Structural and functional characterization of dendritic arbors and GABAergic synaptic inputs on interneurons and principal cells in the rat basolateral amygdala

The basolateral amygdala (BLA) is a complex brain region associated with processing emotional states, such as fear, anxiety, and stress. Some aspects of these emotional states are driven by the network activity of synaptic connections, derived from both local circuitry and projections to the BLA from other regions. Although the synaptic physiology and general morphological characteristics are known for many individual cell types within the BLA, the combination of morphological, electrophysiological, and distribution of neurochemical GABAergic synapses in a three-dimensional neuronal arbor has not been reported for single neurons from this region. The aim of this study was to assess differences in morphological characteristics of BLA principal cells and interneurons, quantify the distribution of GABAergic neurochemical synapses within the entire neuronal arbor of each cell type, and determine whether GABAergic synaptic density correlates with electrophysiological recordings of inhibitory postsynaptic currents. We show that BLA principal neurons form complex dendritic arborizations, with proximal dendrites having fewer spines but higher densities of neurochemical GABAergic synapses compared with distal dendrites. Furthermore, we found that BLA interneurons exhibited reduced dendritic arbor lengths and spine densities but had significantly higher densities of putative GABAergic synapses compared with principal cells, which was correlated with an increased frequency of spontaneous inhibitory postsynaptic currents. The quantification of GABAergic connectivity, in combination with morphological and electrophysiological measurements of the BLA cell types, is the first step toward a greater understanding of how fear and stress lead to changes in morphology, local connectivity, and/or synaptic reorganization of the BLA.

GABAergic excitation in the basolateral amygdala

GABA-containing interneurons are a diverse population of cells whose primary mode of action in the mature nervous system is inhibition of postsynaptic target neurons. Using paired recordings from parvalbumin-positive interneurons in the basolateral amygdala, we show that, in a subpopulation of interneurons, single action potentials in one interneuron evoke in the postsynaptic interneuron a monosynaptic inhibitory synaptic current, followed by a disynaptic excitatory glutamatergic synaptic current. Interneuron-evoked glutamatergic events were blocked by antagonists of either AMPA/kainate or GABA(A) receptors, and could be seen concurrently in both presynaptic and postsynaptic interneurons. These results show that single action potentials in a GABAergic interneuron can drive glutamatergic principal neurons to threshold, resulting in both feedforward and feedback excitation. In interneuron pairs that both receive glutamatergic inputs after an interneuron spike, electrical coupling and bidirectional GABAergic connections occur with a higher probability relative to other interneuron pairs. We propose that this form of GABAergic excitation provides a means for the reliable and specific recruitment of homogeneous interneuron networks in the basal amygdala.

Behavioral traits predicting cocaine-conditioned place reference in mice: role of anxiety adn the basolateral amygdala

Aims. The individual susceptibility to cocaine addiction, a factor of interest in the understanding and prevention of this disorder, may be predicted by certain behavioral traits. However, these are not usually taken into account in research, making it difficult to identify whether they are a cause or a consequence of drug use. Methods. Male C57BL/6J mice underwent a battery of behavioral tests (elevated plus maze, hole-board, novelty preference in the Y maze, episodic-like object recognition memory and forced swimming test), followed by a cocaine-conditioned place preference (CPP) training to assess the reinforcing effect of the drug. In a second study, we aimed to determine the existence of neurobiological differences between the mice expressing high or low CPP by studying the number of neurons in certain addiction-related structures: the medial prefrontal cortex, the basolateral amygdala and the ventral tegmental area. Results. Anxiety-like behaviors in the elevated plus maze successfully predicted the cocaine-CPP behavior, so that the most anxious mice were also more likely to search for cocaine in a CPP paradigm. In addition, these mice exhibited an increased number of neurons in the basolateral amygdala, a key structure in emotional response including anxiety expression, without differences in the others regions analyzed. Conclusions. Our results suggest a relevant role of anxiety as a psychological risk factor for cocaine vulnerability, with the basolateral amygdala as potential common neural center for both anxiety and addiction.

