Chronic ethanol and withdrawal differentially modulate lateral/basolateral amygdala paracapsular and local GABAergic synapses

Abstract

Withdrawal-related anxiety is cited as a major contributor to relapse in recovering alcoholics. Changes in lateral/basolateral amygdala (BLA) neurotransmission could directly influence anxiety-like behaviors after chronic ethanol exposure and withdrawal. We have shown that these treatments enhance BLA glutamatergic function and neurotransmission. However, the BLA GABAergic system tightly controls the expression of anxiety-like behavior, and additional neuroadaptations in this system are potentially important as well. The intrinsic BLA GABAergic system consists of at least two populations of interneurons: local feed-back interneurons scattered throughout the region and feed-forward interneurons concentrated within groups found in the lateral/paracapsular region of the BLA. In the present study, we found that withdrawal from chronic ethanol robustly decreased presynaptic function at feed-forward GABA synapses but did not alter neurotransmitter release from local interneurons. Differential presynaptic changes at these synapses were complemented by decreased zolpidem sensitivity at feed-forward synapses and decreased midazolam sensitivity at local synapses. Consistent with this, chronic ethanol/withdrawal decreased expression of GABA α1-subunit total protein and increased surface expression of α4-subunit protein. We also found transient increases in GABA-receptor-associated protein levels and persistent increases in γ2-subunit and gephyrin proteins that would suggest alterations in GABA(A) receptor trafficking that might help regulate changes in α4-subunit localization. These data together suggest that chronic ethanol and withdrawal differentially modulate local and lateral paracapsular cell GABAergic synapses via distinct presynaptic and postsynaptic mechanisms. These findings extend our understanding of the neurobiological mechanisms governing changes in anxiety-like behavior after chronic ethanol exposure and withdrawal.

Fig. 1.

Chronic ethanol and withdrawal differentially modulate LPC and local interneuron synaptic responses to paired electrical stimuli. A, sample paired-pulse traces from local and LPC neurons across treatment groups. P₁ and P₂ denote the first and second response, respectively. Stimulation artifacts have been removed for clarity. Dotted lines are used to compare the peak of the first response to the second. These continuous traces sampled both local and LPC GABAergic synapses in the same BLA principal neuron using two stimulating electrodes (see Materials and Methods). B, normalized paired-pulse ratios (see Materials and Methods) from LPC interneurons across the treatment groups at 50- and 250-ms interpulse intervals show significant changes in WD-treated neurons (n = 11) relative to CON (n = 21) (one-way ANOVA; *, P < 0.05 with Newman-Keuls multiple comparison post-test). There was no significant difference between CON and CIE neurons (n = 17). C, paired-pulse ratios measured in the same neurons as in B were evoked from local interneurons and were not different between the treatment groups at either stimulus interval.

Fig. 2.

Chronic ethanol and withdrawal differentially modulate presynaptic and postsynaptic properties of mIPSCs. A₁, exemplar traces of mIPSCs collected from individual principal cells at each exposure group (CON, CIE, and WD). A₂ and A₃, mIPSC amplitudes (A₂) and interevent intervals (A₃) were not significantly different between CON (n = 7), CIE (n = 5), or WD (n = 7) BLA neurons. B₁ and B₂, mIPSCs from each neuron were averaged to yield a normalized mIPSC current. B₁, exemplar traces from representative CON and WD neurons are shown. The normalized mIPSC decays were fit to a single exponential; amplitudes from these representative traces have been normalized to emphasize the decay phase of the response. B₂, time constants (Tau) were significantly larger in both CIE and WD mIPSCs relative to CON (one-way ANOVA; **, P < 0.01 relative to CON with Newman-Keuls multiple comparison post-test).

Fig. 3.

