Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are modulated by photic and non-photic stimuli. In rodents, direct photic stimuli reach the SCN mainly through the retinohypothalamic tract (RHT), whereas indirect photic stimuli are mainly conveyed by the geniculohypothalamic tract (GHT). In rodents, retinal cells form a pathway that reaches the intergeniculate leaflet (IGL) where they establish synapses with neurons that express neuropeptide Y (NPY), hence forming the GHT projecting to the SCN. In contrast to the RHT, which has been well described in primates, data regarding the presence or absence of the IGL and GHT in primates are contradictory. Some studies have suggested that an area of the pregeniculate nucleus (PGN) of primates might be homologous to the IGL of rodents, but additional anatomical and functional studies on primate species are necessary to confirm this hypothesis. Therefore, this study investigated the main histochemical characteristics of the PGN and the possible existence of the GHT in the SCN of the primate Cebus, comparing the distribution of NPY immunoreactivity, serotonin (5-HT) immunoreactivity and retinal terminal fibers in these two structures. The results show that a collection of cell bodies containing NPY and serotonergic immunoreactivity and retinal innervations are present within a zone that might be homologous to the IGL of rodents. The SCN also receives dense retinal innervations and we observed an atypical distribution of NPY- and 5-HT-immunoreactive fibers without regionalization in the ventral part of the nucleus as described for other species. These data may reflect morphological differences in the structures involved in the regulation of circadian rhythms among species and support the hypothesis that the GHT is present in some higher primates (diurnal animals). (C) 2009 Elsevier B.V. All rights reserved.

5-HT(1B) receptor in the suprachiasmatic nucleus of the common marmoset (Callithrix jacchus)

Serotonin (5-HT) is involved in the fine adjustments at several brain centers including the core of the mammal circadian timing system (CTS) and the hypothalamic suprachiasmatic nucleus (SCN). The SCN receives massive serotonergic projections from the midbrain raphe nuclei, whose inputs are described in rats as ramifying at its ventral portion overlapping the retinohypothalamic and geniculohypothalamic fibers. In the SCN, the 5-HT actions are reported as being primarily mediated by the 5-HT1 type receptor with noted emphasis for 5-HT(1B) subtype, supposedly modulating the retinal input in a presynaptic way. In this study in a New World primate species, the common marmoset (Callithrix jacchus), we showed the 5-HT(1B) receptor distribution at the dorsal SCN concurrent with a distinctive location of 5-HT-immunoreactive fibers. This finding addresses to a new discussion on the regulation and synchronization of the circadian rhythms in recent primates. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

GABA and nitric oxide in the PVN are involved in arterial pressure control but not in the chemoreflex responses in rats

GABAergic, nitrergic and glutamatergic mechanisms in the PVN on the baseline mean arterial pressure (MAP), heart rate (HR) and on the cardiovascular responses to chemoreflex activation in awake rat were evaluated. Chemoreflex was activated with KCN before and after microinjections into the PVN. Bicuculline into the PVN increased baseline MAP (94+/-3 vs 113+/-5 mmHg) and HR (350+/-9 vs 439+/-18 bpm) but had no effect on the pressor (49+/-5 vs 47+/-6 mmHg) or bradicardic (-213+/-23 vs -256+/-42 bpm) responses (n=7). Kynurenic acid into the PVN (n=6) produced no significant changes in the MAP (98+/-3 vs 100+/-3 mmHg), HR (330+/-5 vs 339+/-12 mmHg) or in the pressor (50+/-4 vs 42+/-4 mmHg) and bradicardic (-252+/-4 vs -285+/-16 bpm) responses to chemoreflex. L-NAME into the PVN (n=8) produced increase in the MAP (94+/-3 vs 113+/-5 mmHg) and HR (350+/-9 vs 439+/-18 bpm) but had no effect on the pressor (52+/-5 vs 47+/-6 mmHg) or bradicardic (-253+/-19 vs -320+/-25 bpm) responses to chemoreflex. We conclude that GABA(A) and nitric oxide in the PVN are involved in the maintenance of the baseline MAP but not in the modulation of the responses to chemoreflex. The results also show that Glutamate receptors in the PVN are not involved in maintenance of the baseline MAP, HR or in the cardiovascular responses to chemoreflex in awake rats. (C) 2008 Elsevier B.V. All rights reserved.