GABAA and NMDA receptors contribute to synaptic integration in lateral amygdala neurons

In classical fear conditioning a neutral conditioned stimulus (CS), is paired with an aversive unconditioned stimulus (US). The CS thereby acquires the capacity to elicit a fear response. This type of associative learning is thought to require co-activation of principal neurons in the lateral nucleus of the amygdala (LA) by two sets of synaptic inputs...

Differences in intrinsic properties of dorsally and ventrally located lateral amygdala neurons of the rat

During Pavlovian auditory fear conditioning a previously neutral auditory stimulus (CS) gains emotional significance through pairing with a noxious unconditioned stimulus (US). These associations are believed to be formed by way of plasticity at auditory input synapses on principal neurons of the lateral nucleus of the amygdala (LA). While the LA has been implicated as a key brain structure for fear learning, how its network of cellular components performs these operations is not yet known...

GABAa receptors contribute to synaptic integration in lateral amygdala neurons

In classical fear conditioning a neutral conditioned stimulus (CS) such as a tone, is paired with an aversive unconditioned stimulus (US) such as a shock. The CS thereby acquires the capacity to elicit a fear response. This type of associative learning is thought to require co-activation of principle neurons in the lateral nucleus of the amygdala (LA) by two sets of synaptic inputs, a weak CS and a strong US...

Central, but not basolateral, amygdala involvement in the anxiolytic-like effects of midazolam in rats in the elevated plus maze

The role of the amygdala in the mediation of fear and anxiety has been extensively investigated. However, how the amygdala functions during the organization of the anxiety-like behaviors generated in the elevated plus maze (EPM) is still under investigation. The basolateral (BLA) and the central (CeA) nuclei are the main input and output stations of the amygdala. In the present study, we ethopharmacologically analyzed the behavior of rats subjected to the EPM and the tissue content of the monoamines dopamine (DA) and serotonin (5-HT) and their metabolites in the nucleus accumbens (NAc), dorsal hippocampus (DH), and dorsal striatum (DS) of animals injected with saline or midazolam (20 and 30 nmol/0.2 mu L) into the BLA or CeA. Injections of midazolam into the CeA, but not BLA, caused clear anxiolytic-like effects in the EPM. These treatments did not cause significant changes in 5-HT or DA contents in the NAc, DH, or DS of animals tested in the EPM. The data suggest that the anxiolytic-like effects of midazolam in the EPM also appear to rely on GABA-benzodiazepine mechanisms in the CeA, but not BLA, and do not appear to depend on 5-HT and DA mechanisms prevalent in limbic structures.

Basolateral amygdala is not critical for cognitive memory of contextual fear conditioning

Evidence that lesions of the basolateral amygdala complex (BLC) impair memory for fear conditioning in rats, measured by lack of “freezing” behavior in the presence of cues previously paired with footshocks, has suggested that the BLC may be a critical locus for the memory of fear conditioning. However, evidence that BLC lesions may impair unlearned as well as conditioned freezing makes it difficult to interpret the findings of studies assessing conditioned fear with freezing. The present study investigated whether such lesions prevent the expression of several measures of memory for contextual fear conditioning in addition to freezing. On day 1, rats with sham lesions or BLC lesions explored a Y maze. The BLC-lesioned rats (BLC rats) displayed a greater exploratory activity. On day 2, each of the rats was placed in the “shock” arm of the maze, and all of the sham and half of the BLC rats received footshocks. A 24-hr retention test assessed the freezing, time spent per arm, entries per arm, and initial entry into the shock arm. As previously reported, shocked BLC rats displayed little freezing. However, the other measures indicated that the shocked BLC rats remembered the fear conditioning. They entered less readily and less often and spent less time in the shock arm than did the control nonshocked BLC rats. Compared with the sham rats, the shocked BLC rats entered more quickly and more often and spent more time in the shock arm. These findings indicate that an intact BLC is not essential for the formation and expression of long-term cognitive/explicit memory of contextual fear conditioning.

