Intracerebroventricular Pretreatment Research Articles

SRI-30827, a novel allosteric modulator of the dopamine transporter, alleviates HIV-1 Tat-induced potentiation of cocaine conditioned place preference in mice.

HIV-1 Tat (transactivator of transcription) protein disrupts dopaminergic transmission and potentiates the rewarding effects of cocaine. Allosteric modulators of the dopamine transporter (DAT) have been shown to reverse Tat-induced DAT dysfunction. We hypothesized that a novel DAT allosteric modulator, SRI-30827, would counteract Tat-induced potentiation of cocaine reward. Doxycycline (Dox)-inducible Tat transgenic (iTat-tg) mice and their G-tg (Tat-null) counterparts were tested in a cocaine conditioned place preference (CPP) paradigm. Mice were treated 14 days with saline, or Dox (100 mg/kg/day, i.p.) to induce Tat protein. Upon induction, mice were place conditioned two days with cocaine (10 mg/kg/day) after a 1-h daily intracerebroventricular (i.c.v.) pretreatment with SRI-30827 (1 nmol) or a vehicle control, and final place preference assessed as a measure of cocaine reward. Dox-treatment significantly potentiated cocaine-CPP in iTat-tg mice over the response of saline-treated control littermates. SRI-30827 treatment eliminated Tat-induced potentiation without altering normal cocaine-CPP in saline-treated mice. Likewise, SRI-30827 did not alter cocaine-CPP in both saline- and Dox-treated G-tg mice incapable of expressing Tat protein. These findings add to a growing body of evidence that allosteric modulation of DAT could provide a promising therapeutic intervention for patients with comorbid HIV-1 and cocaine use disorder (CUD).

Stimulation of brain corticotropin-releasing factor receptor type1 facilitates the rat micturition via brain glutamatergic receptors

Corticotropin-releasing factor (CRF), a representative stress-related neuropeptide, in the central nervous system reportedly both facilitates and suppresses the micturition, therefore, roles of central CRF in regulation of the micturition are still controversial. In this study, we investigated (1) effects of intracerebroventricularly (icv)-administered CRF on the micturition, and (2) brain CRF receptor subtypes (CRFR1/CRFR2) and glutamatergic receptors (NMDA/AMPA subtypes) involved in the CRF-induced effects in male Wistar rats under urethane anesthesia. Intercontraction intervals (ICI), and maximal voiding pressure (MVP), were evaluated by continuous cystometry 45 min before CRF administration or intracerebroventricular pretreatment with other drugs as follows and 3 h after CRF administration. Single-voided volume (Vv), post-voiding residual volume (Rv), bladder capacity (BC), and voiding efficiency (VE) were evaluated by single cystometry 60 min before CRF administration and 60–120 min after the administration. Icv-administered CRF reduced ICI, Vv, and BC without changing MVP, Rv, or VE. The CRF-induced ICI reduction was attenuated by icv-pretreated CP154526 (CRFR1 antagonist), MK-801 (NMDA receptor antagonist), and DNQX (AMPA receptor antagonist), but not by K41498 (CRFR2 antagonist). These results indicate that stimulation of brain CRFR1 can be involved in facilitation of the rat micturition via brain NMDA/AMPA receptors.

Astrocytes in the PVN are involved in corticotropin-releasing factor-induced sympathetic activation in rats

Corticotropin-releasing factor (CRF) plays a key role in stress responses in the brain. CRF activates not only the hypothalamic-pituitary-adrenal axis but also the sympathetic nervous system. We previously reported that both central administration of CRF and exposure to acute restraint stress (RS) increase plasma catecholamine levels and activate presympathetic neurons in the paraventricular hypothalamic nucleus (PVN). However, it is unclear whether glial cells in brain regions including the PVN are involved in CRF- or stress-induced sympathetic activation. In this study, we examined roles of astrocytes in the CRF-induced elevation of plasmas catecholamine levels (noradrenaline and adrenaline) using fluorocitrate which blocks glial metabolic function. Intracerebroventricular pretreatment of fluorocitrate suppressed the CRF-induced elevation of plasma levels of noradrenaline and adrenaline. On the other hand, microinjection of fluorocitrate into the PVN suppressed the CRF-induced elevation of adrenaline, but not of noradrenaline. Furthermore, we tried to examine effects of inhibition of astrocytes on RS-induced sympathetic activation. Our results suggest that astrocytic function in the brain is important for the CRF-induced sympathetic activation and the effects might be brain region-dependent.

