A Functional Switch in Tonic GABA Currents Alters the Output of Central Amygdala Corticotropin Releasing Factor Receptor-1 Neurons Following Chronic Ethanol Exposure

Abstract

The corticotropin releasing factor (CRF) system in the central amygdala (CeA) has been implicated in the effects of acute ethanol and the development of alcohol dependence. We previously demonstrated that CRF receptor 1 (CRF1) neurons comprise a specific component of the CeA microcircuitry that is selectively engaged by acute ethanol. To investigate the impact of chronic ethanol exposure on inhibitory signaling in CRF1+ CeA neurons, we used CRF1:GFP mice subjected to chronic intermittent ethanol (CIE) inhalation and examined changes in local inhibitory control, the effects of acute ethanol, and the output of these neurons from the CeA. Following CIE, CRF1+ neurons displayed decreased phasic inhibition and a complete loss of tonic inhibition that persisted into withdrawal. CRF1- neurons showed a cell type-specific upregulation of both phasic and tonic signaling with CIE, the latter of which persists into withdrawal and is likely mediated by δ subunit-containing GABA_A receptors. The loss of tonic inhibition with CIE was seen in CRF1+ and CRF1- neurons that project out of the CeA and into the bed nucleus of the stria terminalis. CRF1+ projection neurons displayed an increased baseline firing rate and loss of sensitivity to acute ethanol following CIE. These data demonstrate that chronic ethanol exposure produces profound and long-lasting changes in local inhibitory control of the CeA, resulting in an increase in the output of the CeA and the CRF1 receptor system, in particular. These cellular changes could underlie the behavioral manifestations of alcohol dependence and potentially contribute to the pathology of addiction.

Significance statement: The corticotropin releasing factor (CRF) system in the central amygdala (CeA) has been implicated in the effects of acute and chronic ethanol. We showed previously that CRF receptor 1-expressing (CRF1+) neurons in the CeA are under tonic inhibitory control and are differentially regulated by acute ethanol (Herman et al., 2013). Here we show that the inhibitory control of CRF1+ CeA neurons is lost with chronic ethanol exposure, likely by a functional switch in local tonic signaling. The loss of tonic inhibition is seen in CRF1+ projection neurons, suggesting that a critical consequence of chronic ethanol exposure is an increase in the output of the CeA CRF1 system, a neuroadaptation that may contribute to the behavioral consequences of alcohol dependence.

Keywords: CRF; CRF1; GABA; alcohol; amygdala; tonic.

Figure 1.

A, A 10× magnification photomicrograph of a coronal CeA slice indicating recording sites of CRF1+ neurons (asterisks) and anatomical location (bregma, −1.46 mm). B, A 60× magnification of a CRF1+ CeA neuron from an AIR mouse (top) and a CIE mouse (bottom) using fluorescent optics (left) and IR-DIC optics (right). C, Representative current-clamp recordings of CeA cell types and the proportions of CRF1+ and CRF1− neurons of each cell type in AIR, CIE, and CIE-WD groups for low-threshold bursting (left), regular-spiking (middle), and late-spiking neurons (right). D, Summary of membrane characteristics (CRF1+ AIR, n = 34; CRF1− AIR, n = 23; CRF1+ CIE, n = 20; CRF1− CIE, n = 34; CRF1+ CIE-WD, n = 25; CRF1− CIE-WD, n = 31). *p < 0.05 by unpaired t test comparing CRF1+ to CRF1− in each group. Scale bars: A, 100 μm; B, 20 μm.

Figure 2.

