Cleusa V.R. de Oliveira

Cardioacceleratory responses to hypocretin-1 injections into rostral ventromedial medulla

Intracisternal injections of hypocretin-1 (hcrt-1) have been shown to elicit sympathoexciatory responses. However, the location of central sites that may mediate these cardiovascular effects have not been clearly elucidated. This study was done in male Wistar rats to investigate the effects of microinjections of hcrt-1 into the rostral ventromedial medulla (RVMM) on mean arterial pressure (MAP), heart rate (HR) and the arterial baroreflex. An initial series of experiments was done to provide a detailed mapping of the location of hcrt-1- and hcrt-1 receptors (hcrtR-1)-like immunoreactivity (i.r.) in the RVMM region. Hcrt-1 and hcrtR-1 ir were found throughout the RVMM region, but primarily within the magnocellular reticular nucleus and the adjacent nucleus paragigantocellularis lateralis. In the second series, this region containing hcrt-1 and hcrtR-1 ir was explored for sites that elicited changes in MAP and HR in the anaesthetized rat. Microinjection of hcrt-1 (0.5-2.5 pmol) into the region of magnocellular reticular nucleus elicited a dose-dependent increase in HR, with little or no change in MAP. Administration (i.v.) of the muscarinic receptor antagonist atropine methyl bromide significantly attenuated ( approximately 62%) the HR response whereas, the total autonomic blockade abolished the HR response. Finally, unilateral or bilateral microinjection of hcrt-1 into the magnocellular reticular nucleus significantly attenuated the reflex bradycardia resulting from the activation of the baroreflex following the increase in MAP from an iv injection of phenylephrine. These data suggest that hcrt-1 in the RVMM region activates neuronal circuits that both inhibit vagal activity and increase sympathetic activity to the heart, and that it alters the excitability of central circuits that reflexly control the circulation.

Collateral axonal projections from hypothalamic hypocretin neurons to cardiovascular sites in nucleus ambiguus and nucleus tractus solitarius

Hypocretin-1 (hcrt-1)-containing axons have been shown to have an extensive distribution within the central nervous system, although the total number of hypothalamic hcrt-1 neurons has been shown to be small. This suggests that hcrt-1 neurons may innervate central structures with similar function through collateral axonal projections. Retrograde tract-tracing techniques combined with immunohistochemistry were used in this study to investigate whether hypothalamic hcrt-1-containing neurons send collateral axonal projections to cardiovascular sites in the nucleus of the solitary tract (NTS) and in the nucleus ambiguus (Amb) in the rat. Fluorogold- (FG) and/or rhodamine (Rd)-labeled latex microspheres were microinjected into either the NTS or Amb at sites that elicited bardycardia responses (L-glutamate; 0.25 M; 10 nl). After a survival period of 10-15 days, the rats were sacrificed and tissue sections of the hypothalamus were processed immunohistochemically for the identification of hcrt-1-containing cell bodies. After injection of the tract-tracers into the NTS or Amb, retrogradely labeled neurons were observed within several hypothalamic regions; the paraventricular hypothalamic nucleus, lateral hypothalamic area, perifornical hypothalamic area, and posterior hypothalamus, bilaterally, but with an ipsilateral predominance. In addition, after NTS injections, retrogradely labeled neurons were found within the ipsilateral caudal arcuate nucleus. Of the total number (1107+/-97) of hcrt-1-immunoreactive neurons found bilaterally within the lateral and perifornical hypothalamic nuclei, 7.9+/-1.4% were found to be retrogradely labeled from the NTS, 16.4+/-1.8% from the Amb, and 3.1+/-0.5% from both medullary sites. Hcrt-1 neurons projecting to the NTS were found mainly in and around the perifornical hypothalamic region, with a smaller number in the caudal lateral hypothalamic area. On the other hand, those innervating the Amb were primarily observed within the caudal lateral hypothalamic area, with a smaller number in the perifornical hypothalamic area. Neurons with collateral axonal projections to NTS and Amb were observed within two specific hypothalamic areas: one group of neurons was found in the perifornical hypothalamic area, and the other was observed in the lateral hypothalamic region just dorsal to the retrochiasmatic component of the supraoptic nucleus. These data indicate that axons from hcrt-1 neurons bifurcate to innervate functionally similar cardiovascular-responsive sites in the NTS and Amb. Although the function of these hcrt-1-containing hypothalamic-medullary pathways is not known, they likely represent the anatomical substrate by which the lateral hypothalamic hcrt-1 neurons simultaneously coordinate autonomic-cardiovascular responses to different behaviors.

Identification of neurons containing orexin-B (hypocretin-2) immunoreactivity in limbic structures.

