primer. A sequence change from T to G at nucleotide position 1,908 occurred in CAST/Ei relative to inbred strains, such as DBA/2J, C3HeB/FeJ, C57BL/6J, and BALB/cByJ (data not shown); however, this polymorphism does not change the coding of this alanine residue and could account for the non-Lc-specific difference seen in Fig. 2a. , post-NMDG holding currents were very small. In a few Lc/+ cells however, the final holding currents in the presence of NMDG were very large despite the decrease observed in response to this substitution (Fig. 3b, open circles, n ¼ 2). This large residual holding current was probably a sign of compromised recording integrity, evidence of which had initially been masked by the Lc/+ constitutive current. Thus data from these cells were excluded from the mean calculations of both basic Lc/+ membrane properties (holding current and conductance; Fig. 3a) and changes in these values produced by NMDG substitution (Fig. 3c) to avoid artefactually increasing the Lc/+ effect. The values we report here, therefore, were derived only from those Lc/+ cells for which the post-NMDG holding currents were more positive than −230 pA. Electrophysiology in Xenopus oocytes. Two oligonucleotides were designed with homology to the 5Ј and 3Ј ends of the mouse GluRd2 cDNA coding sequence. The Lc mutation was generated using the bridge PCR mutagenesis method 22 . The Lc mutation in mutant clones was verified by sequencing. In vitro translation was used to confirm that both wild-type and mutant constructs yielded the expected products of M r ϳ110K in an SDS-PAGE gel. Concentrations of cRNAs were measured by gel electrophoresis and spectrophotometer. Approximately 65-85 ng per 50 nl of the cRNAs were injected into stage V or VI oocytes from Xenopus laevis (Nasco and Xenopus Express). Electrophysiological recordings were performed 1-3 days postinjection using the two-microelectrode voltage-clamp configuration at room temperature 23 . The recording-bath chamber was ϳ150 l in volume and the flow rate 2-3 ml min −1 . Most recordings were made in a Na/Ca solution containing (in mM): 130 NaCl, 2 CaCl 2 and 10 HEPES, pH 7.3. For NMDG substitution experiments, an equimolar substitution of NMDG for NaCl was made (pH 7.3, adjusted with HCl). Fresh stock solutions were made in the Na/Ca recording solution and kept on ice: L-glutamate (100 mM, Sigma), L-aspartate (100 mM, Sigma), glycine (100 mM, Sigma), kainic acid (1 mM, Research Biochemicals), CNQX (1 mM, Research Biochemicals, AP5 (1 mM, Research Biochemicals), 7-chlorokynurenic acid (10 mM, Research Biochemicals). For resting-potential measurements, close readings between two microelectrodes (less than 5 mV difference) were indicating of good seals of the microelectrodes to the oocyte membrane. When NMDG bath was perfused after Na + bath, the readings of two microelectrodes moved very closely to more hyperpolarized or negative directions until they reached a relatively stable state, between −50 and −90 mV. Some oocytes did not reach such a hyperpolarized state with NMDG,...

Background-Corticotropin-releasing factor (CRF) and gamma-aminobutyric acid (GABA)ergic systems in the central amygdala (CeA) are implicated in the high-anxiety, high-drinking profile associated with ethanol dependence. Ethanol augments CeA GABA release in ethanol-naive rats and mice.

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Acetylcholine (ACh) plays a key role in the transitions between the different phases of sleep: Slow-wave sleep requires low ACh concentrations in the brain, whereas rapid-eye-movement (REM) sleep is associated with high levels of ACh. Also, these phases of sleep are differentially sensitive to a number of endogenous neuropeptides and cytokines, including somatostatin, which has been shown to increase REM sleep without significantly affecting other phases. Here we report the cloning and initial characterization of cortistatin, a neuropeptide that exhibits strong structural similarity to somatostatin, although it is the product of a different gene. Administration of cortistatin depresses neuronal electrical activity but, unlike somatostatin, induces low-frequency waves in the cerebral cortex and antagonizes the effects of acetylcholine on hippocampal and cortical measures of excitability. This suggests a mechanism for cortical synchronization related to sleep.

The central amygdala (CeA) plays a role in the relationship among stress, corticotropin-releasing factor (CRF), and alcohol abuse. In whole-cell recordings, both CRF and ethanol enhanced gamma-aminobutyric acid-mediated (GABAergic) neurotransmission in CeA neurons from wild-type and CRF2 receptor knockout mice, but not CRF1 receptor knockout mice. CRF1 (but not CRF2) receptor antagonists blocked both CRF and ethanol effects in wild-type mice. These data indicate that CRF1 receptors mediate ethanol enhancement of GABAergic synaptic transmission in the CeA, and they suggest a cellular mechanism underlying involvement of CRF in ethanol's behavioral and motivational effects.

