In experiments designed to investigate the relationship between stress and the acquisition of new fear memories, it was found that previous exposure to a restraint session increased fear conditioning in a contextual fear paradigm. Moreover, the infusion of bicuculline, a competitive antagonist of GABA A receptors, into the basolateral amygdala complex (BLA), but not into the central amygdaloid nucleus, induced the same behavioral effect. Pretreatment with midazolam (MDZ), a positive modulator of GABA A sites, prevented the facilitating influence on fear memory of both stress and GABA A receptor blockade in the BLA. These data suggest that facilitation of fear conditioning could be causally related to increased neuronal excitability attributable to depressed GABAergic inhibition in the BLA. To test this hypothesis, evoked potentials were studied in brain slices from stressed animals. Potentials evoked in the BLA by single stimuli applied to the external capsule showed multispike responses, suggestive of GABAergic disinhibition. These multiple responses were no longer evident after the slices were perfused with diazepam or if the stressed animals were pretreated with MDZ. In slices from stressed rats, paired-pulse inhibition (GABA dependent) was suppressed. Also, in stressed animals, long-term potentiation (LTP) was induced with a single train of high-frequency stimulation, which did not induce LTP in control rats. Moreover, MDZ pretreatment prevented the facilitating influence of stress on LTP induction. All of these findings support the hypothesis that previous stress attenuates inhibitory GABAergic control in the BLA, leading to neuronal hyperexcitability and increased plasticity that facilitates fear learning.

Cultures of dissociated hypothalamic cells taken from rat fetuses of 19 days of gestation were studied using time-lapse recording and sequential microphotography from 1 to 5 days in vitro (DIV) and at 7 and 21 DIV. Cultures were seeded with cells taken from fetuses grouped by sex or sexually mixed; experimental cultures were raised in medium containing 17-beta-estradiol 100 nM (E2). Cells were plated on poly-D-lysine-coated coverslips at a culture density of approximately 4,000 cells/cm2. Immunocytochemistry of cell cultures was performed using a Tau monoclonal antibody (clone Tau-1 PC1C6) and a monoclonal antibody against MAP-2 (clone AP-20). Cells started to produce lamellipodia and neuritic processes approximately 4 hr after plating. Forty-eight hours later a few neurons had defined their morphological polarity by the differentiation of an axon-like process that grows faster than the others; at 5 DIV almost all neurons had defined their axons. At this time, monoclonal antibody against MAP-2 clearly stained soma and dendrites, but not axons. Tau immunoreactivity (lots CCA101 and CCA101N from Boeringher Mannheim) was differentially distributed, with a clear predominance in axon and soma. Results on the morphometric analysis of control and E2 treated neurons provide direct evidence for the existence of sex related differences in the neurite outgrowth response of hypothalamic neurons, since cultured neurons taken from female fetuses differentiated axons later and had fewer primary neurites and shorter dendrites than neurons taken from male fetuses or sexually mixed cultures. Also, it was demonstrated in living neurons that E2 effectively enhances outgrowth and elongation in axons. The frequency distribution curves of axonal length for control and E2 treated cultures was unimodal, suggesting that the effect of E2 was a uniform increase in the axonal length of all neurons. The structural differences between neurons from both sexes and the changes induced by E2 may contribute to explain the differences in brain function found between the sexes.

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The slow Ca2+-activated K+ current (sIAHP) was recorded in CA1 pyramidal neurons in hippocampal slices obtained from ovariectomized (OVX) or sham OVX (control) female rats. The sIAHP was significantly larger in cells from OVX rats than in cells from control rats. Superfusion with 5-100 nm 17beta-estradiol (E2) caused a progressive decrease in the sIAHP in cells from OVX rats but not in cells from control rats. In slices from OVX rats injected with 10 microg of E2 24 and 48 hr before they were killed, superfusion with E2 did not modify the sIAHP. In neurons from OVX rats, but not in neurons from control rats, E2 significantly increased both the number of action potentials and the burst duration generated by depolarizing pulses. The inactive isomer 17alpha-estradiol had no effect. The impermeant protein conjugate E2--BSA was as effective as free E2 at decreasing the sIAHP. Ca2+ spikes were also depressed by E2 in neurons from OVX rats, but not in control rats. A decrease in the intracellular Ca2+ signal, correlating with the inhibition of the Ca2+ spike and sIAHP produced by E2, was observed only in neurons from OVX rats. Our results indicate that ovariectomy increases the sIAHP and depresses excitability, whereas bath application or priming with E2 decreases the sIAHP, thus promoting excitability. These effects of E2 on the sIAHP and excitability, which are stereospecific and presumably mediated by membrane-bound receptors, could contribute to the hormonal regulation of synaptic plasticity and epileptiform activity as well as to learning and cognitive abilities dependent on the function of hippocampal neural circuits.