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Evidence suggests that the long-term adaptations in the hippocampus after repeated drug treatment may parallel its role during memory formation. The neuroplasticity that subserves learning and memory is also believed to underlie addictive processes. We have reported previously that repeated morphine administration alters local distribution of endocytic proteins at hippocampal synapses, which could in turn affect expression of glutamate receptors. Glutamatergic systems, including α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), are believed to be involved in opiate-induced neuronal and behavioral plasticity, although the mechanisms underlying these effects are only beginning to be understood. The present study further examines the effects of repeated morphine administration on the expression and composition of AMPARs and the functional ramifications. Twelve hours after the last morphine injection, we observed an increased expression of AMPARs lacking glutamate receptor (GluR) 2 in hippocampal synaptic fractions. Immunoblotting studies show that 12 h after morphine treatment, GluR1 subunits are increased at the postsynaptic density (PSD) and at extrasynaptic sites, whereas GluR3 subunits are only increased at the PSD, and they show how this alters receptor subunit composition. In addition, we provide electrophysiological evidence that AMPARs are switched to Ca2+-permeable (GluR2-lacking) at the synapse 12 h after repeated morphine treatment, affecting the magnitude of long-term depression at hippocampal neurons. We propose that morphine-induced changes in glutamatergic synaptic transmission in the hippocampus may play an important role in the neuroadaptations induced by repeated morphine administration.

Fear conditioning, a behavioral model of fear learning and cue-related anxiety, causes enhanced neuronal transmission in the thalamic to lateral amygdala pathway. 1,2In the expression phase of learned fear, this increased transmission recorded in vitrois revealed in increased amplitudes of excitatory postsynaptic currents (EPSCs) and occlusion of paired-pulse facilitation (PPF) implicating a presynaptic increase in transmitter release. Here we examined the contribution of L-type calcium channels in fear conditioning. We measured the effect of nimodipine (Nim, 1.5-20 mgkg), an L-type calcium channel antagonist, on fear-potentiated startle in which startle was assessed in animals receiving paired or unpaired tone and foot shock. Nim administered intraperitoneally blocked fear-potentiated startle but not baseline startle in a dose-dependent manner. We also analyzed the effect of Nim (10 ?M) in vitroon synaptic facilitation of EPSCs and PPF in slices from naïve control, unpaired control, and fear-conditioned animals. In neurons from naïve control animals, Nim had no effect on EPSC amplitude or PPF, but in slices from fear-conditioned rats, Nim reduced EPSC amplitude, suggesting the recruitment of L-type calcium channels within the fear-conditioning pathway. Nim increased PPF in slices from fear-conditioned animals, suggesting that L-type calcium channels may contribute to increased probability of release in fear conditioning. In slices from unpaired animals, Nim decreased synaptic transmission but had little effect on PPF, suggesting that stress or contextual fear learning may induce L-type channel activity in fear-conditioned and unpaired control animal groups. We also analyzed protein expression of the ?1Cand ?1DL-type calcium channel subunits isolated from the amygdala and found that ?1Cprotein was significantly increased in fear-conditioned animals. These findings suggest that L-type calcium channels play a role in the amygdala in cued fear conditioning and have important implications in the treatment of anxiety and in emotional learning and plasticity.

Stimulating thalamic fibers exiting from the internal capsule evokes a glutamatergic excitatory postsynaptic current (EPSC) recorded in vitro with patch electrodes in neurons of the rat lateral amygdala (LA). The purpose of this study is to compare paired‐pulse facilitation (PPF), a form of short‐term synaptic plasticity, of AMPA and NMDA receptor‐mediated EPSCs. Analysis of PPF at this synapse is important since, in fear‐conditioned animals, PPF reflects an enhanced transmitter release but the amplitude of only AMPA EPSCs is facilitated. PPF magnitude of the composite EPSC is a result of both AMPA and NMDA receptor activation; however, the characteristics of AMPA and NMDA PPF are dissimilar. Specifically, the NMDA EPSC shows greater PPF (NMDA PPF) than does the AMPA EPSC whether measuring the NMDA PPF magnitude in an AMPA antagonist/Mg2+‐free solution or by subtracting the AMPA EPSC from the composite EPSC in normal Mg2+. Presynaptic NMDA receptors neither influence AMPA PPF nor account for the difference between the NMDA and AMPA PPF. Another difference was that removal of inhibitory tone enhanced AMPA PPF, while it had mixed effects on NMDA PPF. Furthermore, AMPA PPF was independent of stimulus intensity and postsynaptic voltage, unlike the NMDA PPF. Another dissimilarity was that the amplitudes of pairs of AMPA EPSCs were not correlated, suggesting presynaptic mechanisms. In contrast, NMDA PPF was dependent on stimulus intensity and postsynaptic voltage and the amplitudes of paired NMDA EPSCs had a positive correlation, suggesting a postsynaptic influence. Both AMPA and NMDA PPF were influenced by GABA inhibition and this could be a factor in the magnitude disparity. These data show that AMPA and NMDA PPF have different characteristics and contribute to the composite PPF in the thalamic to lateral amygdala pathway. Synapse 42:115–127, 2001. © 2001 Wiley‐Liss, Inc.

