Sex Differences in the Physiological Response to Ethanol of Rat Basolateral Amygdala Neurons Following Single-Prolonged Stress.

Females are more likely to develop post-traumatic stress disorder (PTSD) than males. Also, symptoms of PTSD frequently precede alcohol abuse in females. Stressful, threat-related stimuli are evaluated by the amygdala, which is critical for establishing the emotional salience of environmental stimuli. Ethanol and stress have been shown to modify amygdala excitability, but effects of acute ethanol on neurons of the basolateral amygdala (BLA) in both males and females exposed to stress is unknown. The purpose of this study is to determine stress-induced changes in membrane properties of BLA neurons and to determine how ethanol modulates these changes in male and female rats. Whole-cell recordings were obtained from BLA neurons of both male and female rats exposed to single-prolonged stress (SPS). Neuronal excitability, quantified as the number of action potentials, was determined in current clamp mode by applying a series of depolarizing current steps. Hyperpolarization-activated current (Ih) was elicited in voltage clamp. Excitability and Ih amplitude were determined before and during the superfusion of ethanol (EtOH; 30 mM) in BLA neurons from SPS-treated male and female rats. SPS alone did not alter the firing properties of BLA neurons from either males or females. However, following SPS, BLA neurons from males and females respond differently to ethanol. Superfusion of EtOH (30 mM) inhibited spike firing in BLA neurons from rats exposed to SPS, and EtOH-induced inhibition was greater in females than in males exposed to stress. Also, EtOH (30 mM) selectively decreased Ih amplitude in BLA neurons from SPS-treated male rats from 171 ± 46 pA in (pre-EtOH) control to 53 ± 51 pA in the presence of EtOH (30 mM). EtOH did not reduce Ih in BLA neurons from SPS-treated females. Together, these suggest important sex differences in the physiological responses to EtOH in stress disorders such as PTSD, that have high comorbidity with alcohol use disorders.

Depletion of serotonin in the basolateral amygdala elevates glutamate receptors and facilitates fear-potentiated startle.

Our previous experiments demonstrated that systemic depletion of serotonin (5-hydroxytryptamine, 5-HT), similar to levels reported in patients with emotional disorders, enhanced glutamateric activity in the lateral nucleus of the amygdala (LA) and potentiated fear behaviors. However, the effects of isolated depletion of 5-HT in the LA, and the molecular mechanisms underlying enhanced glutamatergic activity are unknown. In the present study, we tested the hypothesis that depletion of 5-HT in the LA induces increased fear behavior, and concomitantly enhances glutamate receptor (GluR) expression. Bilateral infusions of 5,7-dihydroxytryptamine (4 µg per side) into the LA produced a regional reduction of serotonergic fibers, resulting in decreased 5-HT concentrations. The induction of low 5-HT in the LA elevated fear-potentiated startle, with a parallel increase in GluR1 mRNA and GluR1 protein expression. These findings suggest that low 5-HT concentrations in the LA may facilitate fear behavior through enhanced GluR-mediated mechanisms. Moreover, our data support a relationship between 5-HT and glutamate in psychopathologies.

Phenytoin inhibits isolation-induced aggression specifically in rats with low serotonin.

Phenytoin is a widely used anticonvulsant drug that also reduces aggressive behavior. Aggression in humans and animals is often associated with low serotonin levels. This study examined the anti-aggressive properties of phenytoin in rodent isolation-induced aggression using a resident-intruder test to quantify aggression. Chronic treatment with p-chlorophenyl-alanine (PCPA), a competitive inhibitor of serotonin synthesis, significantly enhanced resident attack behavior compared to saline-treated control rats. Phenytoin dose-dependently reduced aggressive behavior specifically in PCPA-treated rats, but had no anti-aggressive properties in saline-treated rats. These data suggest that aggressive behavior in this model may be related to neuronal hyperexcitability that is sensitive to the anticonvulsant effects of phenytoin. Further, these data suggest isolation-induced aggression in PCPA-treated rats may be a useful model to investigate aggression associated with low serotonin in the brain.

LP-BM5 virus-infected mice produce activating autoantibodies to the AMPA receptor.