Stimulation of brain α7-nicotinic acetylcholine receptors suppresses the rat micturition through brain GABAergic receptors

Brain nicotinic acetylcholine receptors (nAChRs) reportedly suppress the micturition, but the mechanisms responsible for this suppression remain unclear. We previously reported that intracerebroventricularly administered (±)-epibatidine (non-selective nAChR agonist) activated the sympatho-adrenomedullary system, which can affect the micturition. Therefore, we investigated (1) whether intracerebroventricularly administered (±)-epibatidine-induced effects on the micturition were dependent on the sympatho-adrenomedullary system, and (2) brain nAChR subtypes involved in the (±)-epibatidine-induced effects in urethane-anesthetized male Wistar rats. Plasma noradrenaline and adrenaline (catecholamines) were measured just before and 5 min after (±)-epibatidine administration. Evaluation of urodynamic parameters, intercontraction intervals (ICI) and maximal voiding pressure (MVP) by cystometry was started 1 h before (±)-epibatidine administration or intracerebroventricular pretreatment with other drugs and continued 1 h after (±)-epibatidine administration. Intracerebroventricularly administered (±)-epibatidine elevated plasma catecholamines and prolonged ICI without affecting MVP, and these changes were suppressed by intracerebroventricularly pretreated mecamylamine (non-selective nAChR antagonist). Acute bilateral adrenalectomy abolished the (±)-epibatidine-induced elevation of plasma catecholamines, but had no effect on the (±)-epibatidine-induced ICI prolongation. The latter was suppressed by intracerebroventricularly pretreated methyllycaconitine (selective α7-nAChR antagonist), SR95531 (GABAA antagonist), and SCH50911 (GABAB antagonist), but not by dihydro-β-erythroidine (selective α4β2-nAChR antagonist). Intracerebroventricularly administered PHA568487 (selective α7-nAChR agonist) prolonged ICI without affecting MVP, similar to (±)-epibatidine. These results suggest that stimulation of brain α7-nAChRs suppresses the rat micturition through brain GABAA/GABAB receptors, independently of the sympatho-adrenomedullary outflow modulation.

Centrally circulating α-klotho inversely correlates with human obesity and modulates arcuate cell populations in mice

ObjectiveOur laboratory recently identified the centrally circulating α-klotho protein as a novel hypothalamic regulator of food intake and glucose metabolism in mice. The current study aimed to investigate novel molecular effectors of central α-klotho in the arcuate nucleus of the hypothalamus (ARC), while further deciphering its role regulating energy balance in both humans and mice. MethodsCerebrospinal fluid (CSF) was collected from 22 adults undergoing lower limb orthopedic surgeries, and correlations between body weight and α-klotho were determined using an α-klotho enzyme-linked immunosorbent assay (ELISA) kit. To investigate the effects of α-klotho on energy expenditure (EE), 2-day intracerebroventricular (ICV) treatment was performed in diet-induced obesity (DIO) mice housed in TSE Phenomaster indirect calorimetry metabolic cages. Immunohistochemical staining for cFOS and patch clamp electrophysiology were used to determine the effects of central α-klotho on proopiomelanocortin (POMC) and tyrosine hydroxylase (TH) neurons. Additional stainings were performed to determine novel roles for central α-klotho to regulate non-neuronal cell populations in the ARC. Lastly, ICV pretreatment with fibroblast growth factor receptor (FGFR) or PI3kinase inhibitors was performed to determine the intracellular signaling involved in α-klotho-mediated regulation of ARC nuclei. ResultsObese/overweight human subjects had significantly lower CSF α-klotho concentrations compared to lean counterparts (1,044 ± 251 vs. 1616 ± 218 pmol/L, respectively). Additionally, 2 days of ICV α-klotho treatment increased EE in DIO mice. α-Klotho had no effects on TH neuron activity but elicited varied responses in POMC neurons, with 44% experiencing excitatory and 56% experiencing inhibitory effects. Inhibitor experiments identified an α-klotho→FGFR→PI3kinase signaling mechanism in the regulation of ARC POMC and NPY/AgRP neurons. Acute ICV α-klotho treatment also increased phosphorylated ERK in ARC astrocytes via FGFR signaling. ConclusionOur human CSF data provide the first evidence that impaired central α-klotho function may be involved in the pathophysiology of obesity. Furthermore, results in mouse models identify ARC POMC neurons and astrocytes as novel molecular effectors of central α-klotho. Overall, the current study highlights prominent roles of α-klotho→FGFR→PI3kinase signaling in the homeostatic regulation of ARC neurons and whole-body energy balance.

Human CYP2D6 in the Brain Is Protective Against Harmine-Induced Neurotoxicity: Evidence from Humanized CYP2D6 Transgenic Mice.