Midazolam sensitivity of local IPSCs and α4-subunit expression are modulated by chronic ethanol exposure/withdrawal. A₁ and A₂, midazolam sensitivity decreases at local GABAergic synapses but not paracapsular GABAergic inputs. A₁, exemplar traces of the effects of midazolam (1 μM) on electrically evoked GABA IPSCs from local interneurons from CON-, CIE-, and WD-treated slices. A₂, summary of data shows that the midazolam sensitivity of local IPSCs (Local) was significantly decreased in principal neurons from WD slices (n = 9; one-way ANOVA; *, P < 0.05 versus CON, Newman-Keuls multiple comparison post-test) relative to CON (n = 10) and CIE (n = 9) BLA neurons. The effect of midazolam on evoked-IPSCs from LPC interneurons was not altered in principal neurons from CIE or WD. B, total α4 protein levels were not significantly changed across treatment groups (n = 4 animals each). Representative immunoreactive bands from α4 total protein and β-actin correspond to the treatment groups in the bar graphs. C, surface-accessible, BS³-sensitive α4-subunit protein levels were significantly elevated in WD BLA (n = 4 animals in each group), compared with CON BLA (one-way ANOVA; **, P < 0.01 with Newman-Keuls multiple comparison posttest) and CIE BLA (#, P < 0.05, Newman-Keuls). Representative aCSF-treated and BS³-treated samples from each treatment are shown below the bar graph. The intracellular, BS³-insensitive pool of α4 subunit protein is shown (see Materials and Methods for details).

Fig. 4.

Zolpidem sensitivity and α1-subunit expression are robustly diminished by chronic ethanol and withdrawal. A₁ and A₂, zopidem (100 nM) modulation of LPC IPSCs is decreased in CIE and WD BLA principal neurons. A₁, sample traces from LPC inputs illustrate the treatment effect. A₂, summary of zolpidem data shows that CIE (LPC: n = 7, local: n = 9) and WD (LPC: n = 9, local: n = 9) significantly diminish modulation at LPC but not local IPSCs relative to CON (LPC: n = 9, local: n = 9; one-way ANOVA; ***, P < 0.001 versus CON, Newman-Keuls multiple comparison post-test). B, total α1-subunit protein levels were significantly reduced in CIE and WD BLA relative to CON (n = 4 animals per group; one-way ANOVA, *, P < 0.05; **, P < 0.01 versus CON with Newman-Keuls post hoc). Representative immunoreactive bands from total α1 protein and β-actin correspond to the treatment groups in the bar graphs. C, BS³-sensitive, surface accessible α1-subunit protein levels were significantly reduced in WD BLA relative to both CON and CIE (n = 4 animals/treatment; one-way ANOVA, *, P < 0.05 compared with CON; #, P < 0.05 compared with CIE, Newman-Keuls post hoc). Representative aCSF-treated and BS³-treated samples from each treatment are shown below the bar graph. The intracellular, BS³-insensitive pool of α1-subunit protein is shown (see Materials and Methods for details).

Fig. 5.

CIE and WD significantly alter protein expression of several GABA_A receptor association proteins. A, total γ2-subunit protein levels were robustly elevated in CIE and WD slices relative to CON (n = 4 animals/treatment; one-way ANOVA, *, P < 0.05 versus CON from Newman-Keuls post-test). B, total gephryin protein levels were also significantly increased in both CIE (**, P < 0.01 relative to CON) and WD BLA slices (*, P < 0.05 versus CON, Newman-Keuls post hoc). C, total GABA-RAP expression was significantly increased in CIE slices (*, P < 0.05, Newman-Keuls post hoc) but returned to CON levels by 24 h of withdrawal. Representative protein and β-actin immunoreactivity from each treatment group is shown at right.

Fig. 6.

CIE and WD treatments do not alter acute ethanol modulation of BLA GABAergic synapses. Graphs demonstrate that the effect of 80 mM acute ethanol on electrically evoked GABAergic transmission from LPC (A) and local (B) synapses is different between CON (n = 6 at each input), CIE (n = 11), and WD (n = 10) principal neurons (P > 0.05, one-way ANOVA). Sample traces correspond to the bar graphs.