Autoantibodies to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors may contribute to chronic hyperexcitability syndromes and neurodegeneration, but their origin is unclear. We examined LP-BM5 murine leukemia virus-infected mice, which manifest excitotoxic brain lesions and hypergammaglobulinemia, for the presence of AMPA-receptor Ab's. Endogenous IgG accumulated upon neurons in the neocortex and caudate/putamen of infected mice and interacted with native and recombinant AMPA-receptor subunits with the following relative abundance: GluR3 > or = GluR1 > GluR2 = GluR4, as determined by immunoprecipitation. In a radioligand assay, IgG preparations from infected mice specifically inhibited [(3)H]AMPA binding to receptors in brain homogenates, an activity that was lost after preadsorbing the IgG preparation to immobilized LP-BM5 virus. These IgGs also evoked currents when applied to hippocampal pyramidal neurons or to damaged cerebellar granule neurons. These currents could be blocked using any of several AMPA receptor antagonists. Thus, anti-AMPA-receptor Ab's can be produced as the result of a virus infection, in part through molecular mimicry. These Ab's may alter neuronal signaling and contribute to the neurodegeneration observed in these mice, actions that may be curtailed by the use of AMPA-receptor antagonists.

Epileptogenesis up-regulates metabotropic glutamate receptor activation of sodium-calcium exchange current in the amygdala.

Postsynaptic metabotropic glutamate (mGlu) receptor-activated inward current mediated by Na(+)-Ca(2+) exchange was compared in basolateral amygdala (BLA) neurons from brain slices of control (naïve and sham-operated) and amygdala-kindled rats. In control neurons, the mGlu agonist, quisqualate (QUIS; 1-100 microM), evoked an inward current not associated with a significant change in membrane slope conductance, measured from current-voltage relationships between -110 and -60 mV, consistent with activation of the Na(+)-Ca(2+) exchanger. Application of the group I selective mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine [(S)-DHPG; 10-1000 microM] or the endogenous agonist, glutamate (10-1000 microM), elicited the exchange current. QUIS was more potent than either (S)-DHPG or glutamate (apparent EC(50) = 19 microM, 57 microM, and 0.6 mM, respectively) in activating the Na(+)-Ca(2+) exchange current. The selective mGlu5 agonist, (R, S)-2-chloro-5-hydroxyphenylglycine [(R,S)-CHPG; apparent EC(50) = 2. 6 mM] also induced the exchange current. The maximum response to (R, S)-DHPG was about half of that of the other agonists suggesting partial agonist action. Concentration-response relationships of agonist-evoked inward currents were compared in control neurons and in neurons from kindled animals. The maximum value for the concentration-response relationship of the partial agonist (S)-DHPG- (but not the full agonist- [QUIS or (R,S)-CHPG]) induced inward current was shifted upward suggesting enhanced efficacy of this agonist in kindled neurons. Altogether, these data are consistent with a kindling-induced up-regulation of a group I mGlu-, possibly mGlu5-, mediated responses coupled to Na(+)-Ca(2+) exchange in BLA neurons.

Differential effects of metabotropic glutamate receptor antagonists on bursting activity in the amygdala.

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 microM) and the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 20 microM) evoked bursting in BLA neurons from amygdala-kindled rats but not in control neurons. Neither the group II agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I, 10 microM) nor the group III agonist L-2-amino-4-phosphonobutyrate (L-AP4, 100 microM) evoked bursting. The agonist-induced bursting was inhibited by the mGluR1 antagonists (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG, 500 microM] and (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG, 300 microM]. 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 microM) 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.

Quisqualate-preferring metabotropic glutamate receptor activates Na(+)-Ca2+ exchange in rat basolateral amygdala neurones.

1. Inward currents evoked by metabotropic glutamate receptor (mGlu) agonists quisqualate and 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) were characterized in the basolateral nucleus of the amygdala. Currents were recorded with whole-cell patch electrodes in the presence of D-2-amino-5-phosphonovaleric acid (D-APV, 50 microM), 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 30 microM) and tetrodotoxin (TTX, 1 microM). 2. When recording with K+ electrodes, quisqualate (10-50 microM) produced an inward current which was not associated with a significant change in membrane slope conductance (Gm) and was insensitive to Ba2+ (0.2 mM) and Cs+ (2 mM). The 1S,3R-ACPD (50-200 microM)-induced inward current was associated with a decreased Gm and reversed polarity around -95 mV. However, in Ba2+ and Cs+, the 1S,3R-ACPD inward current amplitude was enhanced and was not accompanied by a change in Gm, a response similar to that evoked by quisqualate. 3. Glutamate (1 mM) and the group I mGlu specific agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 100 microM) also evoked currents not associated with a change in Gm. 4. When recorded with Cs+ electrodes in external Ba2+ and Cs+ solution, quisqualate activated an inward current more potently than 1S,3R-ACPD, suggesting that this current is preferentially activated by quisqualate. The mGlu agonist-induced inward current was not accompanied by a Gm change under these conditions. 5. Substitution of extracellular Na+ with Li+ (117 or 50 mM) or with 100 mM choline reduced the quisqualate- and 1S,3R-ACPD-induced inward currents, results consistent with mediation by Na(+)-Ca2+ exchange. 6. The quisqualate- and 1S,3R-ACPD-induced inward currents were reduced in Ca(2+)-free EGTA (1 mM) solution and prevented by including the Ca2+ chelating agent BAPTA (10 mM) in the recording electrode. In low-Ca2+ (100 microM)- and Cd2+ (200 microM)-containing solution to block voltage-gated Ca2+ currents, the quisqualate-induced current was not altered, but the 1S,3R-ACPD inward current was blocked. These data suggest that the quisqualate- and 1S,3R-ACPD-induced currents are mediated through a rise in intracellular Ca2+ and require extracellular Ca2+, but that the 1S,3R-ACPD current may depend on Ca2+ influx via voltage-gated Ca2+ channels. 7. The quisqualate current with no Gm change was inhibited by including the Na(+)-Ca2+ exchange inhibitory peptide (XIP; 10 microM) in the K+ recording electrode. XIP did not prevent the outward current evoked by baclofen (10 microM) or the 1S,3R-ACPD-induced inward current associated with decreased conductance. 8. These data are consistent with the hypothesis that quisqualate and 1S,3R-ACPD in Ba2+ and Cs+ solution activate a Na(+)-Ca2+ exchange current not associated with a conductance change. The quisqualate exchange current mediated through a group I mGlu may result from mobilization of Ca2+ from intracellular stores. The 1S,3R-ACPD exchange current requires extracellular Ca2+ passing through voltage-gated Ca2+ channels and may be mediated through a different receptor.