CYP2D6 metabolically inactivates several neurotoxins, including beta-carbolines, which are implicated in neurodegenerative diseases. Variation in CYP2D6 within the brain may alter local inactivation of neurotoxic beta-carbolines, thereby influencing neurotoxicity. The beta-carboline harmine, which induces hypothermia and tremor, is metabolized by CYP2D6 to the non-hypothermic/non-tremorgenic harmol. Transgenic mice (TG), expressing human CYP2D6 in addition to their endogenous mouse CYP2D, experience less harmine-induced hypothermia and tremor compared with wild-type mice (WT). We first sought to elucidate the role of CYP2D in general within the brain in harmine-induced hypothermia and tremor severity. A 4-h intracerebroventricular (ICV) pretreatment with the CYP2D inhibitor propranolol increased harmine-induced hypothermia and tremor in TG and increased harmine-induced hypothermia in WT. We next sought to specifically demonstrate that human CYP2D6 expressed in TG brain altered harmine response severity. A 24-h ICV propranolol pretreatment, which selectively and irreversibly inhibits human CYP2D6 in TG brain, increased harmine-induced hypothermia. This 24-h pretreatment had no impact on harmine response in WT, as propranolol is not an irreversible inhibitor of mouse CYP2D in the brain, thus confirming no off-target effects of ICV propranolol pretreatment. Human CYP2D6 activity in TG brain was sufficient in vivo to mitigate harmine-induced neurotoxicity. These findings suggest that human CYP2D6 in the brain is protective against beta-carboline-induced neurotoxicity and that the extensive interindividual variability in CYP2D6 expression in human brain may contribute to variation in susceptibility to certain neurotoxin-associated neurodegenerative disorders.

Brain nitric oxide induces facilitation of the micturition reflex through brain glutamatergic receptors in rats.

Brain nitric oxide (NO) have been reported in regulation of the sympatho-adrenomedullary system, which can affect voiding and storage functions. Therefore, we investigated effects of intracerebroventricularly (icv) administered 3-(4-morpholinyl)sydnonimine, hydrochloride (SIN-1) (NO donor) on the micturition reflex, focusing on their dependence on the sympatho-adrenomedullary system and on brain N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats. Plasma noradrenaline and adrenaline were measured just before and 5 minutes after SIN-1 administration. Evaluation of urodynamic parameters was started 1 hour before SIN-1 administration or intracerebroventricular pretreatment with other drugs. SIN-1 (100 and 250 µg/animal) elevated plasma adrenaline and reduced intercontraction interval([ICI]values; 110.5% [SIN-1, 0 µg]and 54.9% [SIN-1, 250 µg]during 15 minutes after SIN-1 administration [P < .05;η2 = 0.59]) without affecting plasma noradrenaline or maximal voiding pressure. SIN-1 (250 µg/animal) reduced single-voided volume and bladder capacity without affecting post-voiding residual volume. The SIN-1 (250 µg/animal)-induced adrenaline elevation and ICI reduction were attenuated by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (carboxy-PTIO) (NO scavenger, icv) (ICI values; 44.7% [vehicle + SIN-1]and 77.5% [carboxy-PTIO + SIN-1]during 15 minutes after SIN-1 administration [P < .05;η2 = 0.51]). Acute bilateral adrenalectomyabolished SIN-1-induced adrenaline elevation, while showed no effect on the SIN-1-induced ICI reduction. The ICI reduction was attenuated by MK-801 (NMDA receptor antagonist, icv) (ICI values; 47.0% [vehicle + SIN-1]and 87.6% [MK-801 + SIN-1]during 15 minutes after SIN-1 administration [P < .05;η2 = 0.61]), but not by DNQX (AMPA receptor antagonist, icv). Brain NO is involved in facilitation of the rat micturition reflex through brain NMDA receptors, independently of the sympatho-adrenomedullary outflow modulation.

Thromboxane A2 in the paraventricular hypothalamic nucleus mediates glucoprivation-induced adrenomedullary outflow

Glucoprivation stimulates a rapid sympathetic response to release and/or secrete catecholamines into the bloodstream. However, the central regulatory mechanisms involving adrenoceptors and prostanoids production in the paraventricular hypothalamic nucleus (PVN) that are responsible for the glucoprivation-induced elevation of plasma catecholamines are still unresolved. In this study, we aimed to clarify whether glucoprivation-induced activation of noradrenergic neurons projecting to the PVN can induce α- and/or β-adrenergic receptor activation and prostanoids production in the PVN to elevate plasma catecholamine levels. We examined the effects of α- and β-adrenergic receptor antagonists, a cyclooxygenase inhibitor, a thromboxane A synthase inhibitor, and a PGE2 subtype EP3 receptor antagonist on intravenously administered 2-deoxy-D-glucose (2-DG)-induced elevation of noradrenaline in the PVN and plasma levels of catecholamine in freely moving rats. In addition, we examined whether intravenously administered 2-DG can increase prostanoids levels in the PVN microdialysates. Intracerebroventricular (i.c.v.) pretreatment with phentolamine (a non-selective α-adrenergic receptor antagonist) suppressed the 2-DG-induced increase in the plasma level of adrenaline, whereas i.c.v. pretreatment with propranolol (a non-selective β-adrenergic receptor antagonist) suppressed the 2-DG-induced elevation of the plasma level of noradrenaline. I.c.v. pretreatment with indomethacin (a cyclooxygenase inhibitor) and furegrelate (a thromboxane synthase inhibitor) attenuated the 2-DG-induced elevations of both noradrenaline and adrenaline levels. Furthermore, 2-DG administration elevated the thromboxane B2 level, a metabolite of thromboxane A2 in PVN microdialysates. Our results suggest that glucoprivation-induced activation of α- and β-adrenergic receptor in the brain including the PVN and then thromboxane A2 production in the PVN, which are essential for the 2-DG-induced elevations of both plasma adrenaline and noradrenaline levels.