G-protein-coupled metabotropic glutamate receptors (mGluRs) are being implicated in various forms of neuroplasticity and CNS disorders. This study examined whether the sensitivities of mGluR agonists are modulated in a distinct fashion in different models of synaptic plasticity, specifically, kindling and chronic cocaine treatment. The influence of kindling and chronic cocaine exposure in vivo was examined in vitro on the modulation of synaptic transmission by group II and III metabotropic glutamate receptors using whole cell voltage-clamp recordings of central amygdala (CeA) neurons. Synaptic transmission was evoked by electrical stimulation of the basolateral amygdala (BLA) and ventral amygdaloid pathway (VAP) afferents in brain slices from control rats and from rats treated with cocaine or exposed to three to five stage-five kindled seizures. This study shows that after chemical stimulation with chronic cocaine exposure or after electrical stimulation with kindling the receptor sensitivities for mGluR agonists are altered in opposite ways. In slices from control rats, group II agonists, (2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (LCCG1) and (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), depressed neurotransmission more potently at the BLA-CeA than at the VAP-CeA synapse while group III agonist, L(+)-2-amino-4-phosphonobutyrate (LAP4), depressed neurotransmission more potently at the VAP-CeA synapse than at the BLA-CeA. These agonist actions were not seen (were absent) in amygdala neurons from chronic cocaine-treated animals. In contrast, after kindling, concentration response relationships for LCCG1 and LAP4 were shifted to the left, suggesting that sensitivity to these agonists is increased. Except at high concentrations, LCCG1, LY354740, and LAP4 neither induced membrane currents nor changed current-voltage relationships. Loss of mGluR inhibition with chronic cocaine treatment may contribute to counter-adaptive changes including anxiety and depression in cocaine withdrawal. Drugs that restore the inhibitory effects of group II and III mGluRs may be novel tools in the treatment of cocaine dependence. The enhanced sensitivity to group II and III mGluR agonists in kindling is similar to that recorded at the lateral to BLA synapse in the amygdala where they reduce epileptiform bursting. These findings suggest that drugs modifying mGluRs may prove useful in the treatment of cocaine withdrawal or epilepsy.

Differential effects of metabotropic glutamate receptor antagonists on bursting activity in the amygdala. Metabotropic glutamate receptors (mGluRs) are implicated in both the activation and inhibition of epileptiform bursting activity in seizure models. We examined the role of mGluR agonists and antagonists on bursting in vitro with whole cell recordings from neurons in the basolateral amygdala (BLA) of amygdala-kindled rats. The broad-spectrum mGluR agonist 1S,3R-1-aminocyclopentane dicarboxylate (1S,3R-ACPD, 100 μM) and the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 20 μM) evoked bursting in BLA neurons from amygdala-kindled rats but not in control neurons. Neither the group II agonist (2S,3S,4S)-α-(carboxycyclopropyl)-glycine (l-CCG-I, 10 μM) nor the group III agonistl-2-amino-4-phosphonobutyrate (l-AP4, 100 μM) evoked bursting. The agonist-induced bursting was inhibited by the mGluR1 antagonists (+)-α-methyl-4-carboxyphenylglycine [(+)-MCPG, 500 μM] and (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG, 300 μM]. Kindling enhanced synaptic strength from the lateral amygdala (LA) to the BLA, resulting in synaptically driven bursts at low stimulus intensity. Bursting was abolished by (S)-4C3HPG. Further increasing stimulus intensity in the presence of (S)-4C3HPG (300 μM) evoked action potential firing similar to control neurons but did not induce epileptiform bursting. In kindled rats, the same threshold stimulation that evoked epileptiform bursting in the absence of drugs elicited excitatory postsynaptic potentials in (S)-4C3HPG. In contrast (+)-MCPG had no effect on afferent-evoked bursting in kindled neurons. Because (+)-MCPG is a mGluR2 antagonist, whereas (S)-4C3HPG is a mGluR2 agonist, the different effects of these compounds suggest that mGluR2 activation decreases excitability. Together these data suggest that group I mGluRs may facilitate and group II mGluRs may attenuate epileptiform bursting observed in kindled rats. The mixed agonist–antagonist (S)-4C3HPG restored synaptic transmission to control levels at the LA-BLA synapse in kindled animals. The different actions of (S)-4C3HPG and (+)-MCPG on LA-evoked bursting suggests that the mGluR1 antagonist–mGluR2 agonist properties may be the distinctive pharmacology necessary for future anticonvulsant compounds.