GABAergic mediation of stress-induced secretion of corticosterone and oxytocin, but not prolactin, by the hypothalamic paraventricular nucleus

The hypothalamus-pituitary-adrenal axis (HPA) participates in mediating the response to stressful stimuli. Within the HPA, neurons in the medial parvocellular region of paraventricular nucleus (PVN) of the hypothalamus integrate excitatory and inhibitory signals triggering secretion of corticotropin-releasing hormone (CRH), the main secretagogue of adrenocorticotropic hormone (ACTH). Stressful situations alter CRH secretion as well as other hormones, including prolactin and oxytocin. Most inputs to the PVN are of local origin, half of which are GABAergic neurons, and both GABA-A and GABA-B receptors are present in the PVN. The objective of the present study was to investigate the role of GABA-A and GABA-B receptors in the PVN`s control of stress-induced corticosterone, oxytocin and prolactin secretion. Rats Were microinjected with saline or different doses (0.5, 5 and 50 pmol) of GABA-A (bicuculine) or GABA-B (phaclofen) antagonists in the PVN. Ten minutes later, they were subjected to a stressor (ether inhalation) and blood samples were collected 30 min before and 10, 30, 60, 90 and 120 min after the stressful stimulus to measure hormone levels by radioimmunoassay. Our results indicate that GABA acts in the PVN to inhibit stress-induced corticosterone secretion via both its receptor subtypes, especially GABA-B. In contrast, GABA in the PVN stimulates oxytocin secretion through GABA-B receptors and does not alter prolactin secretion. (C) 2008 Elsevier Inc. All rights reserved.

APM/CD13 and FOS in the hypothalamus of monosodium glutamate obese and food deprived rats

Protein (western blotting) and gene (PCR) expressions, catalytic activity of puromycin-insensitive membrane-bound neutral aminopeptidase (APM/CD13) and in situ regional distribution of CD13 and FOS immunoreactivity (it) were evaluated in the hypothalamus of monosodium glutamate obese (MSG) and/or food deprived (FD) rats in order to investigate their possible interplay with metabolic functions. Variations in protein and gene expressions of CD13 relative to controls coincided in the hypothalamus of MSG and MSG-FD (decreased 2- to 17-fold). Compared with controls, the reduction of hypothalamic CD13 content reflected a negative balance in its regional distribution in the supraoptic, paraventricular, periventricular and arcuate nuclei. CD13-ir increased in the supraoptic nucleus in MSG (2.5-fold) and decreased in the paraventricular nucleus (2-fold) together with FOS-ir (1.5-fold) in FD. In MSG-FD. FOS-ir decreased (7-fold) in the paraventricular nucleus, while CD13-ir decreased in the periventricular (5.6-fold) and the arcuate (3.7-fold) nuclei. It was noteworthy that all these changes of CD13 were not related to catalytic activity of APM. Data suggested that hypothalamic CD13 plays a role in the regulation of energy metabolism not by means of APM enzyme activity. (c) 2010 Elsevier B.V. All rights reserved.