Glucocorticoid enhancement of memory storage involves noradrenergic activation in the basolateral amygdala

Evidence indicates that the modulatory effects of the adrenergic stress hormone epinephrine as well as several other neuromodulatory systems on memory storage are mediated by activation of β-adrenergic mechanisms in the amygdala. In view of our recent findings indicating that the amygdala is involved in mediating the effects of glucocorticoids on memory storage, the present study examined whether the glucocorticoid-induced effects on memory storage depend on β-adrenergic activation within the amygdala. Microinfusions (0.5 μg in 0.2 μl) of either propranolol (a nonspecific β-adrenergic antagonist), atenolol (a β1-adrenergic antagonist), or zinterol (a β2-adrenergic antagonist) administered bilaterally into the basolateral nucleus of the amygdala (BLA) of male Sprague–Dawley rats 10 min before training blocked the enhancing effect of posttraining systemic injections of dexamethasone (0.3 mg/kg) on 48-h memory for inhibitory avoidance training. Infusions of these β-adrenergic antagonists into the central nucleus of the amygdala did not block the dexamethasone-induced memory enhancement. Furthermore, atenolol (0.5 μg) blocked the memory-enhancing effects of the specific glucocorticoid receptor (GR or type II) agonist RU 28362 infused concurrently into the BLA immediately posttraining. These results strongly suggest that β-adrenergic activation is an essential step in mediating glucocorticoid effects on memory storage and that the BLA is a locus of interaction for these two systems.

Risk assessment behaviors associated with corticosterone trigger the defense reaction to social isolation in rats: Role of the anterior cingulate cortex

The extent to which the hypothalamic-pituitary-adrenal axis is activated by short-term and long-term consequences of stress is still open to investigation. This study aimed to determine (i) the correlation between plasma corticosterone and exploratory behavior exhibited by rats subjected to the elevated plus maze (EPM) following different periods of social isolation, (ii) the effects of the corticosterone synthesis blocker, metyrapone, on the behavioral consequences of isolation, and (iii) whether corticosterone produces its effects through an action on the anterior cingulate cortex, area 1 (Cg1). Rats were subjected to 30-min, 2-h, 24-h, or 7-day isolation periods before EPM exposure and plasma corticosterone assessments. Isolation for longer periods of time produced greater anxiogenic-like effects on the EPM. However, stretched attend posture (SAP) and plasma corticosterone concentrations were increased significantly after 30 min of isolation. Among all of the behavioral categories measured in the EPM, only SAP positively correlated with plasma corticosterone. Metyrapone injected prior to the 24 h isolation period reversed the anxiogenic effects of isolation. Moreover, corticosterone injected into the Cg1 produced a selective increase in SAP. These findings indicate that risk assessment behavior induced by the action of corticosterone on Cg1 neurons initiates a cascade of defensive responses during exposure to stressors.

Input from the amygdala to the rat nucleus accumbens : its relationship with tyrosine hydroxylase immunoreactivity and identified neurons.

Both tyrosine hydroxylase-positive fibres from the mesolimbic dopamine system and amygdala projection fibres from the basolateral nucleus are known to terminate heavily in the nucleus accumbens. Caudal amygdala fibres travelling dorsally via the stria terminalis project densely to the nucleus accumbens shell, especially in the dopamine rich septal hook. The amygdala has been associated with the recognition of emotionally relevant stimuli while the mesolimbic dopamine system is implicated with reward mechanisms. There is behavioural and electrophysiological evidence that the amygdala input to the nucleus accumbens is modulated by the mesolimbic dopamine input, but it is not known how these pathways interact anatomically within the nucleus accumbens. Using a variety of neuroanatomical techniques including anterograde and retrograde tracing, immunocytochemistry and intracellular filling, we have demonstrated convergence of these inputs on to medium-sized spiny neurons. The terminals of the basolateral amygdala projection make asymmetrical synapses predominantly on the heads of spines which also receive on their necks or adjacent dendrites, symmetrical synaptic input from the mesolimbic dopamine system. Some of these neurons have also been identified as projection neurons, possibly to the ventral pallidum. We have shown a synaptic level how dopamine is positioned to modulate excitatory limbic input in the nucleus accumbens.