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.

Phasic activation of the locus coeruleus enhances responses of primary sensory cortical neurons to peripheral receptive field stimulation.

In the present study we examined the effects of phasic activation of the nucleus locus coeruleus (LC) on transmission of somatosensory information to the rat cerebral cortex. The rationale for this investigation was based on earlier findings that local microiontophoretic application of the putative LC transmitter, norepinephrine (NE), had facilitating actions on cortical neuronal responses to excitatory and inhibitory synaptic stimuli and more recent microdialysis experiments that have demonstrated increases in cortical levels of NE following phasic or tonic activation of LC. Glass micropipets were used to record the extracellular activity of single neurons in the somatosensory cortex of halothane-anesthetized rats. Somatosensory afferent pathways were activated by threshold level mechanical stimulation of the glabrous skin on the contralateral forepaw. Poststimulus time histograms were used to quantitate cortical neuronal responses before and at various time intervals after preconditioning burst activation of the ipsilateral LC. Excitatory and postexcitatory inhibitory responses to forepaw stimulation were enhanced when preceded by phasic activation of LC at conditioning intervals of 200-500 ms. These effects were anatomically specific in that they were only observed upon stimulation of brainstem sites close to (>150 micron) or within LC and were pharmacologically specific in that they were not consistently observed in animals where the LC-NE system had been disrupted by 6-OHDA pretreatment. Overall, these data suggest that following phasic activation of the LC efferent system, the efficacy of signal transmission through sensory networks in mammalian brain is enhanced.

Mu-opioid and GABA(B) receptors modulate different types of Ca2+ currents in rat nodose ganglion neurons.

Whole-cell patch-clamp recordings were obtained from nodose ganglion neurons acutely dissociated from 10-30-day-old rats to characterize the Ca2+ channel types that are modulated by GABA(B) and mu-opioid receptors. Five components of high-threshold current were distinguished on the basis of their sensitivity to blockade by omega-conotoxin GVIA, nifedipine, omega-agatoxin IVA and omega-conotoxin MVIIC. Administration of the mu-opioid agonist H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol (0.3-1 mM) or the GABA(B) agonist baclofen in saturating concentrations suppressed high-threshold Ca2+ currents by 49.9+/-2.4% (n=69) and 18.7+/-2.1% (n=35), respectively. The inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol exceeded that by baclofen in virtually all neurons that responded to both agonists (67%), and occlusion experiments revealed that responses to mu-opioid and GABA(B) receptor activation were not linearly additive. In addition, administration of staurosporine, a non-selective inhibitor of protein kinase A and C, did not affect the inhibitory responses to either agonist or prevent the occlusion of baclofen-induced current inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol. Blockade of N-type channels by omega-conotoxin GVIA eliminated current suppression by baclofen in all cells tested (n=11). Mu-opioid-induced inhibition in current was abolished by omega-conotoxin GVIA in 12 of 30 neurons tested, but was only partially reduced in the remaining 18 neurons. In the latter cells administration of omega-agatoxin IVA reduced, but did not eliminate the mu-opioid sensitive current component that persisted after blockade of N-type channels. This residual component of mu-opioid-sensitive current was blocked completely by omega-conotoxin MVIIC in nine neurons, whereas responses to H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol were still recorded in the remaining cells after administration of these Ca2+ channel toxins and nifedipine. Dihydropyridine-sensitive (L-type) current was not affected by activation of mu-opioid or GABA(B) receptors in any of the neurons. These data indicate that in nodose ganglion neurons mu-opioid receptors are negatively coupled to N-, P- and Q-type channels as well as to a fourth, unidentified toxin-resistant Ca2+ channel. In contrast, GABA(B) receptors are coupled only to N-type channels. Furthermore, the results do not support a role for either protein kinase C or A in the modulatory pathway(s) coupling mu-opioid and GABA(B) receptors to Ca2+ channels, but rather lend credence to the notion that the signalling mechanisms utilized by these two receptors might simply compete for inhibitory control of a common pool of N-type channels.

Kappa-opioid receptor activation modulates Ca2+ currents and secretion in isolated neuroendocrine nerve terminals.