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 ± 0.39 mL and experimental = 2.71 ± 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 ± 16.73 x 106 and experimental = 144.60 ± 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 ± 43.22 x 106 and experimental = 349.40 ± 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 ± 48.3 ng/mL and experimental = 420.0 ± 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 ± 19.7 and experimental = 116.2 ± 4.6; P < 0.05) and oxidative burst (control = 173.7 ± 40.8 and experimental= 99.58 ± 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Gabaergic mechanisms of hypothalamic nuclei in the expression of conditioned fear

The amygdala, the dorsal periaqueductal gray (dPAG), and the media] hypothalamus have long been recognized to be a neural system responsible for the generation and elaboration of unconditioned fear in the brain. It is also well known that this neural substrate is under a tonic inhibitory control exerted by GABA mechanisms. However, whereas there is a growing body of evidence to suggest that the amygdala and dPAG are also able to integrate conditioned fear, it is still unclear, however, how the distinct hypothalamic nuclei participate in fear conditioning. In this work we aimed to examine the extent to which the gabaergic mechanisms of this brain region are involved in conditioned fear using the fear-potentiated startle (FPS). Muscimol, a GABA-A receptor agonist, and semicarbazide, an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD), were used as an enhancer and inhibitor of the GABA mechanisms, respectively. Muscimol and semicarbazide were injected into the anterior hypothalamus (AHN). the dorsomedial part of the ventromedial nucleus (VMHDM), the dorsomedial (DMH) or the dorsal premammillary (PMD) nuclei of male Wistar rats before test sessions of the fear conditioning paradigm. The injections into the DMH and PMD did not produce any significant effects on FPS. On the other hand, muscimol injections into the AHN and VMHDM caused significant reduction in FPS. These results indicate that injections of muscimol and semicarbazide into the DMH and PMD fail to change the FPS, whereas the enhancement of the GABA transmission in the AHN and VMHDM produces a reduction of the conditioned fear responses. On the other hand, the inhibition of this transmission led to an increase of this conditioned response in the AHN. Thus, whereas DMH and PMD are known to be part of the caudal-most region of the medial hypothalamic defensive system, which integrates unconditioned fear, systems mediating conditioned fear select the AHN and VMHDM nuclei that belong to the rostral-most portion of the hypothalamic defense area. Thus, distinct subsets of neurons in the hypothalamus could mediate different aspects of the defensive responses. (C) 2008 Elsevier Inc. All rights reserved.

Serotonergic mechanisms of the median raphe nucleus-dorsal hippocampus in conditioned fear: Output circuit involves the prefrontal cortex and amygdala

Independent studies have shown that the median raphe nucleus (MRN) and dorsal hippocampus (DH) are involved in the expression of contextual conditioned fear (CFC). However, studies that examine the integrated involvement of serotonergic mechanisms of the MRN-DH are lacking. To address this issue, a CFC paradigm was used to test whether the serotonergic projections from the MRN to DH can influence CFC. Serotoninergic drugs were infused either into the MRN or DH prior to testing sessions in which freezing and startle responses were measured in the same context where 6 h previously rats received footshocks. A reduction of serotonin (5-HT) transmission in the MRN by local infusions of the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) decreased freezing in response to the context but did not reduce fear-potentiated startle. This pattern of results is consistent with the hypothesis that MRN serotonergic mechanisms selectively modulate the freezing response to the aversive context. As for the DH, a decrease in postsynaptic 5-HT receptor activity at projection areas has been proposed to be the main consequence of 5-HT(1A) receptor activation in the MIRN. Intra-DH injections of 8-OH-DPAT inhibited both the freezing and fear-potentiated startle response to the context. To reconcile these findings, an inhibitory mechanism may exist between the incoming 5-HT pathway from the MRN to DH and the neurons of the DH output to other structures. The DH-amygdala or medial prefrontal cortex projections could well be this output circuit modulating the expression of CFC as revealed by measurements of Fos immunoreactivity in these areas. (C) 2009 Elsevier B.V. All rights reserved.