Orexins (hypocretins) are neuropeptides which have recently been identified exclusively within lateral hypothalamic and perifornical neurons, and these orexin (ox) containing neurons appear to have extensive projections to all levels of the neuraxis. In this study, we report the identification of two distinct clusters of neurons containing ox-B-like immunoreactivity within the amygdaloid complex of the rat. A cluster of small to medium size ovoid shaped neurons containing ox-B-like immunoreactivity was found predominantly within the lateral division of the central nucleus of the amygdala (ACe). A second distinct, but smaller group of ox-B labelled neurons with similar shapes and sizes to those in ACe was also identified in the anterior lateral subnucleus of the bed nucleus of the stria terminalis (BST) immediately adjacent the internal capsule, and in an area just ventral to the lateral ventricle. Neurons containing ox-A-like immunoreactivity were not observed in either structure. However, both structures contained ox-A- and ox-B labelled varicose fibers. Unilateral electrolytic lesions of the lateral hypothalamic area that contained ox-A and ox-B neurons did not alter the labelling of either ACe or BST ox-B pericarya. As both the ACe and BST are known to be involved in integrating complex homeostatic mechanisms associated with behaviours, these data suggest that a specific subset of ox-B neurons within the amygdaloid complex may serve as a component of neuronal circuits coordinating these responses.

A Pivotal Role of Lumbar Spinothalamic Cells in the Regulation of Ejaculation via Intraspinal Connections

INTRODUCTION A population of lumbar spinothalamic cells (LSt cells) has been demonstrated to play a pivotal role in ejaculatory behavior and comprise a critical component of the spinal ejaculation generator. LSt cells are hypothesized to regulate ejaculation via their projections to autonomic and motor neurons in the lumbosacral spinal cord. AIM The current study tested the hypothesis that ejaculatory reflexes are dependent on LSt cells via projections within the lumbosacral spinal cord. METHODS Male rats received intraspinal injections of neurotoxin saporin conjugated to substance P analog, previously shown to selectively lesion LSt cells. Two weeks later, males were anesthetized and spinal cords were transected. Subsequently, males were subjected to ejaculatory reflex paradigms, including stimulation of the dorsal penile nerve (DPN), urethrogenital stimulation or administration of D3 agonist 7-OH-DPAT. Electromyographic recordings of the bulbocavernosus muscle (BCM) were analyzed for rhythmic bursting characteristic of the expulsion phase of ejaculation. In addition, a fourth commonly used paradigm for ejaculation and erections in unanesthetized, spinal-intact male rats was utilized: the ex copula reflex paradigm. MAIN OUTCOME MEASURES LSt cell lesions were predicted to prevent rhythmic bursting of BCM following DPN, urethral, or pharmacological stimulation, and emissions in the ex copula paradigm. In contrast, LSt cell lesions were not expected to abolish erectile function as measured in the ex copula paradigm. RESULTS LSt cell lesions prevented rhythmic contractions of the BCM induced by any of the ejaculatory reflex paradigms in spinalized rats. However, LSt cell lesions did not affect erectile function nor emissions determined in the ex copula reflex paradigm. CONCLUSIONS These data demonstrate that LSt cells are essential for ejaculatory, but not erectile reflexes, as previously reported for mating animals. Moreover, LSt cells mediate ejaculation via projections within the spinal cord, presumably to autonomic and motor neurons.

Estrogen alters the bradycardia response to hypocretin-1 in the nucleus tractus solitarius of the ovariectomized female

Experiments were performed to investigate the effect of 17beta-estradiol (E; 30 pg/ml plasma) treatment (15-25 days) in the ovariectomized (OVX) female Wistar rat on the cardiovascular responses to hypocretin-1 (hcrt-1) in the nucleus tractus solitarius (NTS). In an initial series of experiments, the distribution of hcrt-1-like immunoreactivity within the region of the NTS was mapped in both OVX only and OVX+E animals. Hcrt-1 immunoreactivity was found throughout the NTS region in both groups of females, predominantly within the caudal interstitial, commissural, medial and lateral subnuclei of the NTS. The relative density of hcrt-1 immunoreactivity in all NTS subnuclei was similar in both female groups. Microinjections of hcrt-1 (0.5-10 pmol) into the caudal lateral and medial subnuclei of the NTS complex of the alpha-chloralose of the urethane-anaesthetized E-treated OVX rat elicited a dose-related decrease in heart rate (HR). On the other hand, although a dose-response effect on arterial pressure was evident, significant arterial pressure responses were observed only at the higher dose of hcrt-1 (>2.5 pmol). In the OVX only female rat, microinjection of hcrt-1 into similar NTS sites elicited a bradycardia and depressor response only at the highest dose of hcrt-1, and these responses were significantly smaller in magnitude than those elicited in the OVX+E animal. In addition, in the OVX only animals, a few sites within the caudal commissural subnucleus of the NTS complex were found at which hcrt-1 elicited tachycardia and pressor responses. Finally, it was found that the reflex bradycardia to the activation of arterial baroreceptors as a result of increasing systemic arterial pressure with phenylephrine (2-4 microg/kg) was significantly potentiated in the OVX+E animals only. These data suggest that hcrt-1 in the NTS of the female activates a neuronal circuit that controls the circulation and that the circulating level of E alters the sensitivity of these cardiovascular circuits to hcrt-1.

