mu-opioid receptor activation inhibits N- and P-type Ca2+ channel currents in magnocellular neurones of the rat supraoptic nucleus.

1. The whole-cell voltage-clamp technique was used to examine opioid regulation of Ba2+ currents (IBa) through voltage-sensitive Ca2+ channels in isolated magnocellular supraoptic neurones (MNCs). The effects of local application of mu-, delta- or kappa-opioid receptor selective agonists were examined on specific components of high voltage-activated (HVA) IBa, pharmacologically isolated by use of Ca2+ channel-subtype selective antagonists. 2. The mu-opioid receptor selective agonist, DAMGO, suppressed HVA IBa (in 64/71 neurones) in a naloxone-reversible and concentration-dependent manner (EC50 = 170 nM, Emax = 19.5 %). The DAMGO-induced inhibition was rapid in onset, associated with kinetic slowing and voltage dependent, being reversed by strong depolarizing prepulses. Low-voltage activated (LVA) IBa was not modulated by DAMGO. 3. Administration of kappa- (U69 593) or delta-selective (DPDPE) opioid receptor agonists did not affect IBa. However, immunostaining of permeabilized MNCs with an antibody specific for kappa1-opioid receptors revealed the presence of this opioid receptor subtype in a large number of isolated somata. 4. mu-opioid-induced inhibition in IBa was largely abolished after blockade of N-type and P-type channel currents by omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (100 nM), respectively. Quantitation of antagonist effects on DAMGO-induced reductions in IBa revealed that N- and P-type channels contributed roughly equally to the mu-opioid sensitive portion of total IBa. 5. These results indicate that mu-opioid receptors are negatively coupled to N- and P-type Ca2+ channels in the somatodendritic regions of MNCs, possibly via a membrane-delimited G-protein-dependent pathway. They also support a scheme in which opioids may act in part to modulate cellular activity and regulate neurosecretory function by their direct action on the neuroendocrine neurones of the hypothalamic supraoptic neucleus.

Ethanol selectively enhances the hyperpolarizing component of neocortical neuronal responses to locally applied GABA.

Local application of GABA to rat cerebral cortical neurons in brain slices elicited biphasic responses mediated via GABAA receptors. The fast component of the response, which was most apparent with somatic application of GABA, was hyperpolarizing at the normal resting membrane potential (GABAh response). The slower component could be elicited by GABA application to nearly all regions of the cell, and was depolarizing at the resting membrane potential (GABAd response). The reversal potential of evoked IPSCs recorded with whole-cell patch electrodes (-68 mV) was comparable to the reversal potential of the GABAh response (-69 mV), and was significantly different from the reversal potential of the GABAd response (-56 mV). The GABAd response was more sensitive to enhancement by pentobarbital and more readily antagonized by both bicuculline and picrotoxin than the GABAh response. Recording in bicarbonate-free buffer changed the reversal potential of the GABAd response significantly, but had no effect on the GABAh response. In contrast, superfusion with ethanol significantly enhanced the GABAh response, while having no effect on the GABAd component. Although a localized collapse of the Cl- gradient, which has been proposed to underlie the GABAd response, could explain the greater sensitivity of the GABAd response to pentobarbital and the GABAA antagonists, this could not account for the greater sensitivity of the GABAh response to ethanol. Differences in GABAA receptor subunit composition may result in the expression of dendritic and somatic GABAA receptors that have different kinetics, reversal potentials, and sensitivity to pharmacological agents, including ethanol.

mu-Opioid receptor activation decreases N-type Ca2+ current in magnocellular neurons of the rat basal forebrain.

Opioid modulation of calcium currents was studied in acutely dissociated rat basal forebrain neurons using the whole cell patch-clamp recording technique. The mu-opioid receptor agonist DAGO reversibly suppressed high-voltage activated calcium currents and slowed their rate of activation, while neither delta- nor kappa-opioid receptor agonists were effective in modifying calcium current in these neurons. The inhibitory effect of DAGO on calcium current was abolished following irreversible blockade of N-type calcium channels by omega-conotoxin GVIA, whereas DAGO-induced inhibitory responses were not affected following blockade of L-type calcium channels by nifedipine. These findings indicate that mu-opioid receptors are negatively coupled to N-type calcium channels on the postsynaptic membrane of basal forebrain neurons. Calcium currents recorded from a significant number of large, mu-opioid sensitive neurons were also suppressed by muscarinic receptor activation, while smaller, mu-opioid sensitive neurons were not sensitive to muscarinic receptor activation. Thus, the present data demonstrate that voltage-activated calcium influx in several subpopulations of basal forebrain neurons can be regulated by mu-opioid receptor activation. These results suggest that mu-opioid regulation of calcium current may be an important functional mechanism in regulating neuronal excitability and synaptic transmission in the basal forebrain.

Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors.

Gamma-aminobutyric acid A (GABAA) receptors are the principal mediators of synaptic inhibition, and yet when intensely activated, dendritic GABAA receptors excite rather than inhibit neurons. The membrane depolarization mediated by GABAA receptors is a result of the differential, activity-dependent collapse of the opposing concentration gradients of chloride and bicarbonate, the anions that permeate the GABAA ionophore. Because this depolarization diminishes the voltage-dependent block of the N-methyl-D-aspartate (NMDA) receptor by magnesium, the activity-dependent depolarization mediated by GABA is sufficient to account for frequency modulation of synaptic NMDA receptor activation. Anionic gradient shifts may represent a mechanism whereby the rate and coherence of synaptic activity determine whether dendritic GABAA receptor activation is excitatory or inhibitory.

Ethanol differentially modulates GABAA receptor-mediated chloride currents in hippocampal, cortical, and septal neurons in rat brain slices.

Previous electrophysiological studies have reported conflicting results concerning the effects of ethanol on gamma-aminobutyric acid-A (GABAA) receptor-mediated responses in the brain. To examine the variables that might explain these inconsistencies, the present study was designed to determine whether ethanol modulation of synaptically evoked GABA responses is brain region dependent, to identify factors that might regulate ethanol sensitivity, and to investigate the mechanism(s) underlying ethanol modulation of GABA responses. Whole-cell voltage clamp methods were used to examine the effects of ethanol on synaptically evoked GABAA inhibitory postsynaptic currents (IPSCs) recorded from neurons in hippocampus, cerebral cortex, and intermediate lateral and medial septum from rat brain slice preparations. Bicuculline-sensitive IPSCs elicited by local stimulation were pharmacologically isolated by pretreatment with the glutamate specific antagonists, DL-(-)-2-amino-5-phosphonovaleric acid (APV) and 6, 7-dinitroquinoxaline-2, 3-dione (DNQX). Superfused ethanol (80 mM) potentiated evoked GABAA IPSCs in cortical neurons and in intermediate lateral and medial septal neurons but not in CA1 hippocampal neurons. However, the mechanism by which ethanol enhanced GABAA IPSC amplitudes differed between brain regions. In cortex, ethanol induced a hyperpolarizing shift in the GABAA IPSC reversal potential (EIPSC) without modifying the underlying GABAA receptor-mediated conductance (GIPSC). In contrast, ethanol enhanced GABAA IPSC amplitudes differed between brain regions. In cortex, ethanol induced a hyperpolarizing shift in the GABAA IPSC reversal potential (EIPSC) without modifying the underlying GABAA receptor-mediated conductance (GIPSC). In contrast, ethanol enhanced GABAA IPSC amplitudes in lateral and medial septal neurons by increasing the GIPSC without modifying the EIPSC. These results suggest that ethanol differentially modulates responses to endogenous GABA released during synaptic activation and that important differences between various brain regions may reflect multiple mechanisms of ethanol action.

Ethanol enhances synaptically evoked GABAA receptor-mediated responses in cerebral cortical neurons in rat brain slices.

Previous intracellular electrophysiological studies on rat hippocampal brain slices have shown very little effect of acute ethanol application on synaptically evoked GABAA receptor-mediated responses recorded in CA1 pyramidal neurons. The present study was designed to compare the effects of ethanol on pyramidal neurons in the hippocampus and cerebral cortex. Using conventional intracellular microelectrodes (60-80 M omega) to impale cortical neurons in brain slices, 80 mM ethanol application did not affect the membrane input impedance nor evoked EPSPs, but significantly affected the resting membrane potential (usually a 2-5 mV hyperpolarization). When stimulus-evoked GABAA-mediated IPSCs were studied using whole-cell recordings from cortical neurons voltage-clamped at depolarizing potentials, monophasic IPSCs were evoked that were blocked by bicuculline, increased by pentobarbital, and enhanced by ethanol superfusion in a dose dependent manner over the range of 20-160 mM. Hippocampal IPSCs recorded under identical conditions were not enhanced by ethanol. Parallel studies of GABA-stimulated 36Cl- flux measurements in microsacs prepared from hippocampal, cerebral cortical and cerebellar tissue demonstrated that ethanol significantly enhanced (30-50%) 36Cl- flux in microsacs derived from the cerebral cortex and cerebellum, but not in microsacs prepared from the hippocampus. These results demonstrate that there are clear brain region-dependent differences in the way that GABAA receptor function is altered by acute ethanol, and that these differences are apparent not only as an enhancement of responses to exogenous GABA, but also as a facilitation of the responses to endogenous GABA released from inhibitory nerve terminals during synaptic activation.