Chronic ethanol and withdrawal differentially modulate pre- and postsynaptic function at glutamatergic synapses in rat basolateral amygdala

Abstract

Withdrawal anxiety is a significant factor contributing to continued alcohol abuse in alcoholics. This anxiety is long-lasting, can manifest well after the overt physical symptoms of withdrawal, and is frequently associated with relapse in recovering alcoholics. The neurobiological mechanisms governing these withdrawal-associated increases in anxiety are currently unknown. The basolateral amygdala (BLA) is a major emotional center in the brain and regulates the expression of both learned fear and anxiety. Neurotransmitter system alterations within this brain region may therefore contribute to withdrawal-associated anxiety. Because evidence suggests that glutamate-gated neurotransmitter receptors are sensitive to acute ethanol exposure, we examined the effect of chronic intermittent ethanol (CIE) and withdrawal (WD) on glutamatergic synaptic transmission in the BLA. We found that slices prepared from CIE and WD animals had significantly increased contributions by synaptic NMDA receptors. In addition, CIE increased the amplitude of AMPA-receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs), whereas only WD altered the amplitude and kinetics of tetrodotoxin-resistant spontaneous events (mEPSCs). Similarly, the frequency of sEPSCs was increased in both CIE and WD neurons, although only WD increased the frequency of mEPSCs. These data suggest that CIE and WD differentially alter both pre- and postsynaptic properties of BLA glutamatergic synapses. Finally, we show that microinjection of the AMPA-receptor antagonist, DNQX, can attenuate withdrawal-related anxiety-like behavior. Together, our results suggest that increased glutamatergic function may contribute to anxiety expressed during withdrawal from chronic ethanol.

Figure 1

Chronic ethanol and withdrawal increase the NMDA/AMPA current ratio in the BLA measured with low extracellular Mg²⁺. A, Slices were stimulated locally near the boundary of the lateral/basolateral amygdala and the recording electrode was placed in the basolateral amygdala as shown. B, Average synaptic responses for control and 24hr WD cells, in the presence of 10µM bicuculline, show the compound current consisting of the DNQX-sensitive and –resistant components and the current resistant to 20µM DNQX. The DNQX- resistant currents from these neurons are also enlarged below the compound current traces to aid with visual comparisons. The NMDA receptor antagonist APV inhibited >95% of the DNQX-resistant current (see text). C, The NMDA/AMPA ratio, calculated from the amplitude of the DNQX-resistant component (NMDA) divided by the amplitude of the DNQX-sensitive component (AMPA), show a significant increase of 405% in CIE neurons and 342% following 24hr WD (**, P<0.01 and *, P<0.05 compared to CON, one-way ANOVA with Bonferroni posttest). D, NMDA input-output relationships were measured from CON (D₁, top), CIE (D₁, middle), and WD (D₁, bottom) neurons. Note that the amplitude NMDA-mediated (DNQX-resistant) synaptic responses were dependent on the intensity of the stimulation (in µA) and were significantly larger at the greatest intensities for CIE (n=7 neurons from 3 individuals) and WD neurons (n=6 neurons from three individuals) compared to CON neurons (D₂, n=6 neurons from three individuals; *, CIE vs. CON P<0.05, #, WD vs. CON P<0.05, one-way ANOVA with Bonferroni post-test).