Whole-cell patch-clamp recordings were performed together with time-resolved measurements of membrane capacitance (Cm) in nerve terminals acutely dissociated from neurohypophysis of adult rats to investigate modulation of Ca2+ currents and secretion by activation of opioid receptors. Bath superfusion of the kappa-opioid agonists U69,593 (0.3-1 microM), dynorphin A (1 microM), or U50,488H (1-3 microM) reversibly suppressed the peak amplitude of Ca2+ currents 32. 7 +/- 2.7% (in 41 of 56 terminals), 37.4 +/- 5.3% (in 5 of 8 terminals), and 33.5 +/- 8.1% (in 5 of 10 terminals), respectively. In contrast, tests in 11 terminals revealed no effect of the mu-opioid agonist [D-Pen2,5]-enkephalin (1-3 microM; n = 7) or of the delta-agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (1 microM; n = 4) on Ca2+ currents. Three components of high-threshold current were distinguished on the basis of their sensitivity to blockade by omega-conotoxin GVIA, nicardipine, and omega-conotoxin MVIIC: N-, L-, and P/Q-type current, respectively. Administration of U69,593 inhibited N-type current in these nerve terminals on average 32%, whereas L-type current was reduced 64%, and P/Q-type current was inhibited 28%. Monitoring of changes in Cm in response to brief depolarizing steps revealed that the kappa-opioid-induced reductions in N-, L-, or P/Q-type currents were accompanied by attenuations in two kinetically distinct components of Ca2+-dependent exocytotic release. These data provide strong evidence of a functional linkage between blockade of Ca2+ influx through voltage-dependent Ca2+ channels and inhibitory modulation of release by presynaptic opioid receptors in mammalian central nerve endings.

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.

Dynorphin A-mediated reduction in multiple calcium currents involves a G(o) alpha-subtype G protein in rat primary afferent neurons.

We examined the effect of antisera directed at specific G-protein subtype(s) on dynorphin A (Dyn A)-mediated reduction of calcium currents in rat dorsal root ganglia (DRG) neurons. Whole cell patch-clamp recordings were performed on acutely dissociated neurons. Dyn A (1 microM)-mediated decrease in calcium currents was inhibited > 90% by the preferential kappa-receptor antagonist norbinaltorphimine. Dyn A (300-1,000 nM)-mediated reduction in calcium currents was examined during intracellular administration of antisera directed against specific regions of G(o) alpha, G(i) 1 alpha/G(1) 2 alpha, and G(i) 3 alpha subunits. Intracellular dialysis with an antiserum specific for G(o) alpha for 20 min decreased calcium current inhibition by Dyn A (1 microM) in 13 of 15 neurons by an average of 75%. Dialysis with nonimmune serum did not affect Dyn A's action to reduce calcium currents. Intracellular dialysis with either anti-G(i) 1 alpha/G(i) 2 alpha or anti-G(i) 3 alpha antisera did not affect Dyn A-induced changes in calcium currents. In the presence of the N-type calcium channel antagonist omega-conotoxin GVIA, the P-type calcium channel antagonist omega-Aga IVA, and omega-Aga MVIIC applied subsequent to the other toxins, the effect of Dyn A to reduce calcium currents was inhibited by 52, 28, and 16%, respectively. The L channel antagonist nifedipine did not affect the ability to Dyn A to inhibit calcium currents. These results suggest that in rat DRG neurons coupling of kappa-opioid receptors to multiple transient, high-threshold calcium currents involves the G(o) alpha subclass of G proteins.

Nerve growth factor facilitates cholinergic neurotransmission between nucleus basalis and the amygdala in rat: an electrophysiological analysis.

