Maria F.M. Braga

Stress impairs α1A adrenoceptor-mediated noradrenergic facilitation of GABAergic transmission in the basolateral amygdala

Intense or chronic stress can produce pathophysiological alterations in the systems involved in the stress response. The amygdala is a key component of the brains neuronal network that processes and assigns emotional value to lifes experiences, consolidates the memory of emotionally significant events, and organizes the behavioral response to these events. Clinical evidence indicates that certain stress-related affective disorders are associated with changes in the amygdalas excitability, implicating a possible dysfunction of the GABAergic system. An important modulator of the GABAergic synaptic transmission, and one that is also central to the stress response is norepinephrine (NE). In the present study, we examined the hypothesis that stress impairs the noradrenergic modulation of GABAergic transmission in the basolateral amygdala (BLA). In control rats, NE (10 μM) facilitated spontaneous, evoked, and miniature IPSCs in the presence of β and α2 adrenoceptor antagonists. The effects of NE were not blocked by α1D and α1B adrenoceptor antagonists, and were mimicked by the α1A agonist, A61603 (1 μM). In restrain/tail-shock stressed rats, NE or A61603 had no significant effects on GABAergic transmission. Thus, in the BLA, NE acting via presynaptic α1A adrenoceptors facilitates GABAergic inhibition, and this effect is severely impaired by stress. This is the first direct evidence of stress-induced impairment in the modulation of GABAergic synaptic transmission. The present findings provide an insight into possible mechanisms underlying the antiepileptogenic effects of NE in temporal lobe epilepsy, the hyperexcitability and hyper-responsiveness of the amygdala in certain stress-related affective disorders, and the stress-induced exacerbation of seizure activity in epileptic patients.

Pathology and pathophysiology of the amygdala in epileptogenesis and epilepsy

Acute brain insults, such as traumatic brain injury, status epilepticus, or stroke are common etiologies for the development of epilepsy, including temporal lobe epilepsy (TLE), which is often refractory to drug therapy. The mechanisms by which a brain injury can lead to epilepsy are poorly understood. It is well recognized that excessive glutamatergic activity plays a major role in the initial pathological and pathophysiological damage. This initial damage is followed by a latent period, during which there is no seizure activity, yet a number of pathophysiological and structural alterations are taking place in key brain regions, that culminate in the expression of epilepsy. The process by which affected/injured neurons that have survived the acute insult, along with well-preserved neurons are progressively forming hyperexcitable, epileptic neuronal networks has been termed epileptogenesis. Understanding the mechanisms of epileptogenesis is crucial for the development of therapeutic interventions that will prevent the manifestation of epilepsy after a brain injury, or reduce its severity. The amygdala, a temporal lobe structure that is most well known for its central role in emotional behavior, also plays a key role in epileptogenesis and epilepsy. In this article, we review the current knowledge on the pathology of the amygdala associated with epileptogenesis and/or epilepsy in TLE patients, and in animal models of TLE. In addition, because a derangement in the balance between glutamatergic and GABAergic synaptic transmission is a salient feature of hyperexcitable, epileptic neuronal circuits, we also review the information available on the role of the glutamatergic and GABAergic systems in epileptogenesis and epilepsy in the amygdala.

Mechanisms regulating GABAergic inhibitory transmission in the basolateral amygdala: implications for epilepsy and anxiety disorders