Activation of NMDA Receptors in Lumbar Spinothalamic Cells is Required for Ejaculation

INTRODUCTION The sexual reflex ejaculation is controlled by a spinal ejaculation generator located in the lumbosacral spinal cord. A population of spinothalamic (LSt) neurons forms a key component of this generator, as manipulations of LSt cells either block or trigger ejaculation. However, it is currently unknown which afferent signals contribute to the activation of LSt cells and ejaculation. AIM The current study tested the hypothesis that glutamate, via activation of N-Methyl-D-aspartic acid (NMDA) receptors in LSt cells, is a key regulator of ejaculation. METHODS Expression of phosphorylated NMDA receptor subunit 1 (NR1) was investigated following mating, or following ejaculation induced by electrical stimulation of the dorsal penile nerve (DPN) in anesthetized, spinalized male rats. Next, the effects of intraspinal delivery of NMDA receptor antagonist AP-5 on DPN stimulation-induced ejaculation were examined. Moreover, the ability of intraspinal delivery of NMDA to trigger ejaculation was examined. Finally, the site of action of NMDA was determined by studying effects of NMDA in male rats with LSt cell-specific lesions. MAIN OUTCOME MEASURES Expression of NR1 and phosphorylated NR1 in LSt cells was analyzed. Electromyographic recordings of the bulbocavernosus muscle (BCM) were recorded in anesthetized, spinalized rats following stimulation of the DPN and delivery of AP-5 or NMDA. RESULTS Results indicate that the NR1 receptors are activated in LSt cells following ejaculation in mating animals or induced by DPN stimulation in anesthetized, spinalized animals. Moreover, NR1 activation in LSt cells is an essential trigger for rhythmic BCM bursting, as DPN stimulation-induced reflexes were absent following administration of NMDA receptor antagonist in the L3-L4 spinal area, and were triggered by NMDA. NMDA effects were dependent on intact LSt cells and were absent in LSt-lesioned males. CONCLUSION These results demonstrate that glutamate, via activation of NMDA receptors in LSt cells, is a key afferent signal for ejaculation.

Adolescent Cannabinoid Exposure Induces a Persistent Sub-Cortical Hyper-Dopaminergic State and Associated Molecular Adaptations in the Prefrontal Cortex

Abstract Considerable evidence suggests that adolescent exposure to delta‐9‐tetrahydrocanabinol (THC), the psychoactive component in marijuana, increases the risk of developing schizophrenia‐related symptoms in early adulthood. In the present study, we used a combination of behavioral and molecular analyses with in vivo neuronal electrophysiology to compare the long‐term effects of adolescent versus adulthood THC exposure in rats. We report that adolescent, but not adult, THC exposure induces long‐term neuropsychiatric‐like phenotypes similar to those observed in clinical populations. Thus, adolescent THC exposure induced behavioral abnormalities resembling positive and negative schizophrenia‐related endophenotypes and a state of neuronal hyperactivity in the mesocorticolimbic dopamine (DA) pathway. Furthermore, we observed profound alterations in several prefrontal cortical molecular pathways consistent with sub‐cortical DAergic dysregulation. Our findings demonstrate a profound dissociation in relative risk profiles for adolescent versus adulthood exposure to THC in terms of neuronal, behavioral, and molecular markers resembling neuropsychiatric pathology.

Cannabidiol Counteracts Amphetamine-Induced Neuronal and Behavioral Sensitization of the Mesolimbic Dopamine Pathway through a Novel mTOR/p70S6 Kinase Signaling Pathway

