Opioid activation in the lateral parabrachial nucleus induces hypertonic sodium intake

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Endogenous hydrogen peroxide in the hypothalamic paraventricular nucleus regulates sympathetic nerve activity responses to L-glutamate

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Neurochemistry of the afferents to the rat cochlear root nucleus: Possible synaptic modulation of the acoustic startle

Afferents to the primary startle circuit are essential for the elicitation and modulation of the acoustic startle reflex (ASR). In the rat, cochlear root neurons (CRNs) comprise the first component of the acoustic startle circuit and play a crucial role in mediating the ASR. Nevertheless, the neurochemical pattern of their afferents remains unclear. To determine the distribution of excitatory and inhibitory inputs, we used confocal microscopy to analyze the immunostaining for vesicular glutamate and GABA transporter proteins (VGLUT1 and VGAT) on retrogradely labeled CRNs. We also used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to detect and localize specific neurotransmitter receptor subunits in the cochlear root. Our results show differential distributions of VGLUT1- and VGAT-immunoreactive endings around cell bodies and dendrites. The RT-PCR data showed a positive band for several ionotropic glutamate receptor subunits, M1-M5 muscarinic receptor subtypes, the glycine receptor alpha 1 subunit (GlyR alpha 1), GABA(A), GABA(B), and subunits of alpha 2 and beta-noradrenergic receptors. By immunohistochemistry, we confirmed that CRN cell bodies exhibit positive immunoreaction for the glutamate receptor (GluR) 3 and NR1 GluR subunits. Cell bodies and dendrites were also positive for M2 and M4, and GlyR alpha 1. Other subunits, such as GluR1 and GluR4 of the AMPA GluRs, were observed in glial cells neighboring unlabeled CRN cell bodies. We further confirmed the existence of nor-adrenergic afferents onto CRNs from the locus coeruleus by combining tyrosine hydroxylase immunohistochemistry and tract-tracing experiments. Our results provide valuable information toward understanding how CRNs might integrate excitatory and inhibitory inputs, and hence how they could elicit and modulate the ASR. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Antiepileptic effect of acylpolyaminetoxin JSTX-3 on rat hippocampal CA1 neurons in vitro

The Joro spider toxin (JSTX-3), derived from Nephila clavata, has been found to block glutamate excitatory activity. Epilepsy has been studied in vitro, mostly on rat hippocampus, through brain slices techniques. The aim of this study is to verify the effect of the JSTX-3 on the epileptiform activity induced by magnesium-free medium in rat CA1 hippocampal neurons. Experiments were performed on hippocampus slices of control and pilocarpine-treated Wistar rats, prepared and maintained in vitro. Epileptiform activity was induced through omission of magnesium from the artificial cerebrospinal fluid (0-Mg2+ ACSF) superfusate and iontophoretic application of N-methyl-D-aspartate (NMDA). Intracellular recordings were obtained from CA] pyramidal neurons both of control and epileptic rats. Passive membrane properties were analyzed before and after perfusion with the 0-Mg2+ ACSF and the application of toxin JSTX-3. During the ictal-like activity, the toxin JSTX-3 was applied by pressure ejection, abolishing this activity. This effect was completely reversed during the washout period 2. when the slices were formerly perfused with artificial cerebrospinal fluid (ACSF) and again with 0-Mg2+ ACSF. Our results suggest that the toxin JSTX-3 is a potent blocker of induced epileptiform activity. (c) 2005 Elsevier B.V. All rights reserved.