Treatment of rats in vivo with NGF promotes the survival and enhances the neurotransmitter phenotype of basal forebrain cholinergic neurons. We showed recently (Williams et al., 1993) that NGF-induced stimulations of the cholinergic markers ChAT and high-affinity choline uptake are reflected in an enhanced synthesis and release of ACh in terminals fields of basal forebrain cholinergic neurons. The objective of the present study was to determine whether such effects translate into an enhancement in neurotransmission between nucleus basalis neurons and postsynaptic target cells, and therefore are likely to be of physiological significance. Changes in cholinergic neurotransmission after NGF were assessed by comparing the ability of cholinergic pathway activation, produced by electrical stimulation of nucleus basalis or the external capsule, to elicit intracellularly recorded muscarinic responses in basolateral amygdaloid (BLA) neurons in ventral forebrain slice preparations from NGF-treated and control Fischer 344 adult rats. Chronic infusion of NGF for 3 weeks (1.2 micrograms/d, i.c.v.) increased the likelihood of eliciting cholinergic slow depolarizations (slow EPSP) via stimulation of cholinergic pathways in the slice. In addition, the frequency-response curves for generation of the cholinergic slow EPSP by nucleus basalis or external capsule stimulation were shifted approximately twofold to the left and the EF50 values significantly reduced in neurons from NGF-treated slices, compared to those in preparations from vehicle-treated or untreated controls. Treatment with NGF also resulted in a leftward shift in the frequency-response curve for cholinergic pathway-induced blockade of the slow afterhyperpolarization, without change in the maximal inhibitory effect. The NGF-induced enhancement in cholinergic synaptic effectiveness was not accompanied by alterations in the resting membrane properties or intrinsic excitability of BLA pyramidal neurons. Nor did treatment with NGF affect their chemosensitivity or responsiveness to direct postsynaptic applications of the cholinergic carbachol. We conclude from these results that chronic administration of exogenous NGF can facilitate neurotransmission within basal forebrain cholinergic projections in normal adult brain, presumably as a consequence of its ability to stimulate presynaptic mechanisms involved in synthesis and/or release of ACh.

mu-Opioid receptor activation reduces multiple components of high-threshold calcium current in rat sensory neurons.

Whole-cell patch-clamp recordings were used to characterize calcium channel types that are modulated by mu-opioid receptor activation in rat dorsal root ganglion (DRG) neurons. Five distinct components of high-threshold calcium current were isolated on the basis of their sensitivity to the selective channel blockers omega-conotoxin GVIA, nifedipine, omega-conotoxin MVIIC, or omega-agatoxin IVA. The mu-opioid selective agonist Tyr-Pro-NMePhe-D-Pro-NH2 (PLO17) routinely suppressed high-threshold currents and this effect was always reduced by omega-conotoxin GVIA. A fraction of PLO17-sensitive current remained after omega-conotoxin GVIA that was eliminated by application of omega-agatoxin IVA alone or in combination with omega-conotoxin MVIIC. Nifedipine had no effect on mu-opioid responses nor did PLO17 affect the slow component of tail current induced by Bay K 8644. These data suggest that mu-opioid receptors are negatively coupled to three types of calcium channels in rat DRG neurons, including an omega-conotoxin GVIA-sensitive (N-type) channel, an omega-agatoxin IVA-sensitive (P-type) channel and an omega-conotoxin MVIIC-sensitive, nifedipine/GVIA/omega-Aga IVA-resistant (presumptive Q-type) channel.

Mu- and kappa-opioid receptors selectively reduce the same transient components of high-threshold calcium current in rat dorsal root ganglion sensory neurons.

Whole-cell patch-clamp recordings were used to examine the regulation of voltage-dependent calcium channels by mu- and kappa-opioid receptors in acutely isolated rat dorsal root ganglion (DRG) sensory neurons. Agonists selective for either mu- (Tyr-Pro-NMePhe-D-Pro-NH2, PLO17) or kappa-opioid receptors (dynorphin A, U69,593) inhibited high-threshold calcium currents in a reversible and naloxone-sensitive manner, whereas administration of D-Pen2,5-enkephalin, a delta-selective agonist, was without effect. However, none of the opioids reduced low-threshold T-type currents. The inhibitory effects of PLO17 were blocked by the irreversible mu-opioid antagonist beta-funaltrexamine but not the kappa-opioid antagonist nor-binaltorphimine, while responses to kappa-opioid agonists showed the opposite pattern of antagonist sensitivity. In addition, many cells responded to both PLO17 and dynorphin A (or U69,593), and in these neurons the inhibitory response to one agonist was occluded when tested in the presence of the other. These data suggest that mu- and kappa-opioid receptors are coexpressed on at least some DRG neurons and appear to be functionally coupled to a common pool of calcium channels. Both rapidly inactivating (transient) and sustained components of high-threshold current, arising from pharmacologically distinct types of calcium channels, were identified in our neurons. Activation of mu-opioid receptors selectively reduced the transient component of currents evoked at +10 mV from Vh = -80 mV, while sparing the sustained component. The transient component was irreversibly blocked by the N-type channel antagonist omega-conotoxin GVIA (omega-CgTx), and in one-half of the neurons there was a concomitant loss of the response to PLO17. In the remaining neurons, PLO17 continued to reduce a small fraction of omega-CgTx-insensitive current and subsequent administration of the L-type channel blocker nifedipine in saturating concentrations failed to reduce the opioid-induced inhibitory effect. These data demonstrate that mu-opioid receptors are negatively coupled to several pharmacologically distinct types of calcium channels in DRG sensory neurons, one that was blocked by omega-CgTx and thus likely to be N-type, and a second that was resistant to blockade by N- and L-type channel blockers.

