The impact of ageing, fasting and high-fat diet on central and peripheral glucose tolerance and glucose-sensing neural networks in the arcuate nucleus.

Obesity and ageing are risk factors for diabetes. In the present study, we investigated the effects of ageing, obesity and fasting on central and peripheral glucose tolerance and on glucose-sensing neuronal function in the arcuate nucleus of rats, with a view to providing insight into the central mechanisms regulating glucose homeostasis and how they change or are subject to dysfunction with ageing and obesity. We show that, following a glucose load, central glucose tolerance at the level of the cerebrospinal fluid (CSF) and plasma is significantly reduced in rats maintained on a high-fat diet (HFD). With ageing, up to 2 years, central glucose tolerance was impaired in an age-dependent manner, whereas peripheral glucose tolerance remained unaffected. Ageing-induced peripheral glucose intolerance was improved by a 24-hour fast, whereas central glucose tolerance was not corrected. Pre-wean, immature animals have elevated basal plasma glucose levels and a delayed increase in central glucose levels following peripheral glucose injection compared to mature animals. Electrophysiological recording techniques revealed an energy-status-dependent role for glucose-excited, inhibited and adapting neurones, along with glucose-induced changes in synaptic transmission. We conclude that ageing affects central glucose tolerance, whereas HFD profoundly affects central and peripheral glucose tolerance and, in addition, glucose-sensing neurones adapt function in an energy-status-dependent manner.

Endocannabinoid 2-AG and intracellular cannabinoid receptors modulate a low-threshold calcium spike-induced slow depolarizing afterpotential in rat thalamic paraventricular nucleus neurons.

In rat paraventricular thalamic nucleus (PVT) neurons, activation of low-threshold calcium (Ca(2+)) channels triggers a low-threshold spike (LTS) which may be followed by slow afterpotentials that can dramatically influence action potential patterning. Using gluconate-based internal recording solutions, we investigated the properties of a LTS-induced slow afterdepolarization (sADP) observed in a subpopulation of PVT neurons recorded in brain slice preparations. This LTS-induced sADP required T-type Ca(2+) channel opening, exhibited variable magnitudes between neurons and a voltage dependency with a maximum near -50 mV. The area under the sADP remained stable during control monitoring, but displayed gradual suppression in media where strontium replaced Ca(2+). The sADP was suppressed following bath application of 2-APB or ML204, suggesting engagement of transient receptor potential canonical (TRPC)-like channels. Further investigation revealed a reversible suppression during bath applications of membrane permeable cannabinoid receptor (CBR) blockers rimonabant, AM630 or SR144528 suggesting the presence of both CB1Rs and CB2Rs. Similar results were achieved by intracellular, but not bath application of the membrane impermeant CB1R blocker hemopressin, suggesting an intracellular localization of CB1Rs. Data from pharmacologic manipulation of endocannabinoid biosynthetic pathways suggested 2-arachidonlyglycerol (2-AG) as the endogenous cannabinoid ligand, derived via hydrolysis of diacylglycerol (DAG), with the latter formed from the pathway involving phosphatidylcholine-specific phospholipase D and phosphatic acid phosphohydrolase. The sADP suppression observed during recordings with pipettes containing LY294002, a PI3-kinase inhibitor, suggested a role for PI3kinase in the translocation of these TRPC-like channels to the plasma membrane. Drug-induced attenuation of the availability of 2-AG influences the number of action potentials that surmount the LTS evoked in PVT neurons, implying an ongoing intracellular CBR modulation of neuronal excitability during LTS-induced bursting behavior.

Intracellular postsynaptic cannabinoid receptors link thyrotropin-releasing hormone receptors to TRPC-like channels in thalamic paraventricular nucleus neurons.

