caudal levels of the dorsolateral striatum while an inverse distribution was observed for mHTT, suggesting a novel topographic distribution of thalamostriatal projections and mHTT along the rostral-caudal axis of the dorsolateral striatum. These findings are novel in the Q175 HD mouse model and supports the theory of an excitatory: inhibitory imbalance contributing to structural synaptic changes in the dorsal striatum. Further studies of the corticostriatal projections will determine the global extent of this imbalance.

Orexin A and B, also known as hypocretin 1 and 2, are two recently isolated hypothalamic peptides. As orexin-containing neurons are strategically located in the lateral hypothalamus, which has long been suspected to play an important role in feeding behaviors, initial studies were focused on the involvement of orexins in positive food intake and energy metabolism. Recent studies implicate a more diverse biological role of orexins, which can be manifested at different level of the neuraxis. For example, canine narcolepsy, a disorder with close phenotypic similarity to human narcolepsy, is caused by a mutation of hypocretin receptor 2 gene. Results from our immunohistochemical and functional studies, which will be summarized here, suggest that the peptide acting on neurons in the rostral ventrolateral medulla augment sympathoexcitatory outflow to the spinal cord. This finding is discussed in the context of increased sympathetic activity frequently associated with obesity.

Master of Science / Department of Anatomy and Physiology / Michael J. Kenney / Hyperthermia is an environmental stressor that produces marked increases in visceral sympathetic nerve discharge (SND) in young rats. The brainstem in rats contains the essential neural circuitry for mediating visceral sympathetic activation; however, specific brainstem sites involved remain virtually unknown. The rostral ventral lateral medulla (RVLM) is a key central nervous system region involved in the maintenance of basal SND and in mediating sympathetic nerve responses evoked from supraspinal sites. In the present study we tested the hypothesis that inhibition of RVLM synaptic activation at peak hyperthermia (internal body temperature, Tc, increased to 41.5°C) would affect heating-induced visceral sympathetic activation. Experiments were completed in chloralose-urethane anesthetized, baroreceptor-intact and sinoaortic-denervated, 3-6 month-old Sprague-Dawley rats. Bilateral inhibition of RVLM synaptic activation produced by muscimol microinjections (400 and 800 pmol) at 41.5°C resulted in immediate and significant reductions in peak heating-induced renal and splenic sympathoexcitation. Interruption of RVLM synaptic activation and axonal transmission by lidocaine microinjections (40 nmol) at 41.5°C produced significant reductions in hyperthermia-induced sympathetic activation to similar levels produced by RVLM muscimol microinjections. The total amount of SND inhibited by RVLM muscimol and lidocaine microinjections was significantly more during hyperthermia (41.5°C) than normothermia (38°C). These findings demonstrate that maintenance of sympathetic activation at peak hyperthermia is dependent on the integrity of RVLM neural circuits.

La migration des cellules souches / progénitrices neurales (CSPN) dans le cerveau adulte est cruciale pour la réparation cérébrale. Reeline (Rln) est une protéine de la matrice extracellulaire, régulant le positionnement des neurones pendant la croticogénèse. Nous révélons un rôle nouveau de Rln chez l'adulte. In vitro, Rln est chémocinétique mais pas chémoattractante. In vivo, Rln induit le détachement et la dispersion des CSNP de la zone sousventriculaire (SVZ) hors du courant rostral de migration (RMS) où elles sont sinon confinées. Rln potentialise le recrutement spontané des CSPN vers les lésions démyélinisantes où un tiers deviennent oligodendrocytaires. L'expression endogène de Rln est stimulée après lésion. Les animaux sans voie de signalisation Rln ont un recrutement réduit des CSPN vers les lésions.Ces résultats révèlent que Rln est un arbitre clef de la migration post-lésionnelle des CSPN et que permettre au CSPN de sortir du RMS est une stratégie thérapeutique prometteuse. / Neural stem/ progenitor cell (NSPC) migration in the adult brain is crucial for brain repair. Reelin (Rln) is an extracellular matrix protein regulating neuron positioning during coricogenesis. We reveal new roles of Rln in adult NSPC migration. In vitro, Rln promotes detachment, is chemokinetic but not chemoattractant. After Rln ectopic overexpression in the healthly brain, subventricular zone (SVZ) NSPC detach from the rostal migratory stream (RMS) in which they are normally restricted, and disperse in adjacent fiber tracts. Rln over-expression potentiates spontaneous cell recruitment to demyelinated lesion and one third of the NSPC recruited adopt an oligodendrocytic phenotype. Rln expression is spontaneously upregulated after lesion, and disruption of its signaling pathway results in reduced NSPC recruitment toward lesion. Our study reveals that Rln is a key player of post-lesional NSPC migration and that allowing NSPC to escape from RMS is a promising therapeutic approach

Chronic pain is a major healthcare concern that directly affects over one hundred million people in the United States alone. While current treatment options like opioids and NSAIDs are effective, they are with significant drawbacks that prevent long term use. It is important to identify and understand new druggable targets for the treatment of pain. Recent findings have demonstrated substance P functions in the RVM to maintain hypersensitivity to noxious heat stimuli in models of persistent peripheral inflammatory injury in a manner dependent on presynaptic NMDA receptors. What remains unclear is how substance P assumes this pronociceptive role following peripheral inflammatory injury. The experiments detailed in this thesis investigated whether the levels and or release of substance P in the RVM was altered following peripheral inflammatory injury. The effect of peripheral inflammatory injury on levels of substance P in the RVM was tested at several time points. The data show that there were no changes in substance P levels in the ipsilateral or contralateral RVM of CFA injected rats compared to their saline controls at any of the time points tested. To assess whether changes in substance P levels occurred in a subset of neurons within the RVM, computer aided densitometry analysis was used to measure substance P immunoreactivity in sections from the RVM of rats treated with CFA or saline. Substance P immunoreactivity was increased in the ipsilateral RVM of the CFA group compared to the corresponding saline sections at the 4 day, but not the 2 week time point. No other changes were observed. Electron microscopy was used to demonstrate the presence of the NMDA receptor and substance P on the same axon terminals within the RVMs of rats treated with either CFA or saline. This colocalization is significant because it identifies NMDA receptors in position to regulate the release of substance P from axon terminals in the RVM. There were no obvious differences in the degree of colocalization between CFA and saline groups. Functional experiments were devised that tested whether substance P release (basal and evoked) in the RVM was increased following peripheral inflammatory injury, and whether said release was regulated by NMDA receptors. The data show that neither basal nor evoked (potassium or veratridine) release was increased following peripheral inflammatory injury. NMDA was able to facilitate the release of substance P in both the CFA and saline treatment groups, but the facilitation was not different between groups. In the absence of any depolarization stimulus, NMDA was unable to elicit any release of substance P beyond basal values. All told, the data show substance P levels in the RVM are not altered by peripheral inflammatory injury. Additionally, neither basal nor evoked release of substance P is altered by peripheral inflammatory injury. The data provide functional and anatomical evidence for modulation of substance P release by glutamate acting at presynaptic NMDA receptors, but do not support the idea of differential modulation of substance P release following peripheral inflammatory injury.