Role of central nitric oxide in behavioral thermoregulation of toads during hypoxia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

CARDIOVASCULAR EFFECTS OF CENTRAL CLONIDINE IN CONSCIOUS RATS AFTER HYPOTHALAMIC-LESIONS

The central injection of clonidine (an alpha-2-adrenoceptor agonist) in conscious normotensive rats produces hypertensive responses and bradycardia. The present study was performed to investigate the effect of electrolytic lesions in the anteroventral third ventricle (AV3V) region or in the lateral hypothalamus (LH) on the pressor and bradycardic responses induced by central clonidine in rats. Mean arterial pressure and heart rate were recorded in sham or AV3V-lesioned rats with cerebral stainless steel cannulae implanted into the lateral cerebral ventricle (ICV) or LH. and in sham or bilateral LH-lesioned rats with cannulae-implanted ICV. The injection of clonidine (40 nmol) ICV or into the LH of sham rats produced a pressor response (37 +/- 2-48 +/- 3 mmHg) and bradycardia (-45 +/- 10--93 +/- 6 bpm). After AV3V-lesion (3 and 12 days) or LH-lesion (3 days) the pressor response was abolished and a small hypotensive response was induced by the injection of clonidine (-1 +/- 3--16 +/- 3 mmHg). The bradycardia (-27 +/- 6--57 +/- 11 bpm) was reduced, but not abolished by the lesions. These results show that the AV3V region and LH are important cerebral structures that participate in the excitatory pathways involved in the pressor response to central clonidine in rats. They also suggest that, in the absence of these pressor pathways, the hypotensive responses to central clonidine may appear in conscious rats.

Improvement in relaxation response in corpus cavernosum from trained rats

Objectives. To evaluate the contractile and relaxing responses in rat corpus cavernosum (RCC) from rats after 8 weeks of run training, because erectile function is highly dependent on nitric oxide (NO) from nitrergic fibers or endothelium. Physical activity enhances NO production and improves endothelial function, with beneficial effects on cardiovascular disease.Methods. The training program consisted of 8 weeks of run training, 5 days/wk, and each session lasted 60 minutes. The RCC was isolated, and concentration-response curves to NO, acetylcholine, sodium nitroprusside, phenylephrine, and endothelin were obtained. The excitatory and inhibitory effects of electrical field stimulation (2 to 32 Hz) were also evaluated.Results. NO (0.1 to 100 muM) and sodium nitroprusside (0.01 to 1000 muM) produced a relaxing effect in RCC in a dose-dependent manner, with the maximal responses to NO (control 62% +/- 4%, trained 88% +/- 3%) and sodium nitroprusside (control 83% +/- 3%, trained 95% +/- 2%) significantly enhanced after 8 weeks of run training. However, acetylcholine-induced relaxations were not affected by exercise. Similarly, electrical field stimulation-induced relaxations were significantly increased in RCC from trained rats at 2 Hz (control 2.4% +/- 0.3%, trained 4.2% +/- 0.5%) and 4 Hz (control 5.3% +/- 1.2%, trained 12.5% +/- 1.7%). The contractile sensitivity of RCC to phenylephrine (0.01 to 100 AM) and endothelin (0.01 to 100 nM) was not modified by training exercise.Conclusions. Our findings suggest that run training enhances functional responses in rat RCC that involves increases in the NO-cyclic guanosine monophosphate signaling pathway by endothelium-independent mechanisms that is not accompanied by changes in contractile sensitivity. (C) 2004 Elsevier B.V.

