Glutamic acid decarboxylase 65: a link between GABAergic synaptic plasticity in the lateral amygdala and conditioned fear generalization.

An imbalance of the gamma-aminobutyric acid (GABA) system is considered a major neurobiological pathomechanism of anxiety, and the amygdala is a key brain region involved. Reduced GABA levels have been found in anxiety patients, and genetic variations of glutamic acid decarboxylase (GAD), the rate-limiting enzyme of GABA synthesis, have been associated with anxiety phenotypes in both humans and mice. These findings prompted us to hypothesize that a deficiency of GAD65, the GAD isoform controlling the availability of GABA as a transmitter, affects synaptic transmission and plasticity in the lateral amygdala (LA), and thereby interferes with fear responsiveness. Results indicate that genetically determined GAD65 deficiency in mice is associated with (1) increased synaptic length and release at GABAergic connections, (2) impaired efficacy of GABAergic synaptic transmission and plasticity, and (3) reduced spillover of GABA to presynaptic GABAB receptors, resulting in a loss of the associative nature of long-term synaptic plasticity at cortical inputs to LA principal neurons. (4) In addition, training with high shock intensities in wild-type mice mimicked the phenotype of GAD65 deficiency at both the behavioral and synaptic level, indicated by generalization of conditioned fear and a loss of the associative nature of synaptic plasticity in the LA. In conclusion, GAD65 is required for efficient GABAergic synaptic transmission and plasticity, and for maintaining extracellular GABA at a level needed for associative plasticity at cortical inputs in the LA, which, if disturbed, results in an impairment of the cue specificity of conditioned fear responses typifying anxiety disorders.

Differential regulation of glutamic acid decarboxylase gene expression after extinction of a recent memory vs. intermediate memory.

Extinction reduces fear to stimuli that were once associated with an aversive event by no longer coupling the stimulus with the aversive event. Extinction learning is supported by a network comprising the amygdala, hippocampus, and prefrontal cortex. Previous studies implicate a critical role of GABA in extinction learning, specifically the GAD65 isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD). However, a detailed analysis of changes in gene expression of GAD in the subregions comprising the extinction network has not been undertaken. Here, we report changes in gene expression of the GAD65 and GAD67 isoforms of GAD, as measured by relative quantitative real-time RT-PCR, in subregions of the amygdala, hippocampus, and prefrontal cortex 24-26 h after extinction of a recent (1-d) or intermediate (14-d) fear memory. Our results show that extinction of a recent memory induces a down-regulation of Gad65 gene expression in the hippocampus (CA1, dentate gyrus) and an up-regulation of Gad67 gene expression in the infralimbic cortex. Extinguishing an intermediate memory increased Gad65 gene expression in the central amygdala. These results indicate a differential regulation of Gad gene expression after extinction of a recent memory vs. intermediate memory.

GABAergic interneurons in the mouse lateral amygdala: a classification study.

Whole cell patch-clamp recordings were performed in GABAergic interneurons labeled by green fluorescent protein (GFP) in the lateral amygdala (LA) in vitro from glutamic acid decarboxylase 67 (GAD67)-GFP mice. Neurons were characterized by electrotonic and electrogenic parameters. Cytoplasm was collected from individual neurons, and single-cell RT-PCR was used for detection of molecular markers typifying LA interneurons. Hierarchical cluster and multiple discriminant analysis demonstrated the existence of five types of GABAergic interneurons, which can be reliably identified through electrophysiological criteria. Action potentials were of a short duration followed by pronounced fast afterhyperpolarization (AHP) in interneurons of all types, except for type V, which generated broad action potentials and displayed typical spike bursts at the beginning of depolarizing stimuli and prominent anomalous inward rectification. Interneurons of type I and II generated series of action potentials with frequency adaptation on maintained depolarizing current stimulation with overall frequencies at high levels and presented delayed firing, stuttering or fast-spiking behavior. Further distinguishing features of type II interneurons were a medium AHP following spike trains and pronounced anomalous inward rectification. Types III and IV of neurons fired regularly, whereas type IV displayed no prominent spike frequency adaptation. Additionally, interneurons of all five types contained mRNA of glutamic acid decarboxylase 65 and cholecystokinin, whereas only type I interneurons were somatostatin-positive. Overall, these data represent a detailed and reliable classification scheme of LA GABAergic interneurons and will provide a feasible basis for subsequent functional studies.

Alteration of NMDA receptor-mediated synaptic interactions in the lateral amygdala associated with seizure activity in a mouse model of chronic temporal lobe epilepsy.

PURPOSE: Because results from both animal models and human temporal lobe epilepsy (TLE) have pointed to synaptic network alterations in the amygdala, we have tested the hypothesis that glutamatergic transmission in the lateral amygdala (LA) is critically involved. METHODS: Using the pilocarpine mouse model, LA slices were prepared ex vivo in the recurrent phase of TLE (Pilo group), and LA projection neurons (PNs) were recorded using patch-clamp techniques. Intrinsic and synaptic properties of LA PNs were analyzed and compared with those in age-matched saline-injected controls. RESULTS: Only mild changes were observed in intrinsic properties of LA PNs, whereas both spontaneous excitatory postsynaptic currents (sEPSCs) and miniature EPSCs (mEPSCs) were significantly increased in Pilo as compared to saline controls. This difference was sensitive to AP5, but persisted during action of NBQX, indicating mediation by N-methyl-d-aspartate (NMDA) receptors. Moreover, these changes were associated with an increase in frequency but not amplitude of mEPSCs, indicative of a contribution of presynaptic mechanisms. DISCUSSION: In conclusion, dynamic changes seem to occur in glutamatergic transmission within the amygdala during TLE, to which a functional upregulation of presynaptic NMDA receptors in LA PNs makes a significant contribution.