In rat thalamic paraventricular nucleus of thalamus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances excitability via concurrent decrease in G protein-coupled inwardly-rectifying potassium (GIRK)-like and activation of transient receptor potential cation (TRPC)4/5-like cationic conductances. An exploration of intracellular signaling pathways revealed the TRH-induced current to be insensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitors, but reduced by D609, an inhibitor of phosphatidylcholine-specific PLC (PC-PLC). A corresponding change in the I-V relationship implied suppression of the cationic component of the TRH-induced current. Diacylglycerol (DAG) is a product of the hydrolysis of PC. Studies focused on the isolated cationic component of the TRH-induced response revealed a reduction by RHC80267, an inhibitor of DAG lipase, the enzyme involved in the hydrolysis of DAG to the endocannabinoid 2-arachidonoylglycerol (2-AG). Further investigation revealed enhancement of the cationic component in the presence of either JZL184 or WWL70, inhibitors of enzymes involved in the hydrolysis of 2-AG. A decrease in the TRH-induced response was noted in the presence of rimonabant or SR144528, membrane permeable CB1 and CB2 receptor antagonists, respectively. A decrease in the TRH-induced current by intracellular, but not by bath application of the membrane impermeable peptide hemopressin, selective for CB1 receptors, suggests a postsynaptic intracellular localization of these receptors. The TRH-induced current was increased in the presence of arachidonyl-2'-chloroethylamide (ACEA) or JWH133, CB1 and CB2 receptor agonists, respectively. The PI3-kinase inhibitor LY294002, known to inhibit TRPC translocation, decreased the response to TRH. In addition, a TRH-induced enhancement of the low-threshold spike was prevented by both rimonabant, and SR144528. TRH had no influence on excitatory or inhibitory miniature postsynaptic currents, suggesting presynaptic CB receptors are not involved in this situation. Collectively, the data imply that activation of TRH receptors in these midline thalamic neurons engages novel signaling pathways that include postsynaptic intracellular CB1 and CB2 receptors in the activation of TRPC4/5-like channels.

Gastrin-releasing peptide acts via postsynaptic BB2 receptors to modulate inward rectifier K+ and TRPV1-like conductances in rat paraventricular thalamic neurons.

Gastrin-releasing peptide (GRP) is a bombesin-like peptide with a widespread distribution in mammalian CNS, where it has a role in food intake, circadian rhythm generation, fear memory, itch sensation and sexual behaviour. While it has been established that GRP predominantly excites neurons, details of the membrane mechanism involved in this action remain largely undefined. We used perforated patch clamp recording in acute brain slice preparations to investigate GRP-affected receptors and ionic conductances in neurons of the rat paraventricular thalamic nucleus (PVT). PVT is a component of the midline and intralaminar thalamus that participates in arousal, motivational drives and stress responses, and exhibits a prominence of GRP-like immunoreactive fibres. Exposure of PVT neurons to low nanomolar concentrations of GRP induced sustained TTX-resistant membrane depolarizations that could trigger rhythmic burst discharges or tonic firing. Membrane current analyses in voltage clamp revealed an underlying postsynaptic bombesin type 2 receptor-mediated inward current that resulted from the simultaneous suppression of a Ba(2+)-sensitive inward rectifier K(+) conductance and activation of a non-selective cation conductance with biophysical and pharmacological properties reminiscent of transient receptor potential vanilloid (TRPV) 1. A role for a TRPV1-like conductance was further implied by a significant suppressant influence of a TRPV1 antagonist on GRP-induced membrane depolarization and rhythmic burst or tonic firing. The results provide a detailed picture of the receptor and ionic conductances that are involved in GRP's excitatory action in midline thalamus.

Electrophysiological, pharmacological and molecular profile of the transient outward rectifying conductance in rat sympathetic preganglionic neurons in vitro.

Transient outward rectifying conductances or A-like conductances in sympathetic preganglionic neurons (SPN) are prolonged, lasting for hundreds of milliseconds to seconds and are thought to play a key role in the regulation of SPN firing frequency. Here, a multidisciplinary electrophysiological, pharmacological and molecular single-cell rt-PCR approach was used to investigate the kinetics, pharmacological profile and putative K+ channel subunits underlying the transient outward rectifying conductance expressed in SPN. SPN expressed a 4-aminopyridine (4-AP) sensitive transient outward rectification with significantly longer decay kinetics than reported for many other central neurons. The conductance and corresponding current in voltage-clamp conditions was also sensitive to the Kv4.2 and Kv4.3 blocker phrixotoxin-2 (1-10 µM) and the blocker of rapidly inactivating Kv channels, pandinotoxin-Kα (50 nM). The conductance and corresponding current was only weakly sensitive to the Kv1 channel blocker tityustoxin-Kα and insensitive to dendrotoxin I (200 nM) and the Kv3.4 channel blocker BDS-II (1 µM). Single-cell RT-PCR revealed mRNA expression for the α-subunits Kv4.1 and Kv4.3 in the majority and Kv1.5 in less than half of SPN. mRNA for accessory ß-subunits was detected for Kvß2 in all SPN with differential expression of mRNA for KChIP1, Kvß1 and Kvß3 and the peptidase homologue DPP6. These data together suggest that the transient outwardly rectifying conductance in SPN is mediated by members of the Kv4 subfamily (Kv4.1 and Kv4.3) in association with the ß-subunit Kvß2. Differential expression of the accessory ß subunits, which may act to modulate channel density and kinetics in SPN, may underlie the prolonged and variable time-course of this conductance in these neurons.