Multiple effects of long-term morphine treatment on postsynaptic beta-adrenergic receptor function in hippocampus: an intracellular analysis.

We previously reported that beta-adrenergic receptors are increased in cerebral cortex and hippocampus in rats treated chronically with morphine and subsequently down-regulated after morphine withdrawal [22,23]. The changes in receptor density in hippocampus were accompanied by a corresponding super- and subsensitivity, respectively, in beta-adrenergic responsiveness, as assessed electrophysiologically by measuring the ability of isoproterenol to augment population spike responses in the slice. In this study, we compared the ability of isoproterenol to reduce the Ca(2+)-activated K+ slow afterhyperpolarization (slow AHP) in pyramidal neurons in hippocampal slices from opiate-naive and chronic morphine-treated rats to determine whether such changes in beta-adrenergic receptor function are localized postsynaptically. Chronic treatment of rats with morphine produced a 3.5-fold parallel shift to the left in the concentration-response curve for isoproterenol and reduced the EC50 from 4.8 +/- 1.3 to 1.4 +/- 0.5 nM. In contrast, sensitivity and maximal responsiveness to isoproterenol was markedly decreased in pyramidal neurons recorded in slices from morphine withdrawn animals. The concentration-response curves for inhibition of the slow AHP by carbachol or forskolin were not affected by chronic morphine treatment. However, blockade of the slow AHP by forskolin was significantly reduced in pyramidal neurons studied after morphine withdrawal. These data suggest that the increase in electrophysiological responsiveness to beta-adrenergic receptor stimulation found in hippocampus after chronic morphine treatment most likely resulted from an up-regulation in postsynaptic membrane receptors, whereas alterations occurring beyond the receptor level may be involved in the desensitization that is associated with morphine withdrawal.

Metabotropic glutamate receptor agonist ACPD inhibits some, but not all, muscarinic-sensitive K+ conductances in basolateral amygdaloid neurons.

Muscarinic agonists produce membrane depolarization and losses of spike frequency accommodation and the slow afterhyperpolarization (AHP) when applied to neurons of the basolateral amygdala (BLA). Underlying these changes are the muscarinic-induced inhibitions of several K+ conductances, including the voltage-activated M-current (IM), a slowly decaying Ca(2+)-activated current (IAHP), a voltage-insensitive leak current (ILeak), and the hyperpolarization-activated inward rectifier current (IIR). Similar depolarizations and losses of the slow AHP have been observed in other neuronal cell types following stimulation of metabotropic glutamate receptors. Therefore, we tested the effects of the metabotropic glutamate receptor agonist, 1-aminocyclopentane-1s,3R-dicarboxylic acid (ACPD), on pyramidal neurons impaled with a single microelectrode for current- and voltage-clamp recordings in a brain slice preparation of the rat BLA. Application of ACPD (20 or 100 microM) to BLA neurons inhibited IM and IAHP, resulting in membrane depolarization and reductions in the amplitude and duration of the slow AHP. However, ACPD did not inhibit the muscarinic-sensitive current IIR, nor was ILeak blocked in the majority of neurons examined. These findings suggest the possibility that muscarinic cholinergic and metabotropic glutamatergic receptor agonists may activate separate intracellular transduction pathways which have convergent inhibitory effects onto IM and IAHP in BLA pyramidal neurons.

mu-Opioid receptor-mediated reduction of neuronal calcium current occurs via a G(o)-type GTP-binding protein.