Paraventricular nucleus modulates autonomic and neuroendocrine responses to acute restraint stress in rats

The paraventricular nucleus of the hypothalamus (PVN) has been implicated in several aspects of neuroendocrine and cardiovascular control The PVN contains parvocellular neurons that release the corticotrophin release ha mone (CRH) under stress situations In addition this brain area is connected to several limbic structures implicated in defensive behavioral control as well to forebrain and brainst m structures involved in cardiovascular control Acute restraint is an unavoidable stress situation that evokes corticosterone release as well as marked autonomic changes the latter characterized by elevated mean arterial pressure (MAP) intense heart rate (HR) Increases and decrease in the tail temperature We report the effect of PVN inhibition on MAP and HR responses corticosterone plasma levels and tail temperature response during acute restraint in rats Bilateral microinjection of the nonspecific synaptic blocker CoCl(2) (1 mM/100 nL) into the PVN reduced the pressor response it inhibited the increase in plasma corticosterone concentration as well as the fall in tail temperature associated with acute restraint stress Moreover bilateral microinjection of CoCl(2) into areas surrounding the PVN did not affect the blood pressure hormonal and tail vasoconstriction responses to restraint stress The present results show that a local PVN neurotransmission is involved in the neural pathway that controls autonomic and neuroendocrine responses which are associated with the exposure to acute restraint stress (C) 2010 Elsevier B V All rights reservi.d

Oestradiol Potentiates Hormone Secretion and Neuronal Activation in Response to Hypertonic Extracellular Volume Expansion in Ovariectomised Rats

Secretion of vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) is an essential mechanism for the maintenance of hydromineral homeostasis. Secretion of these hormones is modulated by several circulating factors, including oestradiol. However, it remains unclear how oestradiol exerts this modulation. In the present study we investigated the participation of oestradiol in the secretion of VP, OT and ANP and in activation of vasopressinergic and oxytocinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to extracellular volume expansion (EVE). For this purpose, ovariectomised (OVX) rats treated for 7 days with vehicle (corn oil, 0.1 ml/rat, OVX+O group) or oestradiol (oestradiol cypionate, 10 mu g/kg, OVX+E group) were subjected to either isotonic (0.15 m NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 m NaCl, 2 ml/100 g b.w., i.v.) EVE. Blood samples were collected for plasma VP, OT and ANP determination. Another group of rats was subjected to cerebral perfusion, and brain sections were processed for c-Fos-VP and c-Fos-OT double-labelling immunohistochemistry. In OVX+O rats, we observed that both isotonic and hypertonic EVE increased plasma OT and ANP concentrations, although no changes were observed in VP secretion. Oestradiol replacement did not alter hormonal secretion in response to isotonic EVE, but it increased VP secretion and potentiated plasma OT and ANP concentrations in response to hypertonic EVE. Immunohistochemical data showed that, in the OVX+O group, hypertonic EVE increased the number of c-Fos-OT and c-Fos-VP double-labelled neurones in the PVN and SON. Oestradiol replacement did not alter neuronal activation in response to isotonic EVE, but it potentiated vasopressinergic and oxytocinergic neuronal activation in the medial magnocellular PVN (PaMM) and SON. Taken together, these results suggest that oestradiol increases the responsiveness of vasopressinergic and oxytocinergic magnocellular neurones in the PVN and SON in response to osmotic stimulation.

A combined study of behavior and Fos expression in limbic structures after re-testing Wistar rats in the elevated plus-maze

The elevated plus-maze is an animal model used to study anxiety. In a second session, rats show a reduction in the exploratory behavior even when the two sessions are separated by intervals as large as 7 days. The aim of the present study was to investigate whether the reduction in the exploratory behavior is maintained after intervals larger than 7 days. Additionally, we aimed at investigating eventual correlations between behaviors in the plus-maze and activation of limbic structures as measured by Fos protein expression after the second session. Rats were tested for 5 min in the elevated plus-maze and re-tested 3, 9 or 33 days later. Other groups were tested only once. The rat brains were processed for immunohistochemical detection of Fos protein. The results show a decrease in the open arms exploration in the second trial with intervals of 3, 9 and 33 days. The expression of Fos protein in the piriform cortex, septal nucleus and paraventricular hypothalamic nucleus in the groups tested with intervals of 9 and 33 days were statistically different from the other groups. The alterations observed in exploratory behavior in the second session in the plus-maze did not correlate with Fos expression. In conclusion, although the specific test conditions were sufficient to evoke behavioral alterations in exploration in the elevated plus-maze, they were enough to induce significant Fos protein expression in piriform cortex, septal nucleus and thalamic and hypothalamic paraventricular nuclei but not in other areas such as dorsomedial nucleus of the hypothalamus and amygdala nuclei, known to be also active participants in circuits controlling fear and anxiety. (C) 2010 Elsevier Inc. All rights reserved.