Postsynaptic and presynaptic group II metabotropic glutamate receptor activation reduces neuronal excitability in rat midline paraventricular thalamic nucleus.

Drugs that interact with group II metabotropic glutamate receptors (mGluRs) are presently being evaluated for a role in the treatment of anxiety disorders and symptoms of schizophrenia. Their mechanism of action is believed to involve a reduction in excitatory neurotransmission in limbic and forebrain regions commonly associated with these mental disorders. In rodents, the glutamatergic neurons in the midline paraventricular thalamic nucleus (PVT) provide excitatory inputs to the limbic system and forebrain. PVT also displays a high density of group II mGluRs, predominantly the metabotropic glutamate 2 receptor (mGluR2). Because the role of group II mGluRs in regulating cellular and synaptic excitability in this location has yet to be determined, we used whole-cell patch-clamp recording and acute rat brain slice preparations to evaluate PVT neuron responses to a selective group II mGluR agonist, (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY 379268). LY 379268 consistently induced membrane hyperpolarization and suppressed firing by postsynaptic receptor-mediated activation of a barium-sensitive background K(+) conductance. This effect could be blocked by (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid (LY 341495), a selective group II mGluR antagonist. In addition, LY 379268 acted at presynaptic receptors to reduce ionotropic glutamate receptor-mediated excitatory synaptic transmission. An mGluR2-positive allosteric modulator, 2,2,2-trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)ethanesulfonamide hydrochloride (LY 487379), resulted in leftward shifts of the LY 379268 dose-response curve for both postsynaptic and presynaptic actions. The data demonstrate that activation of postsynaptic and presynaptic group II (presumably mGluR2) mGluRs reduces neuronal excitability in midline thalamus, an action that may contribute to the effectiveness of mGluR2-activating drugs in rodent models of anxiety and psychosis.

Effects of VPAC2 receptor activation on membrane excitability and GABAergic transmission in subparaventricular zone neurons targeted by suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN) harbors the master circadian pacemaker. SCN neurons produce the amino acid gamma-aminobutyric acid (GABA) and several peptide molecules for coordination and communication of their circadian rhythms. A subpopulation of SCN cells synthesizes vasoactive intestinal polypeptide (VIP) and provides a dense innervation of the subparaventricular zone (SPZ), an important CNS target of the circadian pacemaker. In this study, using patch-clamp recording techniques and rat brain slice preparations, the contribution of VIP to SCN efferent signaling to SPZ was evaluated by examining membrane responses of SPZ neurons to exogenous VIP receptor ligands. In approximately 50% of the SPZ neurons receiving monosynaptic GABAA receptor-mediated inputs from SCN, bath-applied VIP (0.5-1 microM) resulted in a membrane depolarization caused by tetrodotoxin-resistant inward currents reversing at approximately -23 mV. These data suggest the existence of postsynaptic receptors that activate a nonselective cationic conductance. In addition, a subset of SPZ neurons showed an increase in the amplitude of SCN-evoked GABAergic inhibitory postsynaptic currents (IPSCs) and a decrease in their paired-pulse ratios. This, together with an increase in frequency of spontaneous and miniature IPSCs, implies the presence of presynaptic receptors that facilitate GABA release from SCN and possibly other synaptic terminals. The effects occurred in separate neurons and could be mimicked by the selective VPAC2 receptor agonist BAY 55-9837 (0.2-0.5 microM) and partially blocked by the VIP receptor antagonist VIP(6-28) (5 microM). The results indicate that VIP acts via both post- and presynaptic VPAC2 receptors to differentially modulate SCN GABAergic signaling to distinct subpopulations of SPZ neurons.

Acid-sensitive TASK-like K+ conductances contribute to resting membrane potential and to orexin-induced membrane depolarization in rat thalamic paraventricular nucleus neurons.