Corticotropin-releasing factor (CRF) is an important mediator of stress responses in the brain, and CRF receptors and CRF-containing neurons and terminals are located within the central nucleus of the amygdala (CeA). CeA neurons possess multiple types of Ca++channels, including L, N and Q types and a current resistant to saturating concentrations of dihydropyridine and neurotoxin antagonists. In this study, we used whole-cell patch-clamp techniques to study the effects of CRF on whole-cell Ca++current (ICa) in acutely dissociated CeA neurons and determine components of the current affected. CRF (1–400 nM) increased the peak of the ICain ≈50% of the CeA neurons recorded. In the remaining neurons, CRF had little effect. The CRF-induced increase in the ICawas concentration dependent and the estimated EC50value was 14.9 nM. CRF (50 nM) increased the peak ICaby 25 ± 5% (n= 9). CRF produced an increase in both the transient and the steady state current but did not shift the peak of the current-voltage relationship. CRF did not affect the voltage dependence of activation and inactivation, and the CRF effect on ICas was not significantly different when the neuron was held at −80 or −40 mV. The competitive CRF receptor antagonist (α-helical CRF9–41, 3 μM) blocked the CRF-induced increase in ICa, suggesting that the effect of CRF is receptor mediated. CRF (50 nM) enhanced the ICa(20 ± 3%) in the presence of saturating concentrations of the L-type blocker nimodipine and neurotoxin N- and Q-type blockers ω-conotoxin GVIA and ω-conotoxin MVIIC. We conclude that CRF increased, through a receptor mechanism, dihydropyridine- and neurotoxin-resistant current(s) in CeA neurons.

Interleukin-1 beta (IL-1 beta), a mediator of immune response, is found in the brain and IL-1 binding sites are located in the basolateral amygdala (BLA). Superfusion of IL-1 beta (118 pM) hyperpolarized the membrane and decreased input resistance in most BLA neurons in brain slice preparations. The hyperpolarization was dose dependent, reversible, persisted in tetrodotoxin and had an estimated EC50 of 15.3 pM. Reversal potentials for the hyperpolarization recorded with potassium acetate and KCl electrodes were -74 and -40 mV, respectively. These data suggest involvement of a chloride conductance. The hyperpolarization was not observed in bicuculline or in acutely dissociated BLA neurons, which implicates an indirect mediation through enhancement of endogenous gamma-aminobutyric acid (GABA). Superfusion of IL-1 beta (118 pM) inhibited excitatory and fast and slow inhibitory postsynaptic potentials evoked by stimulating either the stria terminalis or the lateral amygdala. Fast and slow inhibitory postsynaptic potentials elicited by direct stimulation of GABA interneurons in the lateral amygdala were also depressed by IL-1 beta. IL-1 beta did not depress responses to GABA or glutamate receptor agonists in slices or currents induced by glutamate agonists in acutely dissociated BLA neurons. These findings indicate that inhibition of synaptic transmission is presynaptic. The results show that IL-1 beta inhibits excitatory and inhibitory transmission at a presynaptic site and hyperpolarizes the membrane through an indirect action, possibly by enhancing the action of endogenous GABA in the BLA nucleus. The inhibitory effect of IL-1 beta suggests that factors in the immune system play a role in modulating neuronal function in the BLA.

Corticotropin-releasing factor (CRF) is an important mediator of stress responses in the brain, and CRF receptors and CRF-containing neurons and terminals are located within the central nucleus of the amygdala (CeA). CeA neurons possess multiple types of Ca++ channels, including L, N and Q types and a current resistant to saturating concentrations of dihydropyridine and neurotoxin antagonists. In this study, we used whole-cell patch-clamp techniques to study the effects of CRF on whole-cell Ca++ current (ICa) in acutely dissociated CeA neurons and determine components of the current affected. CRF (1-400 nM) increased the peak of the ICa in approximately 50% of the CeA neurons recorded. In the remaining neurons, CRF had little effect. The CRF-induced increase in the ICa was concentration dependent and the estimated EC50 value was 14.9 nM. CRF (50 nM) increased the peak ICa by 25 +/- 5% (n = 9). CRF produced an increase in both the transient and the steady state current but did not shift the peak of the current-voltage relationship. CRF did not affect the voltage dependence of activation and inactivation, and the CRF effect on ICas was not significantly different when the neuron was held at -80 or -40 mV. The competitive CRF receptor antagonist (alpha-helical CRF9-41, 3 microM) blocked the CRF-induced increase in ICa, suggesting that the effect of CRF is receptor mediated. CRF (50 nM) enhanced the ICa (20 +/- 3%) in the presence of saturating concentrations of the L-type blocker nimodipine and neurotoxin N- and Q-type blockers omega-conotoxin GVIA and omega-conotoxin MVIIC. We conclude that CRF increased, through a receptor mechanism, dihydropyridine- and neurotoxin-resistant current(s) in CeA neurons.