Human Milk Oligosaccharide 2'-Fucosyllactose Reduces Neurodegeneration in Stroke Brain.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide in human milk and is present at trace levels in cow milk. 2'-FL reduces inflammation in the gastrointestinal tract. Its action in the central nervous system has not been well characterized. The purpose of this study is to determine 2'-FL-mediated neural protection and repair in culture and stroke brain. In rat primary cortical neuronal cultures, 2'-FL significantly antagonized N-methyl-D-aspartate (NMDA) or glutamate-mediated changes in ATP production, MAP2 immunoreactivity, and TUNEL. The influx of Ca++ (Ca++i) was examined in primary cortical neurons expressing GCaMP5, an endogenous calcium probe. NMDA increased Ca++i; 2'-FL significantly attenuated this reaction. In a rat middle cerebral artery occlusion model of stroke, we found that intracerebroventricular pretreatment or oral posttreatment with 2'-FL significantly reduced brain infarction, mitigated microglial activation, improved locomotor activity, and upregulated brain-derived neurotrophic factor (BDNF) expression. Post-stroke delivery of 2'-FL increased bromodeoxyuridine (BrdU) labeling in the perilesioned area. These BrdU cells co-expressed NeuN, or nestin, or GFAP. Using subventricular Matrigel cultures, we demonstrated that 2'-FL increased cell migration from subventricular zone explant. This response was reduced by anti-BDNF blocking antibody. In conclusion, our data suggest that 2'-FL has neuroprotective action through inhibition of Ca++i, inflammation, and apoptosis. Posttreatment with 2'-FL facilitates neural repair in stroke brain.

Prostaglandin E2 receptor EP3 subtype in the paraventricular hypothalamic nucleus mediates corticotropin-releasing factor-induced elevation of plasma noradrenaline levels in rats

Corticotropin-releasing factor (CRF) plays an important role in sympathetic regulation. Centrally administered CRF elevates plasma catecholamine levels, resulting in CRF-dependent hypertension and tachycardia. We previously reported that brain thromboxane A2 mediates CRF-induced elevation of plasma adrenaline levels, whereas prostanoids other than thromboxane A2 mediate elevations in plasma noradrenaline levels. However, the mechanism by which CRF induces elevations in plasma noradrenaline levels remains unknown. Previous studies have revealed that brain prostaglandin (PG) E2, but not other PGs, causes sympathetic activation. In this study, we examined the roles of brain PGE2 and its receptors in CRF-induced elevation of plasma noradrenaline levels in rats. Our results showed that intracerebroventricular pretreatment with an antagonist of the PGE2 receptor EP3 subtype, but not other subtypes, suppressed CRF-induced elevations in plasma noradrenaline levels. We also examined the role of PGE2 and EP3 receptors in the paraventricular hypothalamic nucleus (PVN), the major integrative center for sympathetic regulation, in CRF-induced elevation of plasma noradrenaline levels. Centrally administered CRF increased PGE2 levels in PVN microdialysates, and microinjection of an EP3 receptor agonist into the PVN elevated plasma noradrenaline levels. Bilateral blockade of EP3 receptors in the PVN suppressed the elevation of plasma noradrenaline levels evoked by intracerebroventricular administration and PVN-microinjection of CRF. Our results suggest that CRF stimulates PGE2 release into the PVN that activates EP3 receptors in the PVN, resulting in the elevation of plasma noradrenaline levels.