Orexin (hypocretin) peptides are known to depolarize rat thalamic paraventricular nucleus (PVT) neurons by suppression of one or more undefined potassium conductances. Here, we investigated a contribution of TWIK-related acid-sensitive K(+) (TASK) channels to the resting membrane potential and orexin-induced depolarization of PVT neurons, using patch clamp recording techniques in brain slice preparations. Upon exposure to an acidic (pH 6.3) extracellular solution, PVT neurons displayed membrane depolarization. Under voltage-clamp and in the presence of tetrodotoxin (TTX, 0.5 microM), low pH solutions induced an inward shift in baseline membrane current, accompanied by a net decrease in membrane conductance, reversing close to the potassium equilibrium potential. By contrast, exposure to alkaline (pH 8.3) solutions resulted in membrane hyperpolarization, induced an outward shift in baseline membrane current and an increase in net conductance that reversed close to the potassium equilibrium potential. A local anesthetic bupivacaine (20-40 microM) and the endocannabinoid anandamide (5-10 microM) mimicked the effects of the acidic solution. Exposure to the volatile anesthetic isoflurane (0.2-0.5 mM) induced changes in resting membrane potential, baseline current and membrane conductance similar to those caused by the alkaline solution. Although responsiveness to orexins was preserved under each of the above conditions, the amplitude of the orexin B (0.5 microM)-induced inward current was depressed in the acidic solution and in the presence of anandamide, remained largely unchanged in the alkaline solution, and was enhanced by isoflurane when compared with that in normal artificial cerebrospinal solution. We conclude that pH-sensitive potassium channels, TASK-1 and TASK-3 channels, contribute substantially to the resting membrane conductance(s) and excitability in PVT neurons. The observations that orexin-induced currents were affected by putative TASK-specific drugs in a manner predictable from their effects on TASK channels also suggest that the orexin-induced excitation in PVT neurons is mediated by closure of TASK channels.

Orexin peptides enhance median preoptic nucleus neuronal excitability via postsynaptic membrane depolarization and enhancement of glutamatergic afferents.

Subpopulations of neurons in the median preoptic nucleus (MnPO) located within the lamina terminalis contribute to thermoregulatory, cardiovascular and hydromineral homeostasis, and sleep-promotion. MnPO is innervated by lateral hypothalamic neurons that synthesize and secrete the arousal-promoting and excitatory orexin (hypocretin) neuropeptides. To evaluate the hypothesis that orexins modulate the excitability of MnPO neurons, we used patch-clamp recording techniques applied in rat brain slice preparations to assess the effects of exogenously applied orexin A and orexin B peptides on their intrinsic and synaptic properties. Whole cell recordings under current-clamp mode revealed that 11/15 tested MnPO neurons responded similarly to either orexin A or B (500-1000 nM) with a slowly rising, prolonged (10-15 min) and reversible membrane depolarization. Under voltage-clamp mode, orexin applications induced a tetrodotoxin-resistant inward current of -7.2+/-1.6 pA, indicating a direct (postsynaptic) activation, with a time course similar to the observed membrane depolarization. The orexin-induced responses in 4/7 neurons were associated with a significant decrease in membrane conductance and the net orexin-induced current that reversed at -99+/-5 mV, suggesting closure of potassium channels. Orexins did not attenuate the properties of excitatory (n=4) or inhibitory (n=7) postsynaptic currents evoked by subfornical organ stimulation. By contrast, orexins applications induce a significant increase in both frequency and amplitude of spontaneous glutamatergic postsynaptic currents (5/7 cells) but had no influence on spontaneous GABAergic currents (6/6 cells). Thus, in addition to a direct postsynaptic receptor-mediated excitation, orexins can also increase the excitability of MnPO neurons via increasing their excitatory inputs, presumably through an orexin receptor-mediated excitation of local glutamatergic neurons whose axons project to MnPO neurons.

Orexin-induced modulation of state-dependent intrinsic properties in thalamic paraventricular nucleus neurons attenuates action potential patterning and frequency.

The thalamic paraventricular nucleus (PVT) receives a dense innervation from orexin-synthesizing lateral hypothalamic neurons. Since PVT neurons display state-dependent tonic or low threshold spike-driven burst firing patterns, we examined how the response to exogenously applied orexins might modulate these features. Data were obtained with whole-cell patch clamp recording techniques in rat brain slices prepared during the subjective lights-on period. PVT neurons displayed a mean resting membrane potential of -61+/-6 mV and input conductance of 1.3+/-0.1 nS (n=60). The majority (90/107) of cells tested responded to orexin A and/or orexin B peptides (100-1000 nM), each inducing similar slowly rising and prolonged membrane depolarizations. We next evaluated associated changes in firing patterns and action potential frequency. Of 17 spontaneously silent neurons, 5 were induced into tonic firing and 4 into burst firing modes. Of nine spontaneously bursting neurons, three displayed an increase in burst frequency and in the number of action potentials within a burst. By contrast, another six cells were induced into tonic firing mode, with a marked decrease in instantaneous firing frequency and a shift in their excitatory postsynaptic potential-evoked responses from burst firing patterns to single action potentials. Under voltage clamp, orexins induced inward current (-21.8+/-2.4 pA at -60 mV) in 20/22 cells. In 13 cells, current-voltage (I-V) plots revealed a decrease in net conductance and reversal at -110+/-9 mV, while 3 cells displayed an increase in net conductance that reversed at -26+/-8 mV. These observations imply suppression of potassium and/or induction of nonselective cationic conductances in orexin-induced depolarization in PVT neurons, permitting these peptides to modulate intrinsic state-dependent properties. In vivo, such changes in firing patterns and frequency of action potential discharges could influence neurotransmission through PVT and activity-dependent synaptic plasticity at target sites of these neurons.