A, Representative voltage-clamp recordings of baseline sIPSCs from a CRF1+ neuron from an AIR mouse (top), a CIE mouse (middle), and a CIE-WD mouse (bottom). B, Representative voltage-clamp recordings of baseline sIPSCs from a CRF1− neuron from an AIR mouse (top), a CIE mouse (middle), and a CIE-WD mouse (bottom). C, Summary of sIPSC frequency (left) and amplitude (right) in LTB CRF1+ neurons from AIR, CIE, and CIE-WD mice (AIR, n = 21; CIE, n = 18; CIE-WD, n = 17). D, Summary of sIPSC frequency (left) and amplitude (right) in regular-spiking CRF1+ neurons from AIR, CIE, and CIE-WD mice (AIR, n = 13; CIE, n = 10; CIE-WD, n = 6). E, Summary of sIPSC frequency (left) and amplitude (right) in late-spiking CRF1− neurons from AIR, CIE, and CIE-WD mice (AIR, n = 18; CIE, n = 12; CIE-WD, n = 16). F, Summary of sIPSC frequency (left) and amplitude (right) in regular-spiking CRF1− neurons from AIR, CIE, and CIE-WD mice (AIR, n = 13; CIE, n = 12; CIE-WD, n = 8). G, Summary of the sIPSC decay in LTB (AIR, n = 21; CIE, n = 18; CIE-WD, n = 17) and regular-spiking (AIR, n = 13; CIE, n = 10; CIE-WD, n = 6) CRF1+ neurons (left) and late-spiking (AIR, n = 18; CIE, n = 12; CIE-WD, n = 16) and regular-spiking (AIR, n = 13; CIE, n = 12; CIE-WD, n = 8) CRF1− neurons (right). *p < 0.05 by one-way ANOVA.

Figure 3.

A, Representative voltage-clamp recordings from CRF1+ neurons from an AIR mouse (top), a CIE mouse (middle), and a CIE-WD mouse (bottom) during superfusion of GBZ (100 μm). B, Representative voltage-clamp recordings from late-spiking CRF1− neurons from an AIR mouse (top), a CIE mouse (middle), and a CIE-WD mouse (bottom) during superfusion of GBZ (100 μm). C, Summary of the tonic current in CRF1+ neurons revealed by superfusion of GBZ in regular-spiking (AIR, n = 5; CIE, n = 4; CIE-WD, n = 2) and low-threshold bursting neurons (AIR, n = 8; CIE, n = 7; CIE-WD, n = 13). *p < 0.05 by one sample t-test; ^#p < 0.001 by treatment. D, Summary of the tonic current in CRF1− neurons revealed by superfusion of GBZ in regular-spiking (n = 4 AIR, n = 6 CIE and n = 8 CIE-WD) and late-spiking neurons (AIR, n = 8; CIE, n = 6; CIE-WD, n = 10). ^#p < 0.01 by treatment; *p < 0.001 by cell type.

Figure 4.

A, Representative voltage-clamp recordings from a regular-spiking (left) and a late-spiking (right) CRF1− neuron from an AIR mouse during superfusion of THIP (5 μm). B, Representative voltage-clamp recordings from a regular-spiking (left) and a late-spiking (right) CRF1− neuron from a CIE mouse during superfusion of THIP (5 μm). C, Summary of the tonic current induced by THIP in regular-spiking (AIR, n = 5; CIE, n = 6; CIE-WD, n = 7) and late-spiking neurons (AIR, n = 8; CIE, n = 6; CIE-WD, n = 9). *p < 0.001 by cell type. D, Summary of the tonic current induced by THIP in regular-spiking (AIR, n = 5; CIE, n = 5; CIE-WD, n = 1) and low threshold bursting neurons (AIR, n = 6; CIE, n = 6; CIE-WD, n = 5).

Figure 5.