It has recently been shown that the activation of mu-opioid receptors inhibits several components of calcium channel current in rat DRG sensory neurons. mu-Opioid receptors, acting through the pertussis toxin (PTX)-sensitive substrate Gi, also reduce the activity of neuronal adenylate cyclase, but the relationship of this effect to changes in calcium channel activity has yet to be determined. Using whole-cell recordings from acutely isolated rat DRG neurons, we examined the ability of the mu-opioid-selective agonist Tyr-Pro-NMe-Phe-D-Pro-NH2 (PLO17) to reduce calcium current after treatment with PTX and in the presence of the nonhydrolyzable GTP analog guanosine 5'-[-thio]triphosphate (GTP gamma S), to assess the role of G-proteins in the coupling of mu-opioid receptors to calcium channels. Inhibition of current by PLO17 was mimicked or rendered irreversible by intracellular administration of GTP gamma S, an activator of G-proteins, and was blocked by pretreatment of neurons with PTX. In contrast, when the catalytic subunit of cAMP-dependent protein kinase was included in the recording pipette, calcium currents increased in magnitude throughout the recording without attenuation of responses to PLO17. Thus, the mu-opioid-induced inhibition of calcium current occurs through activation of a Gi- or G(o)-type G-protein, but independent of changes in adenylate cyclase activity. As a first step in identifying this G-protein, we compared the ability of several antisera directed against specific regions of Gi and G(o)alpha subunits to block the inhibition in current by PLO17. Intracellular dialysis with an antiserum specific for G(o) (GC/2) attenuated calcium current inhibition by PLO17 in five of six neurons by an average of 75%. In contrast, there was no attenuation in the response to PLO17 when neurons were dialyzed with an anti-Gi1 alpha/Gi2 alpha antiserum (AS/7) or antibodies specific for alpha subunits of Gi proteins (Gi1/Gi2 or Gi3) in an identical manner. These results suggest that in rat DRG neurons mu-opioid receptors couple to calcium channels via the PTX-sensitive G(o) subclass of GTP-binding proteins.

Hyperpolarization-activated currents in neurons of the rat basolateral amygdala.

1. A single microelectrode was used to obtain current-clamp or voltage-clamp recordings from two neuronal cell types (pyramidal and late-firing neurons) in the basolateral nucleus of the amygdala (BLA) in slices of the rat ventral forebrain. Conductances activated by hyperpolarizing voltage steps from a holding potential of -70 mV were identified and their sensitivity to muscarinic modulation was determined using bath superfusion of carbachol. 2. Unclamped pyramidal neurons exhibited anomalous rectification, seen as a slowly developing depolarizing sag in the electronic potential in response to a hyperpolarizing current pulse. 3. Stepping voltage-clamped pyramidal neurons to command potentials of between -70 and -100 mV activated a slowly developing inward current (ISlow) that followed a single exponential time course. Larger hyperpolarizing voltage steps evoked a rapidly developing inward current (IFast) that preceded the development of ISlow. 4. The ISlow component reversed at a level positive to the -70 mV holding potential. Its rate of activation accelerated as the hyperpolarizing voltage step was made more negative. The threshold for activation of the conductance underlying ISlow was approximately -60 mV, with half-activation occurring at -90 mV. 5. Extracellular Cs+ (2 mM) blocked ISlow and eliminated anomalous rectification in unclamped pyramidal neurons. The inhibition of ISlow by Cs+ was also associated with membrane hyperpolarization and reduction of the medium afterhyperpolarization. ISlow was unaffected by extracellular Ba2+ (100 microM). The properties of this current appeared similar to that of the mixed cationic H-current previously identified in other neurons. 6. In comparison with pyramidal cells, unclamped late-firing neurons displayed a lesser but more rapidly developing anomalous rectification in response to large hyperpolarizations from rest. In voltage clamp, hyperpolarizing steps to command potentials more negative than -100 mV elicited IFast. Late-firing neurons expressed little or no ISlow. 7. The properties of IFast were identical in both pyramidal and late-firing neurons. This current reversed at a potential negative to -70 mV. Its rate of current activation increased with the magnitude of the hyperpolarizing voltage step. This rate was approximately sevenfold faster than ISlow activation recorded at the same membrane potential. IFast was blocked by 2 mM extracellular Cs+ and reduced by 100 microM extracellular Ba2+. The threshold for activation of the underlying conductance was approximately -85 mV, with half-activation occurring at -112 mV. The properties of IFast were similar to those of the inward rectifier current previously identified in other central neurons. 8. Carbachol (40 microM) largely blocked IFast without affecting its rate of activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic modulation of conductances underlying the afterhyperpolarization in neurons of the rat basolateral amygdala.