Schizophrenia-related psychosis is associated with disturbances in mesolimbic dopamine (DA) transmission, characterized by hyperdopaminergic activity in the mesolimbic pathway. Currently, the only clinically effective treatment for schizophrenia involves the use of antipsychotic medications that block DA receptor transmission. However, these medications produce serious side effects leading to poor compliance and treatment outcomes. Emerging evidence points to the involvement of a specific phytochemical component of marijuana called cannabidiol (CBD), which possesses promising therapeutic properties for the treatment of schizophrenia-related psychoses. However, the neuronal and molecular mechanisms through which CBD may exert these effects are entirely unknown. We used amphetamine (AMPH)-induced sensitization and sensorimotor gating in rats, two preclinical procedures relevant to schizophrenia-related psychopathology, combined with in vivo single-unit neuronal electrophysiology recordings in the ventral tegmental area, and molecular analyses to characterize the actions of CBD directly in the nucleus accumbens shell (NASh), a brain region that is the current target of most effective antipsychotics. We demonstrate that Intra-NASh CBD attenuates AMPH-induced sensitization, both in terms of DAergic neuronal activity measured in the ventral tegmental area and psychotomimetic behavioral analyses. We further report that CBD controls downstream phosphorylation of the mTOR/p70S6 kinase signaling pathways directly within the NASh. Our findings demonstrate a novel mechanism for the putative antipsychotic-like properties of CBD in the mesolimbic circuitry. We identify the molecular signaling pathways through which CBD may functionally reduce schizophrenia-like neuropsychopathology. SIGNIFICANCE STATEMENT The cannabis-derived phytochemical, cannabidiol (CBD), has been shown to have pharmacotherapeutic efficacy for the treatment of schizophrenia. However, the mechanisms by which CBD may produce antipsychotic effects are entirely unknown. Using preclinical behavioral procedures combined with molecular analyses and in vivo neuronal electrophysiology, our findings identify a functional role for the nucleus accumbens as a critical brain region whereby CBD can produce effects similar to antipsychotic medications by triggering molecular signaling pathways associated with the effects of classic antipsychotic medications. Specifically, we report that CBD can attenuate both behavioral and dopaminergic neuronal correlates of mesolimbic dopaminergic sensitization, via a direct interaction with mTOR/p70S6 kinase signaling within the mesolimbic pathway.

Activation of MAP Kinase in Lumbar Spinothalamic Cells Is Required for Ejaculation

INTRODUCTION Ejaculation is a reflex controlled by a spinal ejaculation generator located in the lumbosacral spinal cord responsible for the coordination of genital sensory with autonomic and motor outputs that regulate ejaculation. In the male rat, a population of lumbar spinothalamic cells (LSt cells) comprises an essential component of the spinal ejaculation generator. LSt cells are activated with ejaculation, but the nature of the signal transduction pathways involved in this activation is unknown. Moreover, it is unknown if LSt cell activation is required for expression of ejaculation. AIM The current study tested the hypothesis that ejaculatory reflexes are triggered via activation of the mitogen-activated protein (MAP) kinase signaling pathway in the LSt cells. METHODS Expression of phosphorylated extracellular signal-related kinases 1 and 2 (pERK) was investigated following mating behavior, or following ejaculation induced by electrical stimulation of the dorsal penile nerve (DPN) in anesthetized, spinalized male rats. Next, the effects of intrathecal or intraspinal delivery of Mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor U0126 on DPN stimulation-induced ejaculation was examined. MAIN OUTCOME MEASURES Expression of pERK in LSt cells and associated areas was analyzed. Electromyographic recordings of the bulbocavernosus muscle were recorded in anesthetized, spinalized rats. RESULTS Results indicate that the MAP kinase signaling pathway is activated in LSt cells following ejaculation in mating animals or induced by DPN stimulation in anesthetized, spinalized animals. Moreover, ERK activation in LSt cells is an essential trigger for ejaculation, as DPN stimulation-induced reflexes were absent following administration of MEK inhibitor in the L3-L4 spinal area. CONCLUSION These data provide insight into the nature of the signal transduction pathways involved in the activation of ejaculation through LSt cells. The data demonstrate that ERK activation in LSt cells is essential for ejaculation and contribute to a more detailed understanding of the spinal generation of ejaculation.

Cardiovascular responses to hypocretin-1 in nucleus ambiguus of the ovariectomized female rat

Experiments were done to investigate the effect of chronic estrogen (E; 30 pg/ml plasma) treatment (15-25 days) in the ovariectomized (OVX) female Wistar rat on the cardiovascular responses to hypocretin-1 (hcrt-1) in the nucleus ambiguus (Amb). Microinjections of hcrt-1 (0.5-2.5 pmol) into the external formation of Amb (Ambe) in the urethane anaesthetized, E treated OVX animal or OVX only animal, elicited a dose-related decrease in heart rate (HR). On the other hand, hcrt-1 injections into Ambe did not elicit consistent changes in mean arterial pressure (MAP). The HR response was mediated by vagal excitation as ipsilateral vagotomy abolished the bradycardia response. The bradycardia responses were consistently of greater magnitude and longer duration in the OVX+E animals compared to the OVX only female animals. Finally, it was found that the reflex bradycardia to activation of arterial baroreceptors, as a result of increasing systemic arterial pressure with phenylephrine, was only significantly potentiated in the OVX+E animals. These data suggest that hcrt-1 in the Ambe of the female elicits an increase in vagal cardiomotor neuronal activity to the heart, and that the circulating level of E alters not only the sensitivity of Ambe neurons to hcrt-1 but also the sensitivity of these neurons during activation of baroreceptor afferent inputs.