1. Intracellular electrical recording techniques were used to study the ionic mechanisms of cholinergic synaptic transmission in cat vesical pelvic ganglia (v.p.g.). 2. Orthodromic nerve stimulation as well as ionophoretic application of acetylcholine (ACh) resulted in, first, a fast excitatory post‐synaptic potential (f.e.p.s.p.) and secondly, a slow inhibitory post‐synaptic potential (s.i.p.s.p). These distinct post‐synaptic responses were direct actions of ACh and not mediated through an interneurone. In addition, a slow excitatory post‐synaptic potential (s.e.p.s.p.) was observed in 44% of the cells. 3. The f.e.p.s.p., mediated via nicotinic receptors, had a reversal potential of ‐10 mV and resembled the conventional rapid depolarization in other ganglia. The s.i.p.s.p., mediated by muscarinic receptors, had a reversal potential of about ‐100 mV and resulted from an increase in potassium conductance. 4. The slow muscarinic hyperpolarization could be observed in the absence of antagonists and it was elicited at stimulus frequencies in the physiological range (2‐10 Hz). the s.i.p.s.p. induced orthodromically or ionophoretically inhibited firing in spontaneously active neurones. These observations suggest that the muscarinic hyperpolarization may occur under physiological conditions and has sufficient magnitude to be inhibitory to neuronal activity.

Responses to noradrenaline (NA) applied by superfusion, ionophoresis or pressure pulse were analysed using conventional intracellular recording and voltage‐clamp methods in cat vesical parasympathetic ganglia. NA (1 microM) hyperpolarized 60% of the neurones, depolarized 25%, and produced a biphasic potential, which comprised a membrane hyperpolarization followed by a membrane depolarization, in 10%. About 5% of the neurones did not respond to NA. The NA hyperpolarization was blocked by yohimbine (1 microM), an alpha 2‐adrenoceptor antagonist, whereas the NA depolarization was blocked by prazosin (0.1‐1 microM), an alpha 1‐adrenoceptor antagonist. These data indicated that the NA hyperpolarization was mediated through alpha 2‐adrenoceptors and the NA depolarization through alpha 1‐adrenoceptors. The NA hyperpolarization was accompanied by an increase in conductance, while the NA depolarization was associated with a decrease in conductance measured under manual‐clamp conditions. Similar conductance changes were observed under voltage clamp. NA hyperpolarizations became smaller as the membrane was hyperpolarized and reversed polarity beyond ‐100 mV. NA depolarizations also became smaller at hyperpolarized membrane potentials and reversed polarity around ‐90 mV. The NA responses were enhanced in low‐K media and depressed in high‐K Krebs solution. The NA hyperpolarization was blocked by the Ca antagonists, Cd, Mn and Co. Intracellular injection of EGTA caused a slowly developing, progressive block of the NA hyperpolarization. The NA depolarization was not affected by low Ca concentrations, Ca antagonists or intracellular injection of EGTA. In some neurones the NA depolarization was unmasked in solutions containing Ca antagonists and after intracellular EGTA injection. The NA hyperpolarization was depressed by intracellular injection and extracellular superfusion of Cs but not by TEA. Ba (10‐100 microM) depressed the NA hyperpolarization by 30%. The NA depolarization persisted in the presence of muscarine (10 microM) and was not blocked by Cs or TEA but was depressed 70% by Ba (10 microM). These data are consistent with the hypotheses that alpha 2‐adrenoceptor activation produces a membrane hyperpolarization that is mediated through a Ca‐dependent K conductance, and that alpha 1‐adrenoceptor activation produces a membrane depolarization through closure of a voltage‐insensitive K channel.

1. Intracellular current-clamp recordings were made from neurons of the basolateral nucleus of the amygdala (BLA) of the rat in the in vitro slice preparation. Neurons were identified morphologically after intracellular injection of biocytin, and the electrophysiological properties and morphological characteristics were correlated. 2. Three distinct morphological subtypes were identified: Class I pyramidal neurons, Class I stellate neurons, and Class II neurons. Each morphological subtype could also be distinguished according to its characteristic electrophysiological properties. 3. Class I pyramidal neurons typically had pyramidal perikarya (cross-sectional area = 245 microns2) with spine-laden apical and basal dendrites. The axon originated from the largest basal dendrite and produced several collaterals that ramified throughout the dendritic arborization of the parent cell. These neurons were characterized electrophysiologically by their higher input resistance (65.6 M omega), long time constant of membrane charging tau 0 (27.8 ms), long duration action potential (half-width = 0.85 ms), and regular firing pattern [1st interspike interval ISI) = 91 ms]. 4. Class I stellate neurons differed morphologically from Class I pyramidal neurons only in the size (cross sectional area = 330 microns 2) and stellate appearance of their perikarya. These neurons had characteristic lower input resistance (40.1 M omega), shorter time constant of membrane charging tau 0 (14.5 ms), shorter duration action potential (half-width = 0.7 ms), and a burst firing pattern (1st ISI = 6.0 ms), all of which were statistically different from Class I pyramidal neurons. 5. Class II neurons were multipolar (cross sectional area = 235 microns 2) and were distinguishable from Class I neurons by the almost complete absence of dendritic spines. Class II neurons were characterized electrophysiologically by a midrange input resistance (58 M omega), intermediate time constant of membrane charging tau 0 (19 ms), intermediate action-potential duration (half-width = 0.77 ms), and a burst firing pattern (1st ISI = 6.0 ms). In contrast to Class I neurons, action-potential firing of Class II neurons did not accommodate in response to prolonged depolarizing current injection. 6. In conclusion, BLA neurons may be characterized by their specific electrophysiological properties as well as by their morphological traits. Therefore, permitting assessment of signal transduction in identified populations of neurons within this nucleus.