The excitability level of pyramidal neurons in the basolateral amygdala (BLA) is greatly increased following muscarinic receptor activation, an effect associated with an increased rate of action potential firing and reduction of the afterhyperpolarization (AHP). We impaled BLA pyramidal neurons in slices of rat ventral forebrain with a single microelectrode to examine the currents underlying the AHP and spike frequency accommodation and determine their sensitivities to muscarinic modulation. In voltage-clamp, depolarizing steps were followed by biphasic outward tail currents, consisting of rapidly decaying (IFast) and slowly decaying (ISlow) current components. These corresponded temporally with the medium and slow portions of the AHP, respectively. The reversal potential for the IFast component of the AHP tail current shifted in the depolarizing direction with increases in the extracellular K+ concentration. The amplitude of IFast was reduced during perfusion of 0-Ca2+ medium or by superfusion of TEA (1-5 mM) or carbachol (10-40 microM). It is suggested that IFast was produced by the rapidly decaying Ca(2+)-activated K+ current (IC) and the muscarinic-sensitive M-current (IM). The ISlow tail current component reversed at the estimated values for EK in medium containing either normal or elevated K+ levels. This component was eliminated by perfusion of 0-Ca2+ medium or inclusion of cyclic-AMP in the recording electrode. It was not blocked by TEA (5 mM) or apamin (50-500 nM), but was reduced by carbachol in a dose-dependent manner (IC50 = 0.5 microM). Electrical stimulation of cholinergic afferent pathways to the BLA produced inhibition of ISlow, an effect which was enhanced by eserine and prevented by atropine. Loss of the ISlow component was always accompanied by similar reductions in accommodation and the slow AHP. It was concluded that this tail current component resulted from the slowly decaying Ca(2+)-activated K+ current, IAHP. Thus, the muscarinic inhibition of IAHP contributes to the enhanced excitability exhibited by BLA pyramidal neurons following cholinergic stimulation.

Muscarinic inhibition of M-current and a potassium leak conductance in neurones of the rat basolateral amygdala.

1. Voltage-clamp recordings using a single microelectrode were obtained from pyramidal neurones of the basolateral amygdala (BLA) in slices of the rat ventral forebrain. Slow inward current relaxations during hyperpolarizing voltage steps from a holding potential of -40 mV were identified as the muscarinic-sensitive M-current (IM), a time- and voltage-dependent potassium current previously identified in other neuronal cell types. 2. Activation of IM was voltage dependent with a threshold of approximately -70 mV. At membrane potentials positive to this, the steady-state current-voltage (I-V) relationship showed substantial outward rectification, reflecting the time- and voltage-dependent opening of M-channels. The underlying conductance (gM) also increased sharply with depolarization. 3. The reversal potential for IM was -84 mV in medium containing 3.5 mM K+. This was shifted positively by 27 mV when the external K+ concentration was raised to 15 mM. 4. The time courses of M-current activation and deactivation were fitted by a single exponential. The time constant for IM decay, measured at 24 degrees C, was strongly dependent on membrane potential, ranging from 330 ms at -40 mV to 12 ms at -100 mV. 5. Bath application of carbachol (0.5-40 microM) inhibited IM, as evidenced by the reduction or elimination of the slow inward M-current relaxations evoked during hyperpolarizing steps from a holding potential of -40 mV. The outward rectification of the steady-state I-V relationship at membrane potentials positive to -70 mV was also largely eliminated. The inhibition of IM by carbachol was dose dependent and antagonized by atropine. 6. Carbachol produced an inward current shift at a holding potential of -40 mV that was only partially attributable to inhibition of IM. An inward current shift was also produced by carbachol at membrane potentials negative to -70 mV, where IM is inactive. These effects were dose dependent and antagonized by atropine. They were attributed to the muscarinic inhibition of a voltage-insensitive potassium leak conductance (ILeak). 7. In most cells, carbachol reduced the slope of the instantaneous I-V relationship obtained from a holding potential of -70 mV so that it crossed the control I-V plot at the reversal potential for ILeak. This was found to be -108 mV in 3.5 mM K+ saline, shifting to -66 mV in 15 mM K+ saline.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological and morphological properties of rat basolateral amygdaloid neurons in vitro.