Epileptogenesis in vivo enhances the sensitivity of inhibitory presynaptic metabotropic glutamate receptors in basolateral amygdala neurons in vitro.

Modulation of excitatory synaptic transmission by presynaptic metabotropic glutamate receptors (mGluRs) was examined in brain slices from control rats and rats with amygdala-kindled seizures. Using whole-cell voltage-clamp and current-clamp recordings, this study shows for the first time that in control and kindled basolateral amygdala neurons, two pharmacologically distinct presynaptic mGluRs mediate depression of synaptic transmission. Moreover, in kindled neurons, agonists at either group II- or group III-like mGluRs exhibit a 28- to 30-fold increase in potency and suppress synaptically evoked bursting. The group II mGluR agonist (2S,3S,4S)-2-(carboxycyclopropyl)glycine (L-CCG) dose-dependently depressed monosynaptic EPSCs evoked by stimulation in the lateral amygdala with EC50 values of 36 nM (control) and 1.2 nM (kindled neurons). The group III mGluR agonist L-2-amino-4-phosphonobutyrate (L-AP4) was less potent, with EC50 values of 297 nM (control) and 10.8 nM (kindled neurons). The effects of L-CCG and L-AP4 were fully reversible. Neither L-CCG (0.0001-10 microM) nor L-AP4 (0.001-50 microM) caused membrane currents or changes in the current-voltage relationship. The novel mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)-glycine (MCCG; 100 microM) and (S)-2-methyl-2-amino-4-phosphonobutyrate (MAP4; 100 microM) selectively reversed the inhibition by L-CCG and L-AP4 to 81.3 +/- 12% and 65.3 +/- 6.6% of predrug, respectively. MCCG and MAP4 (100-300 microM) themselves did not significantly affect synaptic transmission. The exquisite sensitivity of agonists in the kindling model of epilepsy and the lack of evidence for endogenous receptor activation suggest that presynaptic group II- and group III-like mGluRs might be useful targets for suppression of excessive synaptic activation in neurological disorders such as epilepsy.

Loss of long-lasting potentiation mediated by group III mGluRs in amygdala neurons in kindling-induced epileptogenesis.

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 -2-amino-4-phosphonobutyrate (-AP4, 10 microM) 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 -AP4, lasted for >45 min, and was not accompanied by postsynaptic membrane changes. -AP4 induced LLP was prevented by the group III mGluR antagonist (S)-2-methyl-2-amino-4-phosphonobutyrate (MAP4; 100 microM, n = 6) but not the group II mGluR antagonist (2S, 3S,4S)-2-methyl-2-carboxycyclopropylglycine (MCCG; 100 microM, n = 3). LLP was not observed after superfusion of the group II mGluR agonist (2S,3S,4S)-2-(carboxycyclopropyl)glycine (-CCG; 1.0 and 10 microM) 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 -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.

Metabotropic glutamate receptor agonist-induced hyperpolarizations in rat basolateral amygdala neurons: receptor characterization and ion channels.