Pharmacological and molecular characterization of ATP-sensitive K(+) conductances in CART and NPY/AgRP expressing neurons of the hypothalamic arcuate nucleus.

The role of hypothalamic ATP-sensitive potassium channels in the maintenance of energy homeostasis has been extensively explored. However, how these channels are incorporated into the neuronal networks of the arcuate nucleus remains unclear. Whole-cell patch-clamp recordings from rat arcuate nucleus neurons in hypothalamic slice preparations revealed widespread expression of functional ATP-sensitive potassium channels within the nucleus. ATP-sensitive potassium channels were expressed in orexigenic neuropeptide Y/agouti-related protein (NPY/AgRP) and ghrelin-sensitive neurons and in anorexigenic cocaine-and-amphetamine regulated transcript (CART) neurons. In 70% of the arcuate nucleus neurons recorded, exposure to glucose-free bathing medium induced inhibition of electrical excitability, the response being characterized by membrane hyperpolarization, a reduction in neuronal input resistance and a reversal potential consistent with opening of potassium channels. These effects were reversible upon re-introduction of glucose to the bathing medium or upon exposure to the ATP-sensitive potassium channel blockers tolbutamide or glibenclamide. The potassium channel opener diazoxide, but not pinacidil, also induced a tolbutamide and glibenclamide-sensitive inhibition of electrical excitability. Single-cell reverse transcription-polymerase chain reaction revealed expression of mRNA for sulfonylurea receptor 1 but not sulfonylurea receptor 2 subunits of ATP-sensitive potassium channels. Thus, rat arcuate nucleus neurons, including those involved in functionally antagonistic orexigenic and anorexigenic pathways express functional ATP-sensitive potassium channels which include sulfonylurea receptor 1 subunits. These data indicate a crucial role for these ion channels in central sensing of metabolic and energy status. However, further studies are needed to clarify the differential roles of these channels, the organization of signaling pathways that regulate them and how they operate in functionally opposing cell types.

Suprachiasmatic nucleus communicates with anterior thalamic paraventricular nucleus neurons via rapid glutamatergic and gabaergic neurotransmission: state-dependent response patterns observed in vitro.

The hypothalamic suprachiasmatic nucleus uniquely projects to the midline thalamic paraventricular nucleus. To characterize this projection, patch clamp techniques applied in acute rat brain slice preparations examined responses of anterior thalamic paraventricular nucleus neurons to focal suprachiasmatic nucleus stimulation. Whole cell recordings from slices obtained during daytime (n=40) revealed neurons with a mean membrane potential of -66+/-1.2 mV, input conductance of 1.5+/-0.1 nS and state-dependent tonic or burst firing patterns. Electrical stimulation (one or four pulses) in suprachiasmatic nucleus elicited monosynaptic excitatory postsynaptic potentials (mean latency of 12.6+/-0.6 ms; n=12), featuring both AMPA and N-methyl-D-aspartate-glutamate receptor-mediated components, and monosynaptic bicuculline-sensitive inhibitory postsynaptic potentials (mean latency of 16.6+/-0.6 ms; n=7) reversing polarity at -72+/-2.6 mV, close to the chloride equilibrium potential. Glutamate microstimulation of suprachiasmatic nucleus also elicited transient increases in spontaneous excitatory or inhibitory postsynaptic currents in anterior thalamic paraventricular neurons. Recordings from rats under reverse light/dark conditions (n=22) yielded essentially similar responses to electrical stimulation. At depolarized membrane potentials, suprachiasmatic nucleus-evoked excitatory postsynaptic potentials triggered single action potentials, while evoked inhibitory postsynaptic potentials elicited a silent period in ongoing tonic firing. By contrast, after manual adjustment of membrane potentials to hyperpolarized levels, neuronal response to the same "excitatory" stimulus was a low threshold spike and superimposed burst firing, while responses to "inhibitory" stimuli paradoxically elicited excitatory rebound low threshold spikes and burst firing. These data support the existence of glutamatergic and GABAergic efferents from the suprachiasmatic nucleus to its target neurons. Additionally, in thalamic paraventricular nucleus neurons, responses to activation of their suprachiasmatic afferents may vary in accordance with their membrane potential-dependent intrinsic properties, a characteristic typical of thalamocortical neurons.