A, Representative voltage-clamp recordings from a regular-spiking (left) and a late-spiking (right) CRF1− neuron from an AIR mouse during superfusion of EtOH (44 mm). B, Representative voltage-clamp recordings from a regular-spiking (left) and a late-spiking (right) CRF1− neuron from a CIE mouse during superfusion of ethanol (44 mm). C, Summary of the tonic current induced by EtOH in regular-spiking (AIR, n = 5; CIE, n = 6; CIE-WD, n = 4) and late-spiking CRF1− neurons (AIR, n = 7; CIE, n = 5; CIE-WD, n = 8). *p < 0.0001 by cell type. D, Summary of the change in sIPSC frequency induced by EtOH in regular-spiking (AIR, n = 5; CIE, n = 6; CIE-WD, n = 4) and late-spiking CRF1− neurons (AIR, n = 7; CIE, n = 5; CIE-WD, n = 8). E, Summary of the tonic current induced by EtOH in regular-spiking (AIR, n = 10; CIE, n = 4; CIE-WD, n = 5) and LTB CRF1+ neurons (AIR, n = 9; CIE, n = 5; CIE-WD, n = 7). F, Summary of the change in sIPSC frequency induced by EtOH in regular-spiking (AIR, n = 4; CIE, n = 4; CIE-WD, n = 3) and LTB CRF1+ neurons (AIR, n = 4; CIE, n = 4; CIE-WD, n = 4).

Figure 6.

A, A 60× magnification of a CRF1− CeA neuron in IR-DIC optics (left) and red fluorescent optics depicting retrogradely transported microspheres (right) indicating a projection to the dlBNST. B, A 60× magnification of a CRF1+ CeA neuron in IR-DIC optics (left), green fluorescent optics (middle), and red fluorescent optics depicting retrogradely transported microspheres (right). C, Representative voltage-clamp recordings from CRF1+ projection neurons from an AIR mouse (top), a CIE mouse (middle), and a CIE-WD mouse (bottom) during superfusion of GBZ (100 μm). D, Summary of the tonic current revealed by gabazine in CRF1+ projection neurons from AIR (n = 11), CIE (n = 8), and CIE-WD (n = 8) mice. E, Summary of the tonic current revealed by gabazine in CRF1− projection neurons from AIR (n = 8), CIE (n = 7), and CIE-WD (n = 6) mice. *p < 0.05 by one-way ANOVA. Scale bars: 20 μm.

Figure 7.

A, Representative cell-attached recordings from CRF1+ neurons containing microspheres from an AIR mouse (left trace) and from a CIE mouse (right trace). B, Summary of the average baseline firing rate in CRF1+ neurons from AIR mice (n = 8) and CRF1+ neurons from CIE mice (n = 5). *p < 0.05 by unpaired t test. C, Representative cell-attached recordings from CRF1+ neurons containing microspheres from an AIR mouse (left traces) and from a CIE mouse (right traces) before (upper traces) and during (lower traces) superfusion of EtOH (44 mm). D, Summary of the average firing rate before and during EtOH superfusion in CRF1+ neurons from AIR mice (n = 8) and CRF1+ neurons from CIE mice (n = 5). *p < 0.05 by paired t test. E, Summary of the average change in firing rate (percentage of control) following EtOH superfusion in CRF1+ neurons from AIR mice (n = 8) and CRF1+ neurons from CIE mice (n = 5). *p < 0.05 by one-sample t test; ^#p < 0.05 by unpaired t test.

Figure 8.

Proposed schematic of the changes in inhibitory signaling in CRF1+ and CRF1− neurons that make up the CeA microcircuitry. In the CeA of naive mice (left) the majority of inhibitory signaling (phasic and tonic) is on CRF1+ neurons, some of which project out of the CeA into the BNST. CRF1− neurons possess the potential for enhanced tonic signaling that can be stimulated by specific GABA_A receptor agonists or by acute ethanol. After CIE vapor exposure (right), there is a switch in inhibitory control such that the dominant inhibitory drive (phasic and tonic) is on late-spiking CRF1− neurons. The enhanced inhibition on this select population of CRF1− neurons results in a reduction in phasic inhibition in CRF1+ neurons and a subsequent loss of the tonic inhibition that was driven by phasic GABA release onto CRF1+ neurons. The net result of this functional switch in inhibitory control is an increase in the baseline activity of CRF1+ neurons that project out of the CeA into the BNST via disinhibition and the loss of sensitivity of these neurons to the effects of acute ethanol on firing.