Electrophysiological and morphological properties of neurons in the rat basolateral amygdala (BLA) were assessed using intracellular recordings in brain slice preparations. The vast majority of cells studied were identified as pyramidal cells on the basis of their accommodation response and by a prominent afterhyperpolarization that followed a current-evoked burst of action potentials. The second class of cells consisted of late-firing neurons that were distinguished electrophysiologically by their very negative resting membrane potential (-82 mV) and conspicuous delay in the onset of spike firing in response to depolarizing current injection. The third class of cells, termed fast-firing neurons, possessed many of the features of intrinsic inhibitory interneurons found elsewhere in the brain. These included very brief action potentials (0.7 msec), a relatively depolarized resting membrane potential (-62 mV), and spontaneous firing at a high rate and the absence of spike frequency accommodation. Intracellular labeling with Lucifer yellow of electrophysiologically identified pyramidal and late-firing cells showed them to have pyramidal to stellate cells bodies and spine-covered dendrites. Although having an overall pyramidal-like morphology, late-firing neurons possessed cells bodies and dendritic fields that were smaller than those of pyramidal cells. Lucifer yellow-labeled fast-firing neurons had a nonpyramidal morphology, with somata that were spherical to multipolar in shape and spine-sparse or aspiny dendrites. The morphological features of these cells corresponded closely to those of GABA-containing interneurons that have been described previously in the rat BLA using immunohistochemical techniques (McDonald, 1985b). Thus, it seems likely that activation of fast-firing neurons underlies inhibitory synaptic events that are recorded in the rat BLA. Our data support the conclusion derived from previous anatomical studies that pyramidal neurons constitute the predominant cell type in the BLA and function as projection neurons in this region of the amygdala. The determination of whether late-firing cells constitute a unique class of projection neurons distinct from pyramidal cells must await the outcome of studies in which the anatomical terminations of this cell type are specified.

Inhibitory responses of rat basolateral amygdaloid neurons recorded in vitro.

The purpose of the present study was to characterize the ionic and pharmacological basis of the actions of synaptically released and exogenously applied GABA in basolateral amygdaloid pyramidal cells in vitro. Stimulation of forebrain afferents to pyramidal neurons in the basolateral amygdala evoked an excitatory postsynaptic potential followed by early and late inhibitory postsynaptic potentials. The early inhibitory postsynaptic potential had a reversal potential near -70 mV, was sensitive to changes in the chloride gradient across the membrane and was blocked by the GABAA antagonists picrotoxin and bicuculline methiodide but not by the GABAB antagonists phaclofen or 2-hydroxysaclofen. In contrast, the late inhibitory postsynaptic potential had a reversal potential of approximately -95 mV and was markedly reduced or abolished by GABAB antagonists. Pressure application of GABA to the surface of the slice typically elicited a triphasic response in basolateral amygdaloid pyramidal neurons consisting of a short-latency hyperpolarization that preceded or was superimposed on a membrane depolarization followed by a longer latency hyperpolarization. Each of the responses was associated with an increase in membrane conductance. Determinations of the reversal potential, ionic dependency and sensitivity to pharmacological blockade of each component of the GABA-induced response revealed that the initial hyperpolarizing (Erev approximately -70 mV) and depolarizing (Erev approximately -55 mV) responses were mediated by a GABAA-mediated increase in chloride conductance, whereas the late hyperpolarizing response (Erev approximately -82 mV) to GABA arose from a GABAB-mediated increase in potassium conductance. Experiments in which GABA was applied at various locations on the cell suggested that the short-latency hyperpolarization resulted from activation of somatic GABA receptors, whereas the depolarizing and late hyperpolarizing responses were generated primarily in the dendrites. In contrast to the complex membrane response profile elicited by GABA, pressure ejection of the GABAB agonist baclofen produced only membrane hyperpolarizations. Taken together, these results suggest that inhibitory responses that are recorded in basolateral amygdaloid pyramidal cells are mediated by activation of both GABAA and GABAB receptors. Consistent with findings elsewhere in the CNS, the early inhibitory postsynaptic potential and initial hyperpolarization and depolarizing response to local GABA application appear to involve a GABAA-mediated increase in chloride conductance, whereas the late inhibitory postsynaptic potential and the late hyperpolarizing response to GABA arise from a GABAB-mediated increase in potassium conductance.