Figure 2

Chronic ethanol and withdrawal increase the frequency of spontaneous excitatory postsynaptic currents (sEPSCs). A, Sample traces illustrate that, under standard conditions (10µM bicuculline; holding potential, −60mV), sEPSCs are mediated by GYKI 53655-sensitive AMPA receptors. B, Sample traces of sEPSCs in the presence of 10µM bicuculline (holding potential, −60mV) are shown for each treatment group. C, The sEPSC amplitude in CIE neurons was significantly larger than CON (*, P<0.05, one-way ANOVA with Bonferroni post-test). Mean±SEM are shown across all cells in a particular treatment group. For these experiments, n=8 neurons for CON (from five rats), n=11 neurons for CIE slices (from four rats), and n=11 for WD neurons (from four animals). D, The charge carried by sEPSCs (amplitude × decay time, expressed in femtoCoulombs) was not significantly affected by CIE or WD. E, The sEPSC inter-event interval was dramatically and significantly decreased (increased frequency) for both CIE and WD neurons (**, P<0.01 compared to CON, one-way ANOVA). F, A cumulative probability plot comparing the inter-event interval from individual CON, CIE, and WD neurons, each neuron represents median values from each treatment group. KS=0.39 for CIE neurons and KS=0.36 for WD neurons (P<0.0001 relative to CON).

Figure 3

Chronic ethanol and withdrawal decrease paired pulse ratio in the BLA. A, Sample traces of paired pulse EPSCs at 50msec inter-pulse interval in the presence of 10µM bicuculline. The amplitude of the second synaptic response has been normalized across treatment groups to emphasize the relative differences between the first synaptic response and the second. B, Paired pulse ratio is significantly decreased at both the 25 and 50msec interval (*P<0.05, **P<0.01 vs. control, one-way ANOVA with Bonferroni’s post-test). These decreased ratios indicate that chronic ethanol and withdrawal may increase presynaptic release of glutamate.

Figure 4

Withdrawal increases the frequency and amplitude of mEPSCs. A, Sample traces demonstrating the efficacy of 1µM tetrodotoxin used in these studies. B, Sample traces of mEPSCs in the presence of 10µM bicuculline and 1µM TTX (holding potential, −60mV) are shown for each treatment group. C, The mEPSC amplitude from WD neurons was significantly greater than CON (*, P<0.05) and CIE neurons (#, P<0.05; one-way ANOVA with Bonferroni’s post-test). D, The charge carried by mEPSCs (amplitude in pA × decay time in milliseconds, expressed as femtoCoulombs) was not significantly different from CON in the CIE and WD treatment groups. E, The inter-event interval of WD mEPSCs was significantly smaller than both control (**, P<0.01) and CIE neurons (#, P<0.05; one-way ANOVA with Bonferroni’s post-test). F, Cumulative probability plot of inter-event interval from individual CON, CIE, and WD neurons; each cell is representative of the median from that particular treatment group. The inter-event interval distribution of the WD neuron (KS=0.19, P<0.0001), but not the CIE neuron (KS=0.06, P≫0.05), was significantly different from the CON neuron.

Figure 5

Withdrawal from chronic ethanol decreases the current-decay time constant of miniature events. A, Average traces from each treatment group illustrate the faster decay kinetics of miniature events in withdrawal neurons. B, Rise-times were not significantly different from each other (one-way ANOVA). C, Using a single-exponential fit of miniature EPSCs, the decay time was significantly smaller than CON for WD events (*, P<0.05, one-way ANOVA) but not CIE events. These results suggest possible postsynaptic alterations of the receptor during withdrawal and provide a mechanism for increased amplitude, but not charge, in these neurons.

Figure 6

Microinjection of the AMPA-type glutamate receptor antagonist, DNQX, into the lateral/basolateral amygdala attenuates anxiety-like behavior expressed during withdrawal. A, Figure (modified from Paxinos and Watson 1997) illustrating the approximate locations for the guide cannulae tips (correct placements=closed circles). Note that some cannulae were placed outside the BLA (open circles, see text). B, Summary of light/dark box behaviors expressed by sham- (WD) and DNQX-injected animals with guide cannulae place within the BLA. While variables like the time spent moving during the assay were not affected (B₁), 100pmol DNQX significantly increased both the total time spent in the light side (B₂) and the number of light/dark transitions (B₃). Intra-BLA DNQX also significantly decreased the latency to re-enter the light-side following initial egress to the dark side (B4). *P<0.05, **P<0.01, t-test.