Central but not systemic inhibition of inducible nitric oxide synthase modulates oxytocin release during endotoxemic shock

Previous studies have shown that immunological challenges as lipopolysaccharide (LPS) administration increases plasma oxytocin (OT) concentration. Nitric oxide (NO), a free radical gas directly related to the immune system has been implicated in the central modulation of neuroendocrine adaptive responses to immunological stress. This study aimed to test the hypothesis that the NO pathway participates in the control of OT release induced by LPS injection. For this purpose, adult male Wistar rats received bolus intravenous (i.v.) injection of LPS, preceded or not by iv. or intracerebroventricular (i.c.v.) injections of aminoguanidine (AG), a selective inducible nitric oxide synthase (iNOS) inhibitor. Rats were decapitated after 2, 4 and 6 h of treatment, for measurement of OT by radioimmunoassay. In a separate set of experiments, mean arterial pressure (MAP) and heart rate (HR) were measured every 15 min over 6 h, using a polygraph. These studies revealed that LPS reduced MAP and increased HR at 4 and 6 h post-injection. LPS significantly increased plasma OT concentration at 2 and 4 h post-injection. Pre-treatment with i.c.v. AG further increased plasma OT concentration and attenuated the LPS-induced decrease in MAP, however, i.v. AG failed to show similar effects. Thus, iNOS pathway may activate a central inhibitory control mechanism that attenuates OT secretion during endotoxemic shock. (C) 2009 Elsevier Inc. All rights reserved.

Hypothalamic oxytocin neurons modulate hypophagic effect induced by adrenalectomy

Glucocorticoids have major effects on food intake, as demonstrated by the decrease of food intake following adrenalectomy (ADX); however, the mechanisms leading to these effects are not well understood. Oxytocin (OT) has been shown to reduce food intake. We evaluated the effects of glucocorticoids on OT neuron activation and OT mRNA expression in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei induced by feeding. We also evaluated the effect of pretreatment with OT-receptor antagonist ([d(CH2)5,Tyr(Me)2,Orn8]-vasotocin, OVT) on food intake in ADX rats. Fos/OT neurons in the posterior parvocellular subdivision of the PVN were increased after refeeding, with a higher number in the ADX group, compared with sham and ADX+corticosterone (B) groups, with no difference in the medial parvocellular and magnocellular subdivisions of the PVN. ADX increased OT mRNA expression in the PVN both in fasting and refeeding condition, compared with sham and ADX+B groups. In the SON, refeeding increased the number of Fos/OT neurons, with a higher number in the ADX+B group. In fasted condition, OT mRNA expression in the SON was increased in ADX and ADX+B, compared with sham group. Pretreatment with OVT reversed the ADX-induced hypophagia, with no difference between sham and ADX+B animals. The present results show that glucocorticoid withdrawal induces a higher activation of PVN OT neurons in response to feeding, and an increase of OT mRNA expression in the PVN and OT-receptor antagonist reverses the anorexigenic effect induced by ADX These data indicate that PVN OT neurons might mediate the hypophagic effect induced by adrenalectomy. (C) 2009 Elsevier Inc. All rights reserved.