Role of hypoxia-inducible factor-1α in regulating oxidative stress and hypothalamic neuropeptides-mediated appetite control

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator responding to hypoxia. Amphetamine (AMPH), however, can activate HIF-1 under normoxic conditions, which is associated with the co-activation of oxidative stress. Hypothalamic neuropeptides and anti-oxidative enzymes have been found to participate in amphetamine (AMPH)-mediated appetite control. The present study examined whether HIF-1 was involved in the oxidative stress and anorectic action of AMPH. Rats were daily treated with AMPH for 4 days, and expression levels of HIF-1α, superoxide dismutase (SOD), catalase, neuropeptide Y (NPY), proopiomelanocortin (POMC), phosphatidylinositol 3-kinase (PI3K), and nuclear factor-kappaB (NF-κB) were assessed and compared. Results revealed that feeding behavior and NPY decreased, whereas HIF-1α/DNA binding activity and SOD, POMC, PI3K, and NF-κB expression levels increased in AMPH-treated rats. Further experiment revealed that intracerebroventricular (i.c.v.) pretreatment with HIF-1α inhibitor modified feeding behavior and expression levels of hypothalamic protein assessed. Another experiment revealed that pretreatment (i.c.v.) with reactive oxygen species scavenger modulated HIF-1α, NPY, POMC, PI3K, and NF-κB expression levels in AMPH-treated rats. It is suggested that HIF-1α plays a functional role in the increase of oxidative stress and the modulation of NFκB/NPY/POMC-mediated appetite control in AMPH-treated rats. These findings advance the knowledge of HIF-1α in the regulation of central dopamine agonist-mediated appetite control.

GABAB receptors in the hypothalamic paraventricular nucleus mediate β-adrenoceptor-induced elevations of plasma noradrenaline in rats

In the brain, various neurotransmitters such as noradrenaline and GABA regulate peripheral sympathetic functions. Previously, it has been reported that both β-adrenoceptor activation and GABAB receptor activation in the brain are involved in the elevation of plasma noradrenaline levels. However, it is unknown whether these pathways interact with each other. In the present study, we examined the relationship between the central actions of β-adrenoceptor activation and GABAB receptor activation with regard to plasma noradrenaline responses using urethane-anesthetized rats. Intracerebroventricular pretreatment with the GABAA receptor antagonist bicuculline did not affect the β-adrenoceptor agonist isoproterenol-induced elevation of plasma noradrenaline levels. In contrast, pretreatment with the GABAB receptor antagonist CGP 35348 suppressed the isoproterenol-induced elevation of noradrenaline levels. Intracerebroventricular pretreatment with the β-adrenoceptor antagonist propranolol did not alter the GABAB receptor agonist baclofen-induced elevation of plasma noradrenaline levels. We next examined the central effects of β-adrenoceptor activation on GABA release in the paraventricular hypothalamic nucleus (PVN), the major integrative center for sympathetic regulation in the brain. Intracerebroventricular administration of isoproterenol increased GABA content in PVN dialysates. In addition, baclofen microinjected unilaterally into the PVN resulted in elevated plasma levels of noradrenaline, but not adrenaline. Finally, unilateral blockade of GABAB receptors in the PVN suppressed the isoproterenol-induced elevation of plasma noradrenaline level. Our results suggest that activation of β-adrenoceptors in the brain, likely in the PVN, induces GABA release in the PVN, which in turn activates GABAB receptors in the PVN, leading to elevated plasma noradrenaline.

Amlexanox Inhibits Cerebral Ischemia-Induced Delayed Astrocytic High-Mobility Group Box 1 Release and Subsequent Brain Damage.

High-mobility group box 1 (HMGB1) is increased in the cerebrospinal fluid (CSF) and serum during the early and late phases of brain ischemia and is known to contribute to brain damage. However, detailed characterization underlying cell type-specific HMGB1 release and pathophysiological roles of extracellularly released HMGB1 in ischemic brain remain unclear. Here, we examined cell type-specific HMGB1 release and the therapeutic potential of amlexanox, an inhibitor of nonclassical release, and of an anti-HMGB1 antibody against ischemic brain damage. HMGB1 depletion from neuronal nuclei was observed within 3 hours after transient middle cerebral artery occlusion (tMCAO), whereas the intracerebroventricular (i.c.v.) pretreatment with amlexanox blocked HMGB1 release from neurons, resulting in HMGB1 redistribution in the nuclei and cytoplasm. HMGB1 was selectively released from astrocytes 27 hours after tMCAO and this HMGB1 release was blocked by late treatment with amlexanox (i.c.v.) 24 hours after tMCAO. Proximity extension assay revealed that the HMGB1 level was elevated in the CSF at 3 and 27 hours after tMCAO. This late treatment with amlexanox significantly protected the brain from ischemic damage, but its pretreatment 30 minutes before tMCAO failed to show any protection. The late treatment (i.c.v.) with anti-HMGB1 antibody 24 hours after tMCAO also ameliorated ischemic brain damage 48 hours after tMCAO. Thus, the inhibition of brain damage by late treatment with amlexanox or anti-HMGB1 antibody indicates that late HMGB1 release plays a role in the maintenance of stroke-induced brain damage, and the inhibition of this release would be a novel therapeutic target for protection of ischemic brain damage.