Role of 5-HT in stress, anxiety, and depression

There are conflicting results on the function of 5-HT in anxiety and depression. To reconcile this evidence, Deakin and Graeff have suggested that the ascending 5-HT pathway that originates in the dorsal raphe nucleus (DRN) and innervates the amygdala and frontal cortex facilitates conditioned fear, while the DRN-periventricular pathway innervating the periventricular and periaqueductal gray matter inhibits inborn fight/flight reactions to impending danger, pain, or asphyxia. To study the role of the DRN 5-HT system in anxiety, we microinjected 8-OH-DPAT into the DRN to inhibit 5 HT release. This treatment impaired inhibitory avoidance (conditioned fear) without affecting one-way escape (unconditioned fear) in the elevated T-maze, a new animal model of anxiety. We also applied three drug treatments that increase 5-HT release from DRN terminals: 1) intra-DRN microinjection of the benzodiazepine inverse agonist FG 4172, 2) intra-DRN microinjection of the excitatory amino acid kainic acid, and 3) intraperitoneal injection of the 5-HT releaser and uptake blocker D-fenfluramine. All treatments enhanced inhibitory avoidance in the T-maze. D-Fenfluramine and intra-DRN kainate also decreased one-way escape. In healthy volunteers, D-fenfluramine and the 5-HT agonist mCPP (mainly 5-HT2C) increased, while the antagonists ritanserin (5-HT2A/(2C)) and SR 46349B (5-HT2A) decreased skin conductance responses to an aversively conditioned stimulus (tone). In addition, D-fenfluramine decreased, whereas ritanserin increased subjective anxiety induced by simulated public speaking, thought to represent unconditioned anxiety. Overall, these results are compatible with the above hypothesis. Deakin and Graeff have suggested that the pathway connecting the median raphe nucleus (MRN) to the dorsal hippocampus promotes resistance to chronic, unavoidable stress. In the present study, we found that 24 h after electrolytic lesion of the rat MRN glandular gastric ulcers occurred, and the immune response to the mitogen concanavalin A was depressed. Seven days after the same lesion, the ulcerogenic effect of restraint was enhanced. Microinjection of 8-OH-DPAT, the nonselective agonist 5-MeO-DMT, or the 5-HT uptake inhibitor zimelidine into the dorsal hippocampus immediately after 2 h of restraint reversed the deficits of open arm exploration in the elevated plus-maze, measured 24 h after restraint. The effect of the two last drugs was antagonized by WAY-100135, a selective 5-HT1A receptor antagonist. These results are compatible with the hypothesis that the MRN-dorsal hippocampus 5-HT system attenuates stress by facilitation of hippocampal 5-HT1A-mediated neurotransmission. Clinical implications of these results are discussed, especially with regard to panic disorder and depression.

Ibotenate lesion of the medial hypothalamus alters the salt intake and pressor responses to activation of the median preoptic nucleus in rats

We investigated the influence of ibotenic acid lesions of the medial hypothalamus (MH) on salt appetite and arterial blood pressure responses induced by angiotensinergic and adrenergic stimulation of the median preoptic nucleus (MnPO) of rats. Previous injection of the adrenergic agonists norepinephrine, clonidine, phenylephrine, and isoproterenol into the MnPO of sham MH-lesioned rats caused no change in the sodium intake induced by ANG II. ANG II injected into the MnPO of MH-lesioned rats increased sodium intake compared with sham-lesioned rats. Previous injection of clonidine and isoproterenol increased, whereas phenylephrine abolished the salt intake induced by ANG II into the MnPO of MH-lesioned rats. Previous injection of norepinephrine and clonidine into the MnPO of sham MH-lesioned rats caused no change in the mean arterial pressure (MAP) induced by ANG II. Under the same conditions, previous injection of phenylephrine increased, whereas isoproterenol reversed the increase in MAP induced by angiotensin II (ANG II). ANG II injected into the MnPO of MH-lesioned rats induce a decrease in MAP compared with sham-lesioned rats. Previous injection of phenylephrine or norepinephrine into the MnPO of MH-lesioned rats induced a negative MAP, whereas pretreatment with clonidine or isoproterenol increased the MAP produced by ANG II injected into the MnPO of sham- or MH-lesioned rats. These data show that ibotenic acid lesion of the MH increases the sodium intake and presser responses induced by the concomitant angiotensinergic, alpha(2) and beta adrenergic activation of the MnPO, whereas alpha(1) activation may have opposite effects. MH involvement in excitatory and inhibitory mechanisms related to sodium intake and MAP control is suggested.