Summary.The amygdala, a temporal lobe structure that is part of the limbic system, has long been recognized for its central role in emotions and emotional behavior. Pathophysiological alterations in neuronal excitability in the amygdala are characteristic features of certain psychiatric illnesses, such as anxiety disorders and depressive disorders. Furthermore, neuronal excitability in the amygdala, and, in particular, excitability of the basolateral nucleus of the amygdala (BLA) plays a pivotal role in the pathogenesis and symptomatology of temporal lobe epilepsy. Here, we describe two recently discovered mechanisms regulating neuronal excitability in the BLA, by modulating GABAergic inhibitory transmission. One of these mechanisms involves the regulation of GABA release via kainate receptors containing the GluR5 subunit (GluR5KRs). In the rat BLA, GluR5KRs are present on both somatodendritic regions and presynaptic terminals of GABAergic interneurons, and regulate GABA release in an agonist concentration-dependent, bidirectional manner. The relevance of the GluR5KR function to epilepsy is suggested by the findings that GluR5KR agonists can induce epileptic activity, whereas GluR5KR antagonists can prevent it. Further support for an important role of GluR5KRs in epilepsy comes from the findings that antagonism of GluR5KRs is a primary mechanism underlying the antiepileptic properties of the anticonvulsant topiramate. Another mechanism regulating neuronal excitability in the BLA by modulating GABAergic synaptic transmission is the facilitation of GABA release via presynaptic α1A adrenergic receptors. This mechanism may significantly underlie the antiepileptic properties of norepinephrine. Notably, the α1A adrenoceptor-mediated facilitation of GABA release is severely impaired by stress. This stress-induced impairment in the noradrenergic facilitation of GABA release in the BLA may underlie the hyperexcitability of the amygdala in certain stress-related affective disorders, and may explain the stress-induced exacerbation of seizure activity in epileptic patients.

Lamotrigine reduces spontaneous and evoked GABAA receptor-mediated synaptic transmission in the basolateral amygdala: implications for its effects in seizure and affective disorders.

Lamotrigine (LTG) is an antiepileptic drug that is also effective in the treatment of certain psychiatric disorders. Its anticonvulsant action has been attributed to its ability to block voltage-gated Na(+) channels and reduce glutamate release. LTG also affects GABA-mediated synaptic transmission, but there are conflicting reports as to whether inhibitory transmission is enhanced or suppressed by LTG. We examined the effects of LTG on GABA(A) receptor-mediated synaptic transmission in slices from rat amygdala, a brain area that is particularly important in epileptogenesis and affective disorders. In intracellular recordings, LTG (100 microM) reduced GABA(A) receptor-mediated IPSPs evoked by electrical stimulation in neurons of the basolateral nucleus. In whole-cell recordings, LTG (10, 50 and 100 microM) decreased the frequency and amplitude of spontaneous IPSCs, as well as the amplitude of evoked IPSCs, but had no effect on the kinetics of these currents. LTG also had no effects on the frequency, amplitude or kinetics of miniature IPSCs recorded in the presence of TTX. These results suggest that in the basolateral amygdala, LTG suppresses GABA(A) receptor-mediated synaptic transmission by a direct and/or indirect effect on presynaptic Ca(++) influx. The modulation of inhibitory synaptic transmission may be an important mechanism underlying the psychotropic effects of LTG.

Expression of miRNAs and Their Cooperative Regulation of the Pathophysiology in Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of injury-related death and disability worldwide. Effective treatment for TBI is limited and many TBI patients suffer from neuropsychiatric sequelae. The molecular and cellular mechanisms underlying the neuronal damage and impairment of mental abilities following TBI are largely unknown. Here we used the next generation sequencing platform to delineate miRNA transcriptome changes in the hippocampus at 24 hours and 7 days following TBI in the rat controlled cortical impact injury (CCI) model, and developed a bioinformatic analysis to identify cellular activities that are regulated by miRNAs differentially expressed in the CCI brains. The results of our study indicate that distinct sets of miRNAs are regulated at different post-traumatic times, and suggest that multiple miRNA species cooperatively regulate cellular pathways for the pathological changes and management of brain injury. The distinctive miRNAs expression profiles at different post-CCI times may be used as molecular signatures to assess TBI progression. In addition to known pathophysiological changes, our study identifies many other cellular pathways that are subjected to modification by differentially expressed miRNAs in TBI brains. These pathways can potentially be targeted for development of novel TBI treatment.