The 5-HT4 receptor-mediated inhibition of visceral nociceptive neurons in the rat caudal ventrolateral medulla

Activation of the serotonin type 4 (5-HT4) receptors has been reported to improve abdominal pain in patients with functional gastrointestinal disorders and reduce visceral nociception in animal models. Earlier studies have proposed that 5-HT4 agonist can produce visceral analgesia by acting at the supraspinal level, but the underlying neuronal mechanisms remain unclear. The caudal ventrolateral medulla (CVLM) is the first site for processing of visceral nociceptive signals ascending via spinal pathways and an important component of the endogenous pain modulatory system. Therefore, the objective of the present study was to examine whether activation of 5-HT4 receptors can affect the visceral pain-related neurons in the CVLM. In urethane-anesthetized adult male Wistar rats, we evaluated the effects of a 5-HT4 receptor agonist, BIMU8 on ongoing firing of the CVLM neurons and their excitatory responses to noxious colorectal distension (CRD, 80mmHg). The drug’s effect was also tested on blood pressure reactions induced by CRD–a general physiological measure of visceral nociception. Intravenous administration of BIMU8 (0.5, 1 or 2mg/kg) produced dose-dependent suppression of both the ongoing and CRD-evoked activities of the CVLM neurons and simultaneously attenuated the depressor hemodynamic reaction to CRD. The compound’s inhibitory effect was almost completely eliminated by intracerebroventricular pretreatment with GR113808, a selective 5-HT4 antagonist, indicating the preferential involvement of supraspinal 5-HT4 receptors. Results indicate that visceral nociceptive transmission through the caudal medulla is negatively modulated by descending 5-HT4-dependent mechanisms. These findings can contribute to a deeper understanding of supraspinal processing of pain signals from the abdomen.

Abstract 041: mTORC1 is Required for Leptin-induced Sympathetic Activation to the Kidney but Not Brown Adipose Tissue

The adipocyte-derived hormone leptin plays a critical role in the regulation of energy homeostasis through its action in the brain to decrease food intake and promote energy expenditure by increasing sympathetic nerve activity (SNA) to the thermogenic brown adipose tissue (BAT). Leptin also increases SNA to cardiovascular organs including the kidney and raises arterial pressure. However, it is unclear whether leptin controls regional SNA via conserved or distinct molecular mechanisms. Multiple intracellular pathways have been associated with leptin signaling including the mechanistic target of rapamycin complex 1 (mTORC1), which has been proposed as a critical determinant of leptin action. Here, we assessed the contribution of mTORC1 signaling to leptin-evoked regional sympathetic activation. Simultaneous multifiber recording of renal and BAT SNA in anesthetized C57BL/6J mice showed that intracerebroventricular (ICV) administration of leptin (2μg, n=5) increased both renal (170±34%) and BAT (208±37%) SNA. Interestingly, ICV pre-treatment with the mTORC1 inhibitor (rapamycin, 5ng, n=6) abolished the leptin-induced increase in renal (10±6%, P&lt;0.05 vs controls) but not BAT (226±31%) SNA. Next, we used conditional knockout mice that lack the critical mTORC1 subunit, Raptor, specifically in leptin receptor (LRb)-expressing cells (LRb Cre /Raptor fl/fl ) to determine the long-term effects of disrupting mTORC1 signaling on leptin-evoked increase in regional SNA. We confirmed the inability of leptin to activate mTORC1 signaling in LRb-expressing cells of LRb Cre /Raptor fl/fl mice relative to controls using immunohistochemical staining of phosphorylated ribosomal S6, a downstream target of mTORC1. We observed a significant increase in renal SNA in response to ICV leptin in control mice (127±16%, n=9), but not in LRb Cre /Raptor fl/fl mice (-4±15%, n=9, P&lt;0.05 vs controls). Conversely, ICV leptin-induced increase in BAT SNA was not different in LRb Cre /Raptor fl/fl mice (109±27%, n=5) vs. littermate controls (173±52%, n=4). Our data suggest a critical role for mTORC1 signaling in selectively mediating the cardiovascular sympathetic but not the thermogenic actions of leptin, with important implications for obesity-associated hypertension.