The cannabinoid receptor CB1 is found in abundance in brain neurons, whereas CB2 is essentially expressed outside the brain. In the neocortex, CB1 is observed predominantly on large cholecystokinin (CCK)-expressing interneurons. However, physiological evidence suggests that functional CB1 are present on other neocortical neuronal types. We investigated the expression of CB1 and CB2 in identified neurons of rat neocortical slices using single-cell RT-PCR. We found that 63% of somatostatin (SST)-expressing and 69% of vasoactive intestinal polypeptide (VIP)-expressing interneurons co-expressed CB1. As much as 49% of pyramidal neurons expressed CB1. In contrast, CB2 was observed in a small proportion of neocortical neurons. We performed whole cell recordings of pyramidal neurons to corroborate our molecular findings. Inhibitory postsynaptic currents (IPSCs) induced by a mixed muscarinic/nicotinic cholinergic agonist showed depolarization-induced suppression of inhibition and were decreased by the CB1 agonist WIN-55212-2 (WIN-2), suggesting that interneurons excited by cholinergic agonists (mainly SST and VIP neurons) possess CB1. IPSCs elicited by a nicotinic receptor agonist were also reduced in the presence of WIN-2, suggesting that neurons excited by nicotinic agonists (mainly VIP neurons) indeed possess CB1. WIN-2 largely decreased excitatory postsynaptic currents evoked by intracortical electrical stimulation, pointing at the presence of CB1 on glutamatergic pyramidal neurons. All WIN-2 effects were strongly reduced by the CB1 antagonist AM 251. We conclude that CB1 is expressed in various neocortical neuronal populations, including glutamatergic neurons. Our combined molecular and physiological data suggest that CB1 widely mediates endocannabinoid effects on glutamatergic and GABAergic transmission to modulate cortical networks.

The central amygdala (CeA) plays a major role in alcohol dependence and reinforcement, and behavioral and neurochemical evidence suggest a role for the endocannabinoid (eCB) system in ethanol binging and dependence. We used the slice preparation to investigate the physiological role of cannabinoids and their interaction with ethanol on inhibitory synaptic transmission in CeA. Superfusion of the cannabinoid receptor (CB1) agonist WIN55212-2 (WIN2) onto CeA neurons decreased evoked GABAA receptor-mediated inhibitory postsynaptic potentials (IPSPs) in a concentration-dependent manner, an effect prevented by the CB1 antagonists Rimonabant (SR141716, SR1) and AM251. SR1 or AM251 applied alone augmented IPSPs, revealing a tonic eCB activity that decreased inhibitory transmission in CeA. Paired-pulse analysis suggested a presynaptic CB1 mechanism. Intracellular BAPTA abolished the ability of AM251 to augment IPSPs, demonstrating the eCB-driven nature and postsynaptic origin of the tonic CB1-dependent control of GABA release. Superfusion of ethanol increased IPSPs and addition of WIN2 reversed the ethanol effect. Similarly, prior superfusion of WIN2 prevented subsequent ethanol effects on GABAergic transmission. The ethanol-induced augmentation of IPSPs was additive to CB1 blockade, ruling out a participation of CB1 in the action of acute ethanol. Our study points to an important role of CB1 in CeA where the eCBs tonically regulate neuronal activity, and suggests a potent mechanism for modulating CeA tone during challenge with ethanol.

The M-current (IM) is a time- and voltage-dependent K+ current that persists at slightly depolarized membrane potentials. IM is reduced by muscarinic cholinergic agonists and certain peptides, and is thought to be responsible in part for the slow and late slow excitatory postsynaptic potentials in sympathetic neurons. Recently, we reported that IM in hippocampal neurons was also augmented by somatostatin-14 and -28 suggesting that two different receptors reciprocally regulate one neuronal channel type. Muscarinic effects on IM may be mediated by various components of the phosphatidylinositol phosphate pathway. We now report the involvement of a different second messenger pathway, that generated by phospholipase A2, in the somatostatin-induced augmentation of IM in hippocampal cells. This pathway generates arachidonic acid from which leukotrienes can be produced by lipoxygenases. We find that the IM-augmenting effects of somatostatin are abolished by two substances that can inhibit phospholipase A2, quinacrine and 4-bromophenacyl bromide, and that both arachidonic acid and leukotriene C4 mimic the effects of somatostatin-14 on hippocampal pyramidal neurons in vitro. Arachidonic and somatostatin effects are blocked by a lipoxygenase inhibitor, implicating an arachidonic acid metabolite, perhaps a leukotriene, in the somatostatin effect.