Topiramate Reduces Excitability in the Basolateral Amygdala by Selectively Inhibiting GluK1 (GluR5) Kainate Receptors on Interneurons and Positively Modulating GABAA Receptors on Principal Neurons

Topiramate [2,3:4,5-bis-O-(1-methylethylidene)-β-d-fructopyranose sulfamate] is a structurally novel antiepileptic drug that has broad efficacy in epilepsy, but the mechanisms underlying its therapeutic activity are not fully understood. We have found that topiramate selectively inhibits GluK1 (GluR5) kainate receptor-mediated excitatory postsynaptic responses in rat basolateral amygdala (BLA) principal neurons and protects against seizures induced by the GluK1 kainate receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA). Here, we demonstrate that topiramate also modulates inhibitory function in the BLA. Using whole-cell recordings in rat amygdala slices, we found that 0.3 to 10 μM topiramate 1) inhibited ATPA-evoked postsynaptic currents recorded from BLA interneurons; 2) suppressed ATPA-induced enhancement of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from BLA pyramidal cells; and 3) blocked ATPA-induced suppression of evoked IPSCs, which is mediated by presynaptic GluK1 kainate receptors present on BLA interneurons. Topiramate (10 μM) had no effect on the AMPA [(R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid]-induced enhancement of spontaneous activity of BLA neurons. Thus, although topiramate inhibits GluK1 kainate receptor-mediated enhancement of interneuron firing, it promotes evoked GABA release, leading to a net inhibition of circuit excitability. In addition, we found that topiramate (0.3–10 μM) increased the amplitude of evoked, spontaneous, and miniature IPSCs in BLA pyramidal neurons, indicating an enhancement of postsynaptic GABAA receptor responses. Taken together with our previous findings, we conclude that topiramate protects against hyperexcitability in the BLA by suppressing the GluK1 kainate receptor-mediated excitation of principal neurons by glutamatergic afferents, blocking the suppression of GABA release from interneurons mediated by presynaptic GluK1 kainate receptors and directly enhancing GABAA receptor-mediated inhibitory currents.

Reduced GABAergic inhibition in the basolateral amygdala and the development of anxiety-like behaviors after mild traumatic brain injury.

Traumatic brain injury (TBI) is a major public health concern affecting a large number of athletes and military personnel. Individuals suffering from a TBI risk developing anxiety disorders, yet the pathophysiological alterations that result in the development of anxiety disorders have not yet been identified. One region often damaged by a TBI is the basolateral amygdala (BLA); hyperactivity within the BLA is associated with increased expression of anxiety and fear, yet the functional alterations that lead to BLA hyperexcitability after TBI have not been identified. We assessed the functional alterations in inhibitory synaptic transmission in the BLA and one mechanism that modulates excitatory synaptic transmission, the α7 containing nicotinic acetylcholine receptor (α7-nAChR), after mTBI, to shed light on the mechanisms that contribute to increased anxiety-like behaviors. Seven and 30 days after a mild controlled cortical impact (CCI) injury, animals displayed significantly greater anxiety-like behavior. This was associated with a significant loss of GABAergic interneurons and significant reductions in the frequency and amplitude of spontaneous and miniature GABAA-receptor mediated inhibitory postsynaptic currents (IPSCs). Decreases in the mIPSC amplitude were associated with reduced surface expression of α1, β2, and γ2 GABAA receptor subunits. However, significant increases in the surface expression and current mediated by α7-nAChR, were observed, signifying increases in the excitability of principal neurons within the BLA. These results suggest that mTBI causes not only a significant reduction in inhibition in the BLA, but also an increase in neuronal excitability, which may contribute to hyperexcitability and the development of anxiety disorders.

Pathological alterations in GABAergic interneurons and reduced tonic inhibition in the basolateral amygdala during epileptogenesis.