Abstract 072: Brain Epithelial Sodium Channels on Organum Vasculosum of the Lamina Terminalis Neurons Mediate Sympathoexcitatory Pressor Responses and Neuronal Excitation by Hypertonic NaCl

High dietary salt intake raises cerebrospinal fluid (CSF) [Na + ] in salt sensitive subjects to elevate sympathetic nerve activity (SNA), and arterial blood pressure (ABP). This occurs through excitation of NaCl-sensitive sites in the brain, including the organum vasculosum of the lamina terminalis (OVLT). Intriguingly, intracerebroventricular (ICV) pretreatment with benzamil (a non-voltage gated Na + channel blocker) also attenuates those systemic responses to central NaCl. Thus, I hypothesized that benzamil acts on NaCl-sensitive OVLT neurons to attenuate neuronal excitation and elevated SNA and ABP in response to hypertonic NaCl. To evaluate this hypothesis, lumbar SNA and ABP were measured in anesthetized adult rats in response to ICV infusion of 0.15, 0.5, and 1.0 M NaCl with and without prior OVLT microinjection of benzamil (5nmol per 20nL). ICV infusion of NaCl produced concentration-dependent increases in lumbar SNA (0.15M: 101±3%, 0.5M: 118±3%, 1.0M: 130±9% n=4 per group, P&lt;0.05) and mean ABP (0.15M: 1±1mmHg; 0.5M: 5±1mmHg, 1.0M: 12±2mmHg). OVLT microinjection of benzamil significantly attenuated the increase in lumbar SNA (0.15M: 100±2%, 0.5M: 108±2%, 1.0M: 115±3% n=4 per group, P&lt;0.05) and mean ABP (0.15M: 1±0mmHg; 0.5M: 2±1mmHg, 1.0M: 6±2mmHg). In a parallel set of experiments, in vitro whole-cell recordings of OVLT neurons in slices were performed to assess whether blockade of epithelial sodium channels (ENaC) attenuated NaCl-induced excitation. Bath application of +7.5mM NaCl increase action potential (AP) discharge of OVLT neurons (n=11) from 0.46±0.16 Hz to 1.09±0.26 Hz (P&lt;0.05). Subsequent addition of an ENaC selective concentration of benzamil (0.5μM) reversed AP discharge (0.75±0.24 Hz, P&lt;0.05). Isotonic 0.5μM benzamil did not significantly change NaCl-sensitive OVLT neuron (n=13) AP discharge from baseline (0.54±0.14 Hz to 0.60±0.16 Hz, P&gt;0.05). Collectively, this data indicates that elevations in CSF NaCl concentrations excite OVLT neurons via ENaC to elevate SNA and ABP.

MTORC1 Signaling Contributes to Drinking But Not Blood Pressure Responses to Brain Angiotensin II.

Mechanistic target of rapamycin complex 1 (mTORC1) is a molecular node that couples extracellular cues to a wide range of cellular events controlling various physiological processes. Here, we identified mTORC1 signaling as a critical mediator of angiotensin II (Ang II) action in the brain. In neuronal GT1-7 cells, we show that Ang II stimulates neuronal mTORC1 signaling in an Ang II type 1 receptor-dependent manner. In mice, a single intracerebroventricular (ICV) injection or chronic sc infusion of Ang II activated mTORC1 signaling in the subfornical organ, a critical brain region in cardiovascular control and fluid balance. Moreover, transgenic sRA mice with brain-specific overproduction of Ang II displayed increased mTORC1 signaling in the subfornical organ. To test the functional role of brain mTORC1 in mediating the action of Ang II, we examined the consequence of mTORC1 inhibition with rapamycin on Ang II-induced increase in water intake and arterial pressure. ICV pretreatment with rapamycin blocked ICV Ang II-mediated increases in the frequency, duration, and amount of water intake but did not interfere with the pressor response evoked by Ang II. In addition, ICV delivery of rapamycin significantly reduced polydipsia, but not hypertension, of sRA mice. These results demonstrate that mTORC1 is a novel downstream pathway of Ang II type 1 receptor signaling in the brain and selectively mediates the effect of Ang II on drinking behavior.

Corticotrophin-releasing factor receptor 2 mediates the enhanced activation of satiety-related responses through oxytocin neurons in the paraventricular nucleus of the hypothalamus after adrenalectomy