SYNAPTONEMAL COMPLEX-ANALYSIS IN SPERMATOCYTES AND OOCYTES OF RAINBOW-TROUT, ONCORHYNCHUS-MYKISS (PISCES, SALMONIDAE) - THE PROCESS OF AUTOSOME AND SEX-CHROMOSOME SYNAPSIS

The surface-spreading synaptonemal complex (SC) technique was employed to analyze spermatocytes and oocytes of rainbow trout in order to visualize the process of autosome and sex chromosome synapsis in this species. The structure of lateral elements (LEs) of the SC and the chromosome synapsis process at the stages of leptotene, zygotene and pachytene are described. Comparative analysis of SCs of spermatocytes and oocytes showed a difference in the synaptic process, i.e. in spermatocytes all LEs were synapsed before the appearance of centromeric regions in the biarmed elements, while in the oocytes some fully synapsed LEs, including the centromeric region of the biarmed elements, were found together with fully or partially unsynapsed LEs. In males the sex chromosome synapsis starts only after all autosomes have synapsed. Irregular synapses involving three or four LEs were found in 3.4% of the cells analyzed in mid or late zygotene. Multivalents were found in males and females. Some aspects of initial meiotic development and their implications in rainbow trout cytogenetics, genetics and evolution are discussed.

EFFECT OF LATERAL HYPOTHALAMUS LESIONS ON THE WATER AND SALT INTAKE, AND SODIUM AND URINE EXCRETION INDUCED BY ACTIVATION OF THE MEDIAN PREOPTIC NUCLEUS IN CONSCIOUS RATS

In this-study we investigated the influence of electrolytic lesion of the lateral hypothalamus (LH) on the water and salt appetite, and the natriuretic, diuretic and cardiovascular effects induced by angiotensinergic, cholinergic and noradrenergic stimulation of the median preoptic nucleus (MnPO) in rats. Male Holtzman rats were implanted with a cannula into the MnPO. Other groups of sham- and LH-lesioned rats received a stainless steel cannula implanted into the MnPO. ANGII injection into the MnPO induced water and sodium intake, and natriuretic, diuretic, presser and tachycardic responses. Carbachol induced water intake, and natriuretic, presser and bradycardic responses, whereas noradrenaline increased urine, sodium excretion and blood pressure, and induced bradycardia. In rats submitted to LH-lesion only, water and sodium intake was reduced compared with sham rats. LH lesion also reduced the sodium ingestion induced by ANGII (12 ng) into the MnPO. In LH-lesioned rats, the dipsogenic, diuretic and presser responses induced by ANGII (12 ng), carbachol (2 nmol) and noradrenaline (20 nmol) injection into the MnPO were reduced. The same occurred with sodium excretion when carbachol (2 nmol) and noradrenaline (20 nmol) were injected into the MnPO of LH-lesioned rats, whereas ANGII(12 ng) induced an increase in sodium excretion. These data show that electrolytic lesion of the LH reduces fluid and sodium intake, and presser responses to angiotensinergic, cholinergic and noradrenergic activation of the MnPO. LH involvement with MnPO excitatory and inhibitory mechanisms related to water and sodium intake, sodium excretion and cardiovascular control is suggested.

Light microscopy and computer three-dimensional reconstruction of the blood capillaries of the enamel organ of rat molar tooth germs

We performed a light microscope and a computer three-dimensional reconstruction study of serial sections of the molar enamel organ of 3- and 5-day-old rats perfused with Indian ink through the arterial system. The tooth germs were fixed in Bouin's solution, embedded in paraffin, sectioned and stained with haematoxylin and eosin. For the three-dimensional reconstruction, light micrographs of the serial sections were digitized, and aligned using the serial EM Align software downloaded from http://synapses.bu.edu/tools/. After alignment, the boundaries of the India-ink-filled blood vessels were manually traced with a mouse using the software IGL trace (version 1.26b), also downloaded from the above website. After tracing, a three-dimensional representation of the blood vessel contours was generated in a VRML format and visualized with the help of the software Cortona Web3D viewer (version 4.0) downloaded from http://www.parallelgraphics.com/products/cortona. Our results showed that in regions where ameloblasts are polarized the capillaries are arranged in three distinct levels: (1) penetrating and leaving capillaries in relation to the outer enamel epithelium; (2) capillaries crossing and branching inside the stellate reticulum; and (3) capillaries branching and anastomosing profusely within the stratum intermedium, thereby forming an extensive capillary plexus intimately associated with the cells of the stratum intermedium. The existence of a conspicuous capillary plexus intermingled with cells of the stratum intermedium, as shown in our results, suggests that some molecules produced by cells of the stratum intermedium could be released into the capillary plexus and thereafter carried to the dental follicle.