Neuropeptide Y activates a G-protein-coupled inwardly rectifying potassium current and dampens excitability in the lateral amygdala.

Neuropeptide Y (NPY) reduces anxiety-related behavior in various animal models. Since activity in the lateral amygdala (LA) seems crucial for fear expression of behavior, we studied the mechanisms of NPY in LA projection neurons using whole-cell patch-clamp recordings in slices of the rat amygdala in vitro. Application of NPY activated a membrane K(+) current with inwardly rectifying properties in 92% of tested neurons. Pharmacological properties were indicative of mediation via Y1 receptors. Nonhydrolyzable analogues of guanine nucleotides and SCH23390 blocked the NPY-activated current. Single-cell RT-PCR demonstrated expression of G-protein-coupled inwardly rectifying K(+) channel (GIRK) subunits GIRK1, GIRK2 and GIRK3, suggesting mediation of the NPY response through GIRK type channels. The NPY-activated current depressed action potential firing in LA projection neurons, through membrane hyperpolarization and decreased input resistance. Functionally, the dampening of excitability in projection neurons of the amygdala may contribute to the decrease in anxiogenic behavior during action of NPY.

Neuropeptide S-mediated control of fear expression and extinction: role of intercalated GABAergic neurons in the amygdala.

A deficient extinction of memory is particularly important in the regime of fear, where it limits the beneficial outcomes of treatments of anxiety disorders. Fear extinction is thought to involve inhibitory influences of the prefrontal cortex on the amygdala, although the detailed synaptic mechanisms remain unknown. Here, we report that neuropeptide S (NPS), a recently discovered transmitter of ascending brainstem neurons, evokes anxiolytic effects and facilitates extinction of conditioned fear responses when administered into the amygdala in mice. An NPS receptor antagonist exerts functionally opposing responses, indicating that endogenous NPS is involved in anxiety behavior and extinction. Cellularly, NPS increases glutamatergic transmission to intercalated GABAergic neurons in the amygdala via presynaptic NPS receptors on connected principal neurons. These results identify mechanisms of NPS in the brain, a key role of intercalated neurons in the amygdala for fear extinction, and a potential pharmacological avenue for treating anxiety disorders.

Postsynaptic mechanisms underlying responsiveness of amygdaloid neurons to cholecystokinin are mediated by a transient receptor potential-like current.

Projection neurons of mouse basolateral amygdala responded to CCK with an inward current at a holding potential of -70 mV. This response was mediated by CCK2 receptors as indicated by agonist and antagonist effectiveness, and conveyed via G-proteins of the G(q/11) family as it was abolished in gene knockout mice. Maximal current amplitude was insensitive to extracellular potassium, cesium, and calcium ions, respectively, whereas amplitude and reversal potential critically depended upon extracellular sodium concentration. The current reversed near -20 mV consistent with activation of a mixed cationic channel reminiscent of transient receptor potential (TRP) channels. Extracellular application of the non-selective TRP channel blockers 2-APB, flufenamic acid, Gd3+, and ruthenium red, respectively, inhibited CCK induced inward currents. Single cell PCR confirmed the expression of TRPC1,4,5 and coexpression of TRPC1 with TRPC4 or TRPC5 in some cells. CCK responses were associated with depolarization leading to an increase in cell excitability.

Classification of projection neurons and interneurons in the rat lateral amygdala based upon cluster analysis.

Neurons in the rat lateral amygdala in situ were classified based upon electrophysiological and molecular parameters, as studied by patch-clamp, single-cell RT-PCR and unsupervised cluster analyses. Projection neurons (class I) were characterized by low firing rates, frequency adaptation and expression of the vesicular glutamate transporter (VGLUT1). Two classes were distinguished based upon electrotonic properties and the presence (IB) or absence (IA) of vasointestinal peptide (VIP). Four classes of glutamate decarboxylase (GAD67) containing interneurons were encountered. Class III reflected "classical" interneurons, generating fast spikes with no frequency adaptation. Class II neurons generated fast spikes with early frequency adaptation and differed from class III by the presence of VIP and the relatively rare presence of neuropeptide Y (NPY) and somatostatin (SOM). Class IV and V were not clearly separated by molecular markers, but by membrane potential values and spike patterns. Morphologically, projection neurons were large, spiny cells, whereas the other neuronal classes displayed smaller somata and spine-sparse dendrites.

Mechanisms of somatostatin-evoked responses in neurons of the rat lateral amygdala.

The effects of somatostatin in the rat lateral amygdala (LA) in vitro were investigated through whole cell recording techniques. Somatostatin induced an inwardly rectifying K+ current in approximately 98% of LA projection neurons. Half-maximal effects were obtained by 189 nM somatostatin. The effects of somatostatin were insensitive to tetrodotoxin, reduced by Ba2+, occluded or abolished by the presence of nonhydrolysable GTP or GDP analogues, respectively, and blocked or mimicked by a somatostatin receptor type 2 antagonist (BIM-23627) or somatostatin receptor type 2 agonist (L-779,976), respectively, while somatostatin receptor type 1, 3 and 4 agonists were ineffective (L-797,591, L-796,778, L-803,087). Responses to somatostatin were associated with membrane hyperpolarization and decrease in input resistance, resulting in a dampening of cell excitability. It is suggested that these cellular mechanisms contribute to the role of somatostatin in decreasing anxiety behaviour as well as to anticonvulsant and antiepileptogenic actions of somatostatin or somatostatin agonists in the amygdala.