Electrogenesis of the slow excitatory post‐synaptic current (slow e.p.s.c.) was analysed with voltage‐clamp methods in curarized sympathetic ganglion cells of bull‐frogs. Three types of slow e.p.s.c. were observed from B neurones of sympathetic ganglia. The type I slow e.p.s.c. was associated with a decrease in membrane conductance, was depressed by membrane hyperpolarization and nullified at ‐60 to ‐70 mV. It was observed in 65% of the sympathetic neurones studied. The type II slow e.p.s.c. was associated with an increase in membrane conductance, was depressed by membrane depolarization and nullified at around +5 mV. It was observed in 14% of the neurones studied. A third type of slow e.p.s.c. was recorded from 21% of the sympathetic neurones in this study. This slow e.p.s.c. was a mixed type having characteristics of both type I and type II slow e.p.s.c.s. Activation of muscarinic cholinergic receptors by application of acetylcholine (ACh) also produced two types of inward currents. The nature of each type of muscarinic slow ACh current was similar to that of each type of slow e.p.s.c. The time course of the falling phase of type I and type II slow e.p.s.c.s was dependent on the membrane potential. The type I slow e.p.s.c. was primarily dependent on extracellular K+ and appeared to be produced by a suppression of the M‐current (Brown & Adams, 1980). The type II slow e.p.s.c. was due to an increased conductance, probably to Na+, and other cations.

The effect of vasoactive intestinal polypeptide (VIP) on the neuronal membranes of isolated cat vesical pelvic ganglia and its underlying ionic mechanism were examined by means of intracellular recording and voltage‐clamp techniques. Application of VIP (0.05‐50 microM) to the neurones by pressure 'puff' ejection through a micropipette placed close to the neurones produced a depolarizing response (2‐15 mV) in 83% of neurones tested; this effect was concentration dependent. The VIP‐induced depolarization frequently evoked spontaneous action potentials in quiescent neurones and increased the frequency of action potentials in spontaneously firing neurones. The VIP depolarization was not blocked in a Ca2+‐free, high‐Mg2+ solution or in a solution containing hexamethonium (1 mM) and atropine (1 microM). Tetrodotoxin (TTX; 1 microM) also did not affect the VIP depolarization. The VIP depolarization was associated with an increase in membrane resistance and the slope of a current‐voltage relation (I‐V curve) was increased by VIP. Conditioning hyperpolarization and depolarization of the membrane increased and decreased the amplitude of the VIP depolarization, respectively. The VIP depolarization reversed polarity around‐‐100 mV. The reversal potential shifted about 20 mV to a more positive level in a high‐K+ (10 mM) solution in accord with the Nernst equation. Substituting Cl‐ with isethionate in the superfusate did not affect the reversal potential of the VIP depolarization. Closure of M‐channels does not underlie VIP action since the VIP depolarization was enhanced by muscarine (10 microM) and unchanged in the presence of Ba (5 mM), or intracellular or extracellular Cs+, conditions known to block the M‐channels (Adams, Brown & Constanti, 1982a, b). Tetraethylammonium (TEA; 20 mM) also did not affect the VIP depolarization. Voltage‐clamp analyses showed that VIP applied by pressure ejection produced an inward current of 80‐110 pA associated with a decrease in membrane conductance (from 2.8 to 3.5 nS) at a holding potential of‐‐60 mV. VIP inward current was diminished by either repetitive or continuous application of VIP (5 microM) suggesting desensitization of the VIP receptor. It is concluded that VIP produces a depolarization in neurones of bladder parasympathetic ganglia by decreasing a K+ conductance, the pharmacological characteristics of which are unlike previously described K+ conductance mechanisms.

The metabotropic glutamate receptor (mGluR) antagonist, (RS)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM), was tested on intracellularly recorded epileptiform activity induced by 4-aminopyridine (4-AP) in amygdala neurons. Superfusing 4-AP (1 mM) produced interictal spiking followed by ictal bursting. MCPG prevented the progressive transition from interictal spiking to ictal bursting but affected neither induction of interictal spiking nor maintenance of ongoing ictal bursting. These data suggest that mGluRs may be involved in the induction of ictal seizure events.

Glutamatergic excitatory postsynaptic potentials (EPSPs) in the basolateral amygdala (BLA) are reduced in amplitude following agonist activation of presynaptic metabotropic glutamate receptors (mGluR). In this study, the effect of a presumed mGluR antagonist, (RS)-α-methyl-4-carboxyphenylglycine (MCPG), was investigated on the EPSP recorded intracellularly in BLA neurons. Superfusion of MCPG (500 μM) significantly reduced the amplitude of evoked EPSPs. In the presence of MCPG, postsynaptic responses to α-amino-3-hydroxy-5- methyl-4-isoxazoleproprionic acid (AMPA, 1 μM) were unaltered while responses to N-methyl-D-aspartate (NMDA, 3–5 μM) were potentiated. These data suggest that the MCPG-induced reduction of EPSP amplitude is due to a mGluR agonist action at a presynaptic mGluR 'autoreceptor'.