An acute brain insult such as traumatic head/brain injury, stroke, or an episode of status epilepticus can trigger epileptogenesis, which, after a latent, seizure-free period, leads to epilepsy. The discovery of effective pharmacological interventions that can prevent the development of epilepsy requires knowledge of the alterations that occur during epileptogenesis in brain regions that play a central role in the induction and expression of epilepsy. In the present study, we investigated pathological alterations in GABAergic interneurons in the rat basolateral amygdala (BLA), and the functional impact of these alterations on inhibitory synaptic transmission, on days 7 to 10 after status epilepticus induced by kainic acid. Using design-based stereology combined with glutamic acid decarboxylase (GAD) 67 immunohistochemistry, we found a more extensive loss of GABAergic interneurons compared to the loss of principal cells. Fluoro-Jade C staining showed that neuronal degeneration was still ongoing. These alterations were accompanied by an increase in the levels of GAD and the alpha1 subunit of the GABA(A) receptor, and a reduction in the GluK1 (previously known as GluR5) subunit, as determined by Western blots. Whole-cell recordings from BLA pyramidal neurons showed a significant reduction in the frequency and amplitude of action potential-dependent spontaneous inhibitory postsynaptic currents (IPSCs), a reduced frequency but not amplitude of miniature IPSCs, and impairment in the modulation of IPSCs via GluK1-containing kainate receptors (GluK1Rs). Thus, in the BLA, GABAergic interneurons are more vulnerable to seizure-induced damage than principal cells. Surviving interneurons increase their expression of GAD and the alpha1 GABA(A) receptor subunit, but this does not compensate for the interneuronal loss; the result is a dramatic reduction of tonic inhibition in the BLA circuitry. As activation of GluK1Rs by ambient levels of glutamate facilitates GABA release, the reduced level and function of these receptors may contribute to the reduction of tonic inhibitory activity. These alterations at a relatively early stage of epileptogenesis may facilitate the progress towards the development of epilepsy.

The GluK1 (GluR5) Kainate/α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Antagonist LY293558 Reduces Soman-Induced Seizures and Neuropathology

The possibility of mass exposure to nerve agents by a terrorist attack necessitates the availability of antidotes that can be effective against nerve agent toxicity even when administered at a relatively long latency after exposure, because medical assistance may not be immediately available. Nerve agents induce status epilepticus (SE), which can cause brain damage or death. Antagonists of kainate receptors that contain the GluK1 (formerly known as GluR5) subunit (GluK1Rs) are emerging as a new potential treatment for SE and epilepsy from animal research, whereas clinical trials to treat pain have shown that the GluK1/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist LY293558 [(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid] is safe and well tolerated. Therefore, we tested whether LY293558 is effective against soman-induced seizures and neuropathology, when administered 1 h after soman exposure, in rats. LY293558 stopped seizures induced by soman and reduced the total duration of SE, monitored by electroencephalographic recordings within a 24 h-period after exposure. In addition, LY293558 prevented neuronal loss in the basolateral amygdala (BLA) and the CA1 hippocampal area on both days 1 and 7 after soman exposure and reduced neuronal degeneration in the CA1, CA3, and hilar hippocampal regions, entorhinal cortex, amygdala, and neocortex on day 1 after exposure and in the CA1, CA3, amygdala, and neocortex on day 7 after exposure. It also prevented the delayed loss of glutamic acid decarboxylase-67 immuno-stained BLA interneurons on day 7 after exposure. LY293558 is a potential new emergency treatment for nerve agent exposure that can be expected to be effective against seizures and brain damage even with late administration.

Primary brain targets of nerve agents: the role of the amygdala in comparison to the hippocampus.

Exposure to nerve agents and other organophosphorus acetylcholinesterases used in industry and agriculture can cause death, or brain damage, producing long-term cognitive and behavioral deficits. Brain damage is primarily caused by the intense seizure activity induced by these agents. Identifying the brain regions that respond most intensely to nerve agents, in terms of generating and spreading seizure activity, along with knowledge of the physiology and biochemistry of these regions, can facilitate the development of pharmacological treatments that will effectively control seizures even if administered when seizures are well underway. Here, we contrast the pathological (neuronal damage) and pathophysiological (neuronal activity) findings of responses to nerve agents in the amygdala and the hippocampus, the two brain structures that play a central role in the generation and spread of seizures. The evidence so far suggests that exposure to nerve agents causes significantly more damage in the amygdala than in the hippocampus. Furthermore, in in vitro brain slices, the amygdala generates prolonged, seizure-like neuronal discharges in response to the nerve agent soman, at a time when the hippocampus generates only interictal-like activity. In vivo experiments are now required to confirm the primary role that the amygdala seems to play in nerve agent-induced seizure generation.