Hypophagia induced by glucocorticoid deficiency is associated with an increased activation of satiety-related responses

Uchoa ET, Sabino HA, Ruginsk SG, Antunes-Rodrigues J, Elias LL. Hypophagia induced by glucocorticoid deficiency is associated with an increased activation of satiety-related responses. J Appl Physiol 106: 596-604, 2009. First published November 20, 2008; doi: 10.1152/japplphysiol.90865.2008.-Glucocorticoids have major effects on food intake, demonstrated by the decrease of food intake following adrenalectomy. Satiety signals are relayed to the nucleus of the solitary tract (NTS), which has reciprocal projections with the arcuate nucleus (ARC) and paraventricular nucleus (PVN) of the hypothalamus. We evaluated the effects of glucocorticoids on the activation of hypothalamic and NTS neurons induced by food intake in rats subjected to adrenalectomy (ADX) or sham surgery 7 days before the experiments. One-half of ADX animals received corticosterone (ADX + B) in the drinking water (B: 25 mg/l). Fos/tyrosine hydroxylase (TH), Fos/corticotrophin-releasing factor (CRF) and Fos immunoreactivity were assessed in the NTS, PVN, and ARC, respectively. Food intake and body weight were reduced in the ADX group compared with sham and ADX + B groups. Fos and Fos/TH in the NTS, Fos, and Fos/CRF immunoreactive neurons in the PVN and Fos in the ARC were increased after refeeding, with higher number in the ADX group, compared with sham and ADX + B groups. CCK administration showed no hypophagic effect on ADX group despite a similar increase of Fos/TH immunoreactive neurons in the NTS compared with sham and ADX + B groups, suggesting that CCK alone cannot further increase the anorexigenic effect induced by glucocorticoid deficiency. The present data indicate that glucocorticoid withdrawal reduced food intake, which was associated with higher activation of ARC, CRF neurons of the PVN, and catecholaminergic neurons of the NTS. In the absence of glucocorticoids, satiety signals elicited during a meal lead to an augmented activation of brain stem and hypothalamic pathways.

Are l-glutamate and ATP cotransmitters of the peripheral chemoreflex in the rat nucleus tractus solitarius ?

Peripheral chemoreflex activation in awake rats or in the working heart-brainstem preparation (WHBP) produces sympathoexcitation, bradycardia and an increase in the frequency of phrenic nerve activity. Our focus is the neurotransmission of the sympathoexcitatory component of the chemoreflex within the nucleus of the tractus solitarius (NTS), and recently we verified that the simultaneous antagonism of ionotropic glutamate and purinergic P(2) receptors in the NTS blocked the pressor response and increased thoracic sympathetic activity in awake rats and WHBP, respectively, in response to peripheral chemoreflex activation. These previous data suggested the involvement of ATP and L-glutamate in the NTS in the processing of the sympathoexcitatory component of the chemoreflex by unknown mechanisms. For a better understanding of these mechanisms, here we used a patch-clamp approach in brainstem slices to evaluate the characteristics of the synaptic transmission of NTS neurons sending projections to the ventral medulla, which include the premotor neurons involved in the generation of the sympathetic outflow. The NTS neurons sending projections to the ventral medulla were identified by previous microinjection of the membrane tracer dye, 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate (DiI), in the ventral medulla and the spontaneous (sEPSCs) and tractus solitarius (TS)-evoked excitatory postsynaptic current (TS-eEPSCs) were recorded using patch clamp. With this approach, we made the following observations on NTS neurons projecting to the ventral medulla: (i) the sEPSCs and TS-eEPSCs of DiI-labelled NTS neurons were completely abolished by 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), an antagonist of ionotropic non-NMDA glutamatergic receptors, showing that they are mediated by L-glutamate; (ii) application of ATP increased the frequency of appearance of spontaneous glutamatergic currents, reflecting an increased exocytosis of glutamatergic vesicles; and (iii) ATP decreased the peak of TS-evoked glutamatergic currents. We conclude that L-glutamate is the main neurotransmitter of spontaneous and TS-evoked synaptic activities in the NTS neurons projecting to the ventral medulla and that ATP has a dual modulatory role on this excitatory transmission, facilitating the spontaneous glutamatergic transmission and inhibiting the TS-evoked glutamatergic transmission. These data also suggest that ATP is not acting as a cotransmitter with L-glutamate, at least at the level of this subpopulation of NTS neurons studied.