Vasopressin induces depolarization and state-dependent firing patterns in rat thalamic paraventricular nucleus neurons in vitro.

The thalamic midline paraventricular nucleus (PVT) is prominently innervated by vasopressin-immunoreactive neurons from the suprachiasmatic nucleus (SCN), site of the brain's biological clock. Using patch-clamp recordings in slice preparations taken from Wistar rats during the subjective day, we examined 90 PVT neurons for responses to bath-applied AVP (0.5-2 microM; 1-3 min). In current clamp at resting membrane potentials (-65 +/- 1 mV), PVT neurons displayed low-threshold spikes (LTSs) and burst firing patterns. In 50% of cells tested, AVP induced a slowly rising, prolonged membrane depolarization and tonic firing, returning to burst firing upon recovery. AVP modulated hyperpolarization-activated LTSs by decreasing the time to the initial sodium spike at the onset of LTS, also increasing the duration of the afterdepolarization. Responses were blockable with a V(1a) receptor antagonist (Manning compound). Under voltage clamp, AVP induced a TTX-resistant, slowly rising, and prolonged (approximately 15 min) inward current (<40 pA). Current-voltage relationship (I-V) analyses of the AVP responses revealed a decrease in membrane conductance to 73.1 +/- 6.2% of control, with net AVP current reversing at -106 +/- 4 mV, and decreased inward rectification at negative potentials. These observations are consistent with an AVP-induced closure of an inwardly rectifying potassium conductance. On the basis of these in vitro observations, we suggest that the SCN vasopressinergic innervation of PVT is excitatory in nature, possibly releasing AVP with circadian rhythmicity and contributing to state-dependent firing patterns in PVT neurons over the sleep-wake cycle.

Lesions of the diagonal band of broca enhance drinking in the rat.

This study examined the role of the diagonal band of Broca (DBB) in drinking behaviour and vasopressin release. Adult male rats were anaesthetized (pentobarbital 50 mg/kg) and received DBB injections of either ibotenic acid (0.5 microl of 5 micro g/ microl) or vehicle (0.5 microl of phosphate-buffered saline). Although baseline drinking and urine output were not affected, drinking to 30% polyethylene glycol (MW 8000; 1 ml/100 g s.c.) and angiotensin II (0, 1.5 and 3.0 mg/kg s.c.) were significantly increased in ibotenic acid in phosphate-buffered saline (DBBX) rats. Drinking to hypertonic saline (0.9, 4 and 6%; 1 ml/100 g), and water deprivation were not significantly affected. DBBX rats had significantly lower basal heart rates than controls but the cardiovascular responses to infusions of angiotensin II (100 ng/kg/min i.v. for 45 min) were not affected. DBBX rats had significantly higher basal vasopressin, but angiotensin-stimulated vasopressin release was not significantly different. Although the DBB is not involved in basal water intake, it is involved in dipsogenic responses to hypovolemic stimuli and possibly basal autonomic function and basal vasopressin release.

Pre- and postsynaptic GABA(B) receptors modulate rapid neurotransmission from suprachiasmatic nucleus to parvocellular hypothalamic paraventricular nucleus neurons.