1. Metabotropic glutamate receptor (mGluR)-agonist-induced hyperpolarizations and corresponding outward currents were analyzed in basolateral amygdala (BLA) neurons in rat brain slice preparations with current-clamp and single-electrode voltage-clamp recording to characterize the mGluR subtype(s) and the ion channel(s) mediating this response. 2. The mGluR agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) induced a membrane hyperpolarization or outward current in BLA neurons in a concentration-dependent manner (median effective concentration = 34 microM; range = 10-200 microM); the 1S,3R-ACPD hyperpolarizations are recorded in 89% of neurons that accommodate or cease firing in response to a 400-ms depolarizing current injection (0.5 nA). 3. mGluR agonists elicited hyperpolarizations or outward currents in a concentration-dependent manner in the following rank order of potency: (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I) > 1S,3R-ACPD > (s)-4-carboxyphenylglycine = (RS)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) > L-aminophosphonobutyric acid > (1S,3S)-1-amino-cyclopentane-1,3-dicarboxylic acid. In contrast, the mGluR agonists quisqualate and ibotenate induced only depolarizations in the presence of D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione in BLA neurons. 4. The 1S,3R-ACPD-induced outward current is mediated through a large-conductance calcium-dependent potassium (BK) conductance. The BK channel blockers iberiotoxin and charybdotoxin blocked the response, as did the potassium channel blockers tetraethylammonium and 4-aminopyridine; the small-conductance calcium-activated potassium channel blocker apamin did not affect the response. 5. The mGluR-agonist-induced hyperpolarization is blocked in amygdala slices from animals pretreated with pertussis toxin (PTX). 1S,3R-ACPD hyperpolarizations were recorded in neurons contralateral but not ipsilateral to the site of PTX injection. 6. The antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM) reduced significantly the 1S,3R-ACPD-induced hyperpolarization. 7. In conclusion, the relative potency of L-CCG-I and 4C3HPG in evoking only hyperpolarizations (outward currents) in accommodating neurons, and the observation that MCPG (500 microM) reduces the hyperpolarization, suggest that a group-II-like mGluR underlies the hyperpolarizing response. The mGluR-induced response is sensitive to iberiotoxin and to pretreatment with PTX, suggesting activation of BK channels through a group II mGluR linked to a PTX-sensitive G protein in BLA neurons.

Loss of mGluR-mediated hyperpolarizations and increase in mGluR depolarizations in basolateral amygdala neurons in kindling-induced epilepsy.

1. Intracellular recordings were made from neurons of the basolateral amygdala (BLA) in in vitro slice preparations to determine long-term differences in metabotropic glutamate receptor (mGluR) agonist-induced membrane responses in control and amygdala-kindled rats. 2. (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine-1 (L-CCG-I; 100 microM) typically evoked a hyperpolarization/outward current in control BLA neurons; the hyperpolarization is mediated through a group-II-like mGluR subtype of receptor and is recorded in accommodating neurons that cease firing in the presence of a long (400 ms) depolarizing current injection (0.5 nA). In amygdala-kindled slices, L-CCG-I (100 microM) hyperpolarized only 1 of 13 BLA neurons. 3. 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (100 microM) elicited a hyperpolarization/depolarization (outward/inward current) in control neurons and evoked only a membrane depolarization (inward current) in kindled BLA neurons; this depolarization is similar to that mediated by group I mGluR activation in other neurons. 4. In control nonaccommodating neurons the concentration-response relationship for the 1S,3R-ACPD-induced inward current had a median effective concentration (EC50) of 49 microM and a maximum amplitude of 182 +/- 30 (mean +/- SE) pA. In kindled nonaccommodating neurons the EC50 of the concentration-response relationship for 1S,3R-ACPD was shifted to 29 microM and the maximum value increased to 265 +/- 15 pA, reflecting an increase in efficacy. 5. These data suggest that amygdala kindling causes lasting changes in mGluR responses in the BLA reflecting a downregulation of a group-II-like mGluR subtype mediating the hyperpolarizing response and an upregulation of a group I mGluR1 or 5 subtype. The hyperpolarizing response reduced by kindling and the increase in the group I mGluR response may reflect an alteration in the balance between inhibition and excitation and may contribute to the transition to epileptiform bursting in kindled neurons.

Agonist action of (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) in the amygdala.

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)-alpha-methyl-4-carboxyphenylglycine (MCPG), was investigated on the EPSP recorded intracellularly in BLA neurons. Superfusion of MCPG (500 microM) significantly reduced the amplitude of evoked EPSPs. In the presence of MCPG, postsynaptic responses to alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA, 1 microM) were unaltered while responses to N-methyl-D-aspartate (NMDA, 3-5 microM) 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'.

The functional role of metabotropic glutamate receptors in epileptiform activity induced by 4-aminopyridine in the rat amygdala slice.

The metabotropic glutamate receptor (mGluR) antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM), 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.