Muscarinic responses of rat basolateral amygdaloid neurons recorded in vitro.

1. Intracellular recordings were obtained from pyramidal-type neurons in the basolateral amygdaloid nucleus (BLA) in slices of rat ventral forebrain and used to compare the actions of exogenously applied cholinomimetics to the effects produced by electrical stimulation of amygdalopetal cholinergic afferents from basal forebrain. 2. Bath application of carbachol depolarized pyramidal cells with an associated increase in input resistance (Ri), reduced the slow after-hyperpolarization (AHP) that followed a series of current-evoked action potentials and blocked spike frequency accommodation. All of these effects were reversed by the muscarinic antagonist atropine but not by the nicotinic antagonist hexamethonium. 3. Electrical stimulation of amygdaloid afferents within the external capsule evoked a series of synaptic potentials consisting of a non-cholinergic fast excitatory postsynaptic potential (EPSP), followed by early and late inhibitory postsynaptic potentials (IPSPs). Each of these synaptic potentials was reduced by carbachol in an atropine-sensitive manner. 4. Local application of carbachol to pyramidal cells produced a short-latency hyperpolarization followed by a prolonged depolarization. The hyperpolarization and depolarization to carbachol were blocked by atropine but not hexamethonium. 5. The carbachol-induced hyperpolarization was associated with a decrease in Ri and had a reversal potential nearly identical to that of the early IPSP. The inhibitory response was blocked by perfusion of medium containing tetrodotoxin (TTX), bicuculline or picrotoxin, while the subsequent depolarization was unaffected. On the basis of these data, it is concluded that the muscarinic hyperpolarization is mediated through the rapid excitation of presynaptic GABAergic interneurons in the slice. 6. The findings that the carbachol-induced depolarization was associated with an increase in Ri, often had a reversal potential below -80 mV, was sensitive to changes in extracellular potassium concentration and was blocked by intracellular ionophoresis of the potassium channel blocker caesium suggest that it resulted from a muscarinic blockade of one or more potassium conductances. 7. Repetitive stimulation of sites within the slice containing cholinergic afferents evoked a series of fast EPSPs followed by IPSPs. These non-cholinergic potentials were followed by a slow EPSP that lasted from 10 s-4 min. The slow EPSP was enhanced by eserine and blocked by atropine. It was also blocked by TTX or cadmium, indicating that it was dependent on spike propagation and calcium-dependent release of acetylcholine (ACh). 8. Stimulation of cholinergic afferents in the slice mimicked other effects produced by carbachol including blockade of the slow AHP and accommodation of action potential discharge and these actions were potentiated by eserine and blocked by atropine.(ABSTRACT TRUNCATED AT 400 WORDS)

Exogenous NGF affects cholinergic transmitter function and Y-maze behavior in aged Fischer 344 male rats.

Chronic ICV administration of NGF stimulates the activity of the cholinergic neuronal markers, HACU and ChAT, as well as the evoked release of both endogenous and newly synthesized acetylcholine in the brain of aging Fischer 344 male rats. However, the pattern of cholinergic phenotype stimulation indicates an age-related differential regulation of ChAT, HACU, and ACh release between specific brain areas, with the largest effects found in the striatum. NGF treatment also increases the effectiveness of neurotransmission between basal forebrain cholinergic neurons and postsynaptic amygdaloid target neurons. The stimulation of central cholinergic transmitter function after NGF treatment affects behavior in a Y-maze brightness discrimination paradigm. NGF treatment does not affect the cognitive measure of brightness discrimination, but reduces the number of avoidance attempts, a measure of motor function.