The suprachiasmatic nucleus (SCN), the dominant circadian pacemaker in mammalian brain, sends axonal projections to the hypothalamic paraventricular nucleus (PVN), a composite of magno- and parvocellular neurons. This neural network likely offers SCN output neurons a means to entrain diurnal rhythmicity in various autonomic and neuroendocrine functions. Earlier investigations using patch-clamp recordings in slice preparations have suggested differential innervation by SCN efferents to magnocellular versus parvocellular PVN cells. In magnocellular PVN, cells respond to focal electrical stimulation in SCN with a GABA(A) receptor-mediated postsynaptic inhibition whose magnitude can be modulated by presynaptic GABA(B) receptors. By contrast, SCN-evoked responses in parvocellular PVN neurons typically involve both GABA(A)- and glutamate-receptor-mediated components. In the present patch-clamp study, 69/85 periventricular parvocellular PVN cells displayed SCN-evoked inhibitory and/or excitatory postsynaptic currents (IPSCs; EPSCs). In the presence of selective receptor antagonists, we sought evidence for their modulation by GABA acting at pre- and/or postsynaptic GABA(B) receptors. Cells responded to bath-applied baclofen (5-10 microM) with a tetrodotoxin-resistant membrane hyperpolarization associated with a reduction in input resistance and/or outward current, due to increase in a potassium conductance, blockable with 2-hydroxysaclofen (300 microM). At 1 microM where baclofen had no significant postsynaptic effect, evidence of activation of presynaptic GABA(B) receptors included reduction in SCN-evoked IPSCs and EPSCs with no change in their kinetics, and paired-pulse depression that was sensitive to both baclofen and saclofen. Baclofen also induced significant reductions in frequency but not amplitudes of miniature IPSCs and EPSCs. These observations suggest that levels of synaptically released GABA from the terminals of SCN output neurons can influence the relative contribution of pre- versus postsynaptic GABA(B) receptors in modulating both excitatory and inhibitory SCN innervation to parvocellular PVN neurons.

Electrophysiological evidence for vasopressin V(1) receptors on neonatal motoneurons, premotor and other ventral horn neurons.

Prominent arginine-vasopressin (AVP) binding and AVP V(1) type receptors are expressed early in the developing rat spinal cord. We sought to characterize their influence on neural excitability by using patch-clamp techniques to record AVP-induced responses from a population of motoneurons and interneurons in neonatal (5-18 days) rat spinal cord slices. Data were obtained from 58 thoracolumbar (T(7)-L(5)) motoneurons and 166 local interneurons. A majority (>90%) of neurons responded to bath applied AVP (10 nM to 3 microM) and (Phe(2), Orn(8))-vasotocin, a V(1) receptor agonist, but not V(2) or oxytocin receptor agonists. In voltage-clamp, postsynaptic responses in motoneurons were characterized by slowly rising, prolonged (7-10 min) and tetrodotoxin-resistant inward currents associated with a 25% reduction in a membrane potassium conductance that reversed near -100 mV. In interneurons, net AVP-induced inward currents displayed three patterns: decreasing membrane conductance with reversal near -100 mV, i.e., similar to that in motoneurons (24 cells); increasing conductance with reversal near -40 mV (21 cells); small reduction in conductance with no reversal within the current range tested (41 cells). A presynaptic component recorded in most neurons was evident as an increase in the frequency but not amplitude (in motoneurons) of inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs), in large part due to AVP-induced firing in inhibitory (mainly glycinergic) and excitatory (glutamatergic) neurons synapsing on the recorded cells. An increase in frequency but not amplitude of miniature IPSCs and EPSCs also indicated an AVP enhancement of neurotransmitter release from axon terminals of inhibitory and excitatory interneurons. These observations provide support for a broad presynaptic and postsynaptic distribution of AVP V(1) type receptors and indicate that their activation can enhance the excitability of a majority of neurons in neonatal ventral spinal cord.

Norepinephrine acts via alpha(2) adrenergic receptors to suppress N-type calcium channels in dissociated rat median preoptic nucleus neurons.

The median preoptic (MnPO) nucleus, a key CNS site for hydromineral and cardiovascular homeostasis, receives a dense norepinephrine innervation from brainstem autonomic centers. Since norepinephrine is known to influence neuronal excitability by modulating calcium channel function, we applied whole cell patch clamp techniques to study calcium currents in 116 dissociated MnPO neurons, including 30 cells identified by a retrograde label as projecting to the hypothalamic paraventricular nucleus. Norepinephrine (3-50 microM) suppressed high-voltage-activated calcium currents (HVA I(Ca)) in 80% of cells, selectively blockable by yohimbine and mimicked by UK14,304 and clonidine. The norepinephrine effect was relieved by strong prior depolarization, indicating a voltage-dependent component. Intracellular GTP-gamma-S blocked the effect. Blockade by extracellular NEM suggested involvement of pertussis-toxin sensitive G-proteins. Based on pharmacological properties, these HVA I(Ca)s had the following composition: 40-45% N-type (blockable by omega-conotoxin GVIA); 20-25% L-type (blockable by nimodipine); 15-20% P/Q-type (blockable by omega-agatoxin IVA). Since approximately 75% of the norepinephrine effect was blockable with omega-conotoxin GVIA, we conclude that postsynaptic alpha(2) adrenoceptors preferentially suppress N-type calcium channels, revealing a novel mechanism whereby norepinephrine can modulate excitability in MnPO neurons.