Intracellular current- and voltage-clamp recordings were obtained from basolateral (BLA) and central (ACe) neurones of the rat amygdala in vitro. The effect of superfusion of rat corticotropin releasing factor (rCRF) was examined on the ACe and BLA, nuclei with a high density of CRF-immunoreactive cell bodies and CRF receptors, respectively. rCRF (12.5-250 nM) had no effect on either resting membrane potential or input resistance in BLA neurones. In contrast, at low nanomolar concentrations rCRF (10-50 nM) caused hyperpolarisation and decreased membrane input resistance in the majority of ACe neurones. The equilibrium potential for this effect was -84 mV in the presence of tetrodotoxin (0.5 microM), suggesting that this response may be mediated by a postsynaptic increase in K+ conductance. Furthermore, rCRF consistently reduced the amplitude of, and the current mediating, the slow after hyperpolarisation following evoked action potential firing in both nuclei. This effect was concentration-dependent with an EC50 of 40 nM in BLA neurones and was insensitive to tetrodotoxin and noradrenergic antagonists. In addition, the putative CRF antagonist alpha-helical CRF9-41 (5 microM) blocked the effect of rCRF and showed partial agonist action on slow after hyperpolarisations. In BLA neurones, rCRF (50 nM) prolonged the Ca(2+)-spike evoked in the presence of tetrodotoxin and tetraethylammonium (2 mM), suggesting that the blockade of the slow after hyperpolarisation occurs subsequent to Ca2+ entry. These results suggest that rCRF has a differential effect on the membrane properties of BLA and ACe neurones possibly by activation of a heterogeneous population of CRF receptors.

Intracellular recordings were obtained from the basolateral amygdala in in vitro rat brain slice preparations to examine whether gamma-aminobutyric acid (GABA)B receptors are altered after in vivo kindling-induced epileptogenesis. Stimulating the stria terminalis evoked excitatory (EPSPs) and inhibitory (IPSPs) postsynaptic potentials in control neurons, and epileptiform bursting or enhanced EPSPs, but no IPSPs, in neurons from animals, 4 to 8 weeks after the last kindled seizure. Baclofen (0.1 nM-100 microM) depressed EPSPs in control and kindled basolateral amygdala neurons, but the EC50 appeared to be shifted 100-fold from 5 nM in control to 500 nM in kindled neurons. Further analysis suggested a high-affinity component may be affected in kind led neurons. The absence of IPSPs in kindled neurons could not account for this shift, because effects of baclofen on EPSP amplitude were reduced in kindled animals even when GABAA receptors were blocked with bicuculline methiodide (30 microM) and postsynpatic GABAB receptors with intracellular guanosine 5'-O-3-thiotriphosphate (10 mM); 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (10 microM) was also present to block bicuculline methiodide-induced bursting. Membrane responses to exogenously applied N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid were not affected by baclofen. Baclofen also hyperpolarized basolateral amygdala neurons and reduced membrane input resistance with an EC50 of 1 microM in control and kindled neurons. Post- but not presynaptic effects of baclofen were blocked by 2-hydroxy-saclofen (100 microM) and pertussis toxin pretreatment. In conclusion, kindling-induced epileptogenesis reduces the sensitivity of presynaptic GABAB receptors, an effect which may contribute to the enhancement of excitatory transmission in kindled animals. Furthermore, different pharmacological properties of pre- and postsynaptic receptors in the amygdala suggest two distinct populations of GABAB receptors whose long-lasting responses to kindling-induced seizures are different.

The subsynaptic actions of theophylline were studied by employing intracellular recording techniques in the isolated cat tenuissimus muscle. Theophylline produced a dose-dependent increase in miniature end-plate potential (MEPP) amplitude. The maximal increase (30%) in MEPP amplitude occurred at the 1.8 mM concentration of theophylline with a concomitant depolarization of the resting membrane potential. Depolarizations induced by acetylcholine were potentiated by 1.8 mM theophylline. This effect was less evident with a carbachol-induced depolarization, which indicates a possible anticholinesterase action. Input resistance decreased significantly; this effect correlated with the decline in resting membrane potential produced by theophylline. The equilibrium potential of the EPP was shifted to a more positive value in a reversible manner. In addition, the conductance change due to MEPPs, acetylcholine and carbachol was increased by theophylline treatment. These results suggest that the shift in equilibrium potential of the EPP coupled with a slight anticholinesterase action may account for the increase in MEPP amplitude observed with theophylline treatment.