Adrenalectomy (ADX)-induced hypophagia is associated with increased activation of corticotrophin-releasing factor (CRF) and oxytocin (OT) neurons in the paraventricular nucleus of the hypothalamus (PVN) after refeeding. CRF2- and OT-receptor antagonists abolish the hypophagia and the augmented activation of the nucleus of the solitary tract neurons induced by feeding after ADX. In addition, OT-receptor antagonist reversed CRF-induced anorexia. We evaluated the effect of intracerebroventricular pretreatment with CRF2-receptor antagonist, antisauvagine-30 (AS30), on the activation of OT neurons of the PVN in response to refeeding of sham, adrenalectomized (ADX) and ADX rats replaced with corticosterone (ADX+B). In vehicle-pretreated animals, refeeding increased the number of Fos+OT double labeled neurons in the posterior parvocellular subdivision of the PVN (PaPo) of sham, ADX and ADX+B animals, with higher Fos expression and OT neuronal activation in the ADX group. AS30 reversed refeeding-induced increased activation of OT and non-OT neurons in the PaPo in the ADX group. In the medial parvocellular subdivision of the PVN (PaMP) of vehicle-pretreated animals, the number of Fos- and Fos+OT-immunoreactive neurons was increased after refeeding in ADX group. AS30 in the ADX group attenuated the enhanced Fos expression but not the number of Fos+OT double labeled neurons in the PaMP. In conclusion, CRF2-receptor antagonist reverses the increased activation of OT neurons in the PaPo induced by feeding in ADX animals, suggesting that OT neurons might be downstream mediators of CRF effects on satiety-related responses after ADX.

Contribution of serotonin and dopamine to changes in core body temperature and locomotor activity in rats following repeated administration of mephedrone.

The psychoactive effects of mephedrone are commonly compared with those of 3,4-methylenedioxymethamphetamine, but because of a shorter duration of action, users often employ repeated administration to maintain its psychoactive effects. This study examined the effects of repeated mephedrone administration on locomotor activity, body temperature and striatal dopamine and 5-hydroxytryptamine (5-HT) levels and the role of dopaminergic and serotonergic neurons in these responses. Adult male Lister hooded rats received three injections of vehicle (1 ml/kg, i.p.) or mephedrone HCl (10 mg/kg) at 2 h intervals for radiotelemetry (temperature and activity) or microdialysis (dopamine and 5-HT) measurements. Intracerebroventricular pre-treatment (21 to 28 days earlier) with 5,7-dihydroxytryptamine (150 µg) or 6-hydroxydopamine (300 µg) was used to examine the impact of 5-HT or dopamine depletion on mephedrone-induced changes in temperature and activity. A final study examined the influence of i.p. pre-treatment (-30 min) with the 5-HT1A receptor antagonist WAY-100635 (0.5 mg/kg), 5-HT1B receptor antagonist GR 127935 (3 mg/kg) or the 5-HT7 receptor antagonist SB-258719 (10 mg/kg) on mephedrone-induced changes in locomotor activity and rectal temperature. Mephedrone caused rapid-onset hyperactivity, hypothermia (attenuated on repeat dosing) and increased striatal dopamine and 5-HT release following each injection. Mephedrone-induced hyperactivity was attenuated by 5-HT depletion and 5-HT1B receptor antagonism, whereas the hypothermia was completely abolished by 5-HT depletion and lessened by 5-HT1A receptor antagonism. These findings suggest that stimulation of central 5-HT release and/or inhibition of 5-HT reuptake play a pivotal role in both the hyperlocomotor and hypothermic effects of mephedrone, which are mediated in part via 5-HT1B and 5-HT1A receptors.

Effect of Brain CYP2B Inhibition on Brain Nicotine Levels and Nicotine Self-Administration.

The CYP2B enzyme is expressed in human and rat brain, and metabolizes many CNS-acting drugs. The gene that encodes human CYP2B6 is highly polymorphic, where the variation in brain enzyme levels could result in altered brain drug levels. CYP2B can metabolize nicotine, the main psychoactive ingredient in cigarettes; if altered brain CYP2B activity can influence nicotine brain levels, it could influence nicotine-mediated behaviors. To investigate this, a mechanism-based inhibitor selective for CYP2B, C8-xanthate (20 μg), was administered intracerebroventricularly (ICV) into the brain of rats, and 22 h later, nicotine levels were measured by in vivo microdialysis following nicotine (150 μg/kg intravenous). Brain nicotine levels from 15 to 30 min and the AUC0-45 min were both twofold higher (p<0.05) with C8-xanthate vs vehicle pretreatment; there was no difference in peripheral nicotine levels. Rats were then given ICV pretreatment with C8-xanthate/ASCF and underwent intravenous nicotine self-administration with 3.75-30 μg/kg per infusion dose. C8-xanthate pretreatment increased responding in progressive ratio (15 μg/kg per infusion dose, p<0.05). In a separate cohort, C8-xanthate increased the percentage of rats that acquired self-administration (7.5 μg/kg per infusion dose, p<0.05) from 40% after vehicle pretreatment to 100%, with no difference in peripheral nicotine levels measured at the end of behavior. In a third cohort, C8-xanthate increased the number of sessions required to meet extinction criteria (p<0.05). Together these data demonstrate that the brain CYP2B activity can influence nicotine brain levels and subsequent behaviors independent of hepatic metabolism. This suggests that human smokers with variable CYP2B brain levels could have different nicotine levels and reinforcement, which might have a role in smoking behaviors and dependence.