Glutamate and GABA mediate suprachiasmatic nucleus inputs to spinal-projecting paraventricular neurons.

We used patch-clamp recordings in slice preparations from Sprague-Dawley rats to evaluate responses of 20 spinal-projecting neurons in the dorsal paraventricular nucleus (PVN) to electrical stimulation in suprachiasmatic nucleus (SCN). Neurons containing a retrograde label transported from the thoracic (T(1)-T(4)) intermediolateral column displayed three intrinsic properties that collectively allowed distinction from neighboring parvocellular or magnocellular cells: a low-input resistance, a hyperpolarization-activated time-dependent inward rectification, and a low-threshold calcium conductance. Twelve of fifteen cells tested responded to electrical stimulation in SCN. All of 10 cells tested in media containing 2,3,-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (5 microM) and D(-)-2-amino-5-phosphonopentanoic acid (20 microM) responded with constant latency (11.4 +/- 0.7 ms) inhibitory postsynaptic potentials, able to follow 20- to 50-Hz stimulation and blockable with bicuculline (20 microM). By contrast, all eight cells tested in the presence of bicuculline demonstrated constant latency (9.8 +/- 0.6 ms) excitatory postsynaptic potentials that followed at 20-50 Hz and featured both non-N-methyl-D-aspartate (NMDA) and NMDA receptor-mediated components. We conclude that both GABAergic and glutamatergic neurons in SCN project directly to spinal-projecting neurons in the dorsal PVN.

Presynaptic GABA(B) receptors modulate organum vasculosum lamina terminalis-evoked postsynaptic currents in rat hypothalamic supraoptic neurons.

Whole-cell patch-clamp recordings obtained from 36 hypothalamic supraoptic nucleus neurons in explant preparations evaluated a role for GABA(B) receptors in modulating postsynaptic inhibitory and excitatory currents evoked by electrical stimulation in the organum vasculosum of the lamina terminalis. At a holding current of -65 mV, application of baclofen (1-10 microM) induced a dose-dependent reduction in the amplitude of pharmacologically isolated inhibitory and excitatory postsynaptic currents, converted paired-pulse depression in inhibitory postsynaptic currents to paired-pulse facilitation, and enhanced paired-pulse ratios for excitatory postsynaptic currents. In media containing 2-hydroxysaclofen (200-400 microM), baclofen-associated events were blocked and paired-pulse depression in evoked inhibitory postsynaptic currents was abolished. In addition, a progressive increase in the amplitude of inhibitory postsynaptic currents implied that GABA was endogenously active at presynaptic GABA(B) receptors. In contrast, no paired-pulse depression was observed for inhibitory postsynaptic currents evoked in six non-magnocellular neurons. Neither baclofen nor 2-hydroxysaclofen altered holding currents or input resistances in supraoptic neurons, or altered the kinetics of the evoked responses.These observations imply that the terminals of both inhibitory (GABAergic) and excitatory (glutamatergic) afferents to supraoptic nucleus neurons from organum vasculosum lamina terminalis neurons are subject to modulation by presynaptic GABA(B) receptors, and that this modulation is preferentially directed to the inhibitory inputs.

GABA(B) presynaptically modulates suprachiasmatic input to hypothalamic paraventricular magnocellular neurons.

This study used whole cell patch clamp recordings in rat hypothalamic slice preparations to evaluate the effects of GABA(B) receptor activation on GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) in paraventricular nucleus magnocellular neurons evoked by electrical stimulation in the suprachiasmatic nucleus (SCN). Baclofen induced a dose-dependent (1-10 microM) and reversible reduction in SCN-evoked IPSC amplitude (11/11 cells), blockable with 2-hydroxysaclofen (300 microM; 3/3 cells). IPSCs displayed paired-pulse depression (PPD), attenuated by both baclofen and 2-hydroxysaclofen, but neither altered resting membrane conductances or IPSC time constants of decay. Baclofen induced a significant dose-dependent (1-100 microM) reduction in frequency, but not amplitude, of spontaneous IPSCs and miniature IPSCs, reversible with 2-hydroxysaclofen pretreatment. Baclofen effects and PPD persisted in slices pretreated with pertussis toxin (PTX) and N-ethylmaleimide, implying that these GABA(B) receptors are coupled to PTX-insensitive G proteins. Responses were unaltered by barium (2 mM) or nimodipine, ruling out involvement of K(+) channels and L-type Ca(2+) channels. Thus pre- and postsynaptic GABA(B) and GABA(A) receptors participate in SCN entrainment of paraventricular neurosecretory neurons.