We investigated the proposed catecholamine receptor in the superior cervical ganglion of the rabbit with the sucrose-gap technique to characterize the receptor pharmacologically. It has been suggested that this receptor is involved in the slow inhibitory postsynaptic potential in sympathetic ganglia. Epinephrine, norepinephrine and dopamine consistently hyperpolarized the ganglion membrane (N = 60). The order of potency was epinephrine greater than or equal to norepinephrine much greater than dopamine. Clonidine (10(-5) M), phenylephrine (10(-4) M) and isoproterenol (10(-3) M) also hyperpolarized the ganglion. However, apomorphine, even at millimolar concentration, did not hyperpolarize the membrane. The alpha antagonists phentolamine (10(-6) M) and yohimbine (10(-6) M) depressed the response to all catecholamines and shifted the catecholamine concentration-response curve to the right; dopamine and beta antagonists and the alpha-1 antagonist prazosin had no effect on the catecholamine hyperpolarizations. In contrast, the nerve-evoked slow inhibitory postsynaptic potential was selectively depressed only by atropine (10(-7) M). In conclusion, we characterized an alpha-2 adrenergic receptor in the rabbit superior cervical ganglion responsible for the catecholamine hyperpolarization and found that the slow inhibitory postsynaptic potential does not appear to be mediated by the same receptor.

Sympathetic reflexes in an in vitro frog spinal cord appeared to be qualitatively similar to those observed in cats in vivo. The present study ws conducted to determine whether or not this similarity extended to catecholamine effects on spinal sympathetic reflexes and to examine the adrenoceptor mechanisms responsible for these effects. Norepinephrine inhibited spinal sympathetic reflexes elicited by supramaximal electrical stimulation of the second spinal nerve in a concentration-dependent manner. This inhibition could be mimicked by other alpha adrenoceptor agonists and was antagonized by phentolamine. No effect of norepinephrine was observed when submaximal stimuli were employed. Epinephrine facilitated reflex activity elicited by both submaximal and supramaximal stimulation; however, in the presence of propranolol, an inhibitory effect of epinephrine was observed. At high concentrations, dopamine inhibited spinal sympathetic reflex activity and this inhibition was antagonized by phentolamine. l-Dopa produced a slow-onset, long-lasting inhibition which could be blocked by pretreatment with a decarboxylase inhibitor. It is concluded that alpha adrenoceptor agonists acting on alpha receptors can inhibit spinal sympathetic reflex activity and that the inhibition is dependent on the intensity of stimulation. Activation of beta adrenoceptors facilitates, whereas activation of dopamine receptors has no effect on, spinal sympathetic reflexes. These alpha and beta adrenoceptor mechanisms may function in the modulation of sympathetic preganglionic activity in the intact animal.

1. Intracellular recording techniques were used to study individual neurons in the cat parasympathetic vesical pelvic ganglion (VPG). 2. Active and passive electrical properties were determined from 140 ganglion cells (35 preparations). 3. Three types of ganglion cells were distinguished. Type I (A and B) cells were nonaccommodating cells in response to depolarizing current pulses. Type II cells showed accommodation to depolarizing current pulses. Other cells, presumably glia, were also impaled. 4. Type IB cells exhibited two kinds of nonsynaptic spontaneous activity, spontaneous action potentials (60-70 mV) and small spontaneous potentials (up to 5 mV). Characteristics of the spontaneous activity were examined. 5. The duration of the spikes' afterhyperpolarization resulting from either orthodromic or antidromic train stimulation was dependent on the frequency of train stimulation. No long-lasting posttrain hyperpolarization was observed. 6. Chlorisondamine (10(-6) M), d-tubocurarine (10(-5) M), and hexamethonium (10(-5) M) reversibly blocked orthodromic responses. 7. The VPG is a useful model to study parasympathetic ganglionic transmission at the cellular level.

Neugebauer, Volker, N. Bradley Keele, and Patricia Shinnick-Gallagher.Loss of long-lasting potentiation mediated by group III mGluRs in amygdala neurons in kindling-induced epileptogenesis. J. Neurophysiol.78: 3475–3478, 1997. Long-lasting modifications of synaptic transmission can be induced in the amygdala by electrical stimulation as done in the long-term potentiation (LTP) model of learning and memory and the kindling model of epilepsy. The present study reports for the first time a long-lasting potentiation (LLP) of synaptic transmission that is induced pharmacologically by the activation of group III metabotropic glutamate receptors (mGluRs) in basolateral amygdala (BLA) neurons. In whole cell voltage-clamp mode, BLA neurons were recorded in brain slices from control rats and rats with amygdala-kindled seizures. The group III mGluR agonist l-2-amino-4-phosphonobutyrate (l-AP4, 10 μM) induced LLP of monosynaptic excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation in the lateral amygdala (maximum 258 ± 50% of predrug control; means ± SE) in control (n= 7) but not in kindled neurons(n= 6). LLP was measured 15 min after the superfusion of l-AP4, lasted for >45 min, and was not accompanied by postsynaptic membrane changes. l-AP4 induced LLP was prevented by the group III mGluR antagonist (S)-2-methyl-2-amino-4-phosphonobutyrate (MAP4; 100 μM, n= 6) but not the group II mGluR antagonist (2S,3S,4S)-2-methyl-2-carboxycyclopropylglycine (MCCG; 100 μM, n= 3). LLP was not observed after superfusion of the group II mGluR agonist (2S,3S,4S)-2-(carboxycyclopropyl)glycine (l-CCG; 1.0 and 10 μM) in either control (n= 13) or kindled (n= 10) neurons. If the underlying mechanisms and the functional significance of pharmacologically induced LLP are similar to those of LTP, the loss of l-AP4 induced LLP in kindled neurons may be a neurobiological correlate of learning and memory deficits in kindled animals and long-term alterations of brain functions in patients with epilepsies.