ROLE OF LATERAL HYPOTHALAMUS ON FLUID, ELECTROLYTE, AND CARDIOVASCULAR-RESPONSES TO ACTIVATION OF THE MSA

In this study we investigated the influence of electrolytic lesion or of opioid agonist injections into the lateral hypothalamus (LH) on the dipsogenic, natriuretic, kaliuretic, antidiuretic, presser, and bradycardic effects of cholinergic stimulation of the medial septal area (MSA) in rats. Sham- and LH-lesioned male Holtzman rats received a stainless steel cannula implanted into the LH. Other groups of rats had cannulas implanted simultaneously into the MSA and LH. Carbachol (2 nmol) injection into the MSA induced water intake, presser, and bradycardic responses. LH lesion reduced all of these effects (1-3 and 15-18 days). Previous injection of synthetic opiate agonist, FK-33824 (100 ng), into the LH reduced the water intake, natriuresis, kaliuresis, and presser responses induced by carbachol injected into the MSA. These data show that both electrolytic lesion or injection of an opiate agonist in the LH reduces the fluid-electrolyte and cardiovascular responses to cholinergic activation of the MSA. The involvement of LH with central excitatory and inhibitory mechanisms related to fluid-electrolytic and cardiovascular control is suggested.

Commissural NTS contributes to pressor responses to glutamate injected into the medial NTS of awake rats

In the present study we investigated whether interruption of the chemoreceptor reflex by an electrolytic lesion of the commissural subnucleus of the nucleus tractus solitarii (commNTS) influenced presser and bradycardic responses induced by microinjection of L-glutamate (L-Glu) into the medial NTS (mNTS) of conscious rats. Seven days after sham lesions, seven rats demonstrated significant presser [change in mean arterial pressure (MAP) = +33 +/- 3 mmHg] and bradycardic [change in heart rate (HR) = -74 +/- 8 beats/min (bpm)] responses to chemoreceptor reflex activation by intravenous injection of KCN. Likewise, L-Glu (1 nmol in 100 nl) injected into the mNTS in sham rats induced presser (+29 +/- 2 mmHg) and bradycardic responses (-90 +/- 8 bpm). However, in 11 rats with lesions in commNTS, presser and bradycardic chemoreceptor reflex responses were abolished, and injection of L-Glu into the mNTS decreased MAP (-14 +/- 6 mmHg) and HR (-59 +/- 16 bpm) as is reported in anesthetized control rats. We conclude that presser responses induced by L-Glu microinjected into the baroreceptor reflex region of mNTS in conscious rats depend on the integrity of the commNTS, which plays an important role in central chemoreceptor reflex pathways.

An animal model for the study of Chamomilla in stress and depression: pilot study

The behavioral and hematological effects of treatment with Chamomilla 6cH in mice subjected to experimental stress are described. Swiss mice were randomly divided into pairs, one animal was inoculated with Ehrlich's tumor, the other was treated daily with Chamomilla 6cH or control or received no treatment. After 7 days, the animals were observed in an open-field arena and blood samples taken. Mice who cohabitated with a sick cage-mate showed a decrease in their general activity, but those treated with Chamomilla 6cH were less severely affected (p = 0.0426). No hematologicall changes were observed.In a second experiment, the forced swimming test was applied to mice pre-treated with Chamomilla 6cH, controls were: water, 10% ethanol or amitriptyline. Only the amitriptyline and ethanol treated groups showed significant excitatory behavior (p = 0.0020), Chamomilla 6cH treated animals' scores intermediate between water control and ethanol or amitriptyline. A decrease in the leukocyte count was observed in the amitriptyline and Chamomilla 6cH treated groups (p = 0.039). These data suggest that treatment with Chamomilla 6cH is related to the recovery of basal behavioral conditions in mice subjected to stressful conditions.