Yu, Baojian and Patricia Shinnick-Gallagher.Dihydropyridine- and neurotoxin-sensitive and -insensitive calcium currents in acutely dissociated neurons of the rat central amygdala. J. Neurophysiol.77: 690–701, 1997. The central amygdala (CeA) is an area involved in emotional learning and stress, and identification of Ca2+currents is essential to understanding interneuronal communication through this nucleus. The purpose of this study was to separate and characterize dihydropyridine (DHP)- and neurotoxin-sensitive and -resistant components of the whole cell Ca2+current (ICa) in acutely dissociated rat CeA neurons with the use of whole cell patch-clamp recording. Saturating concentrations of nimodipine (NIM, 5 μM), a DHP antagonist, blocked 22% of ICa; this NIM-sensitive (L-type) current was recorded in 68% of CeA neurons. The DHP agonist Bay K 8644 (5 μM) produced a 36% increase in ICain a similar proportion of CeA neurons (70%). ω-Conotoxin GVIA (CgTx GVIA, 1 μM) in saturating concentrations inhibited 30% of ICa, whereas ω-agatoxin IVA (Aga IVA, 100 nM), in concentrations known to block P-type currents, did not affect ICa. Higher concentrations of Aga IVA (1 μM) alone reduced ICaby 34%, but in the presence of NIM (5 μM) and CgTx GVIA (1 μM) blocked only 18% of ICa. ω-Conotoxin MVIIC (CgTx MVIIC, 250 nM) reduced ICaby 13% in the presence of CgTx GVIA (1 μM). Application of NIM (5 mM), CgTx GVIA (1 μM), and Aga IVA (1 μM) blocked ∼67% of ICa. A similar portion (63%) of Ca2+current was blocked with CgTx MVIIC (250 nM) in the presence of NIM (5 μM) and CgTx GVIA (1 μM). The current resistant to NIM and the neurotoxins represented 37% of ICa, whereas in neurons not having L-type currents the resistant current made up ∼53% of ICa(49 ± 2%, mean ± SE). The resistant current activated at around −40 mV and peaked at ∼0 mV with half-activation and -inactivation potentials of −17 and −58 mV and slopes for activation and inactivation of −5 and 13 mV, respectively. The resistant current was sensitive to Cd2+(IC50= 2.5 μM) and Ni2+(IC50= 86 μM), was larger in Ca2+than in Ba2+(ratio = 1.31:1), and showed a moderate rate of decay. In summary, our results show that the high-voltage-activated calcium current in rat CeA neurons is composed of at least four pharmacologically distinct components: L-type current (NIM sensitive, 22%), N-type current (CgTx GVIA sensitive, 30%), Q-type current [Aga IVA (1 μM) and CgTx MVIIC sensitive, ∼13–18%], and a resistant current (Non-L, -N, and -Q current, 33 ∼ 37%), amounting to 37–53% of the total current. The resistant current has some electrophysiological and pharmacological characteristics in common with doe-1, α1E, and R-type calcium currents, but remains unclassified.

Responses to gamma-aminobutyric acid (GABA) and 13 structurally related analogs were recorded under voltage clamp conditions from isolated cat dorsal root ganglia (DRG). All of the analogs were applied by superfusion at a concentration of 1 mM. Of the 13 structurally related compounds, only muscimol was more effective than GABA in its ability to produce an inward current at the DRG membrane. Complete (1 microM-10 mM) concentration-response relationships were obtained for muscimol, GABA and 3-aminopropane sulfonic acid. Muscimol was most potent at all concentrations, however, the relative order of potency for GABA and 3-aminopropane sulfonic acid was concentration-dependent. In addition to producing differences in their peak currents, the various agonists demonstrated different time courses in their respective rates of decay. When the glial uptake system for GABA in cat DRG was inhibited by substituting lithium for sodium or by the addition of 1 mM nipecotic acid to the normal Krebs' solution, the membrane responses to GABA, SL-75102 and isoguvacine were facilitated, whereas the responses induced by the other analogs remained unchanged. Although the process of uptake altered significantly the decay phase of GABA responses when GABA was applied by superfusion or by iontophoresis, nipecotic acid inhibition of uptake did not appear to affect GABA-induced desensitization that was apparent when GABA was applied either by superfusion or with long iontophoretic pulses. GABA and all of the analogs in this study demonstrated cross-desensitization, each being able to depress the action of the other. Our results suggest that all of the active GABA analogs appear to be acting at a similar GABA receptor on cat DRG and this GABA receptor is coupled to a chloride channel.