Cell-type-specific tonic GABA signaling in the rat central amygdala is selectively altered by acute and chronic ethanol

Abstract

The central nucleus of the amygdala (CeA) is an important site for the reinforcing effects of ethanol and has been implicated in the development of alcohol dependence. The CeA GABAA receptor system is particularly vulnerable to the effects of acute and chronic ethanol exposure. Previous work in the CeA focused on ethanol and phasic GABAA receptor signaling, but tonic GABAA receptor signaling in the rat CeA remains understudied. In the present study, we found that the CeA contains two types of tonic conductance that are expressed in a cell-type-specific manner. Low threshold bursting (LTB) and some regular spiking (RS) neurons have an ongoing tonic conductance that is mediated by the α1-GABAA receptor subunit and is insensitive to acute ethanol exposure. Late spiking (LS) and a separate population of RS neurons do not display a persistent tonic conductance but have the potential for tonic signaling that is mediated by the δ-GABAA receptor subunit and can be activated by increasing the ambient GABA concentration or by acute ethanol exposure. Acute ethanol exposure differentially alters the firing discharge of different CeA cell types. Chronic ethanol exposure produces a switch in tonic signaling such that the tonic conductance in LTB and some RS neurons is lost and an ongoing tonic conductance is present in LS and a separate population of RS neurons. Collectively, these data demonstrate cell-type-specific tonic signaling in the CeA and provide new insight into how acute and chronic ethanol exposure differentially alter specific aspects of inhibitory circuitry in the CeA.

Keywords: Alcohol; GABAA; delta.

Figure 1

A. Representative voltage-clamp recordings from a Low Threshold Bursting (LTB, left panel) and Regular Spiking (RS, right panel) CeA neuron during focal application of gabazine (GBZ, 100 μM). Dashed lines indicate average holding current. B. Representative voltage-clamp recordings from a Late Spiking (LS, left panel) and Regular Spiking (RS, right panel) CeA neuron during superfusion of gabazine (GBZ, 100 μM). Dashed lines indicate average holding current. C. Summary of the tonic current by cell type in CeA neurons revealed by focal application of GBZ [n = 13 (LTB); n = 5 (RS); n = 6 (LS); n = 7 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. D. Summary of the tonic current by cell type in CeA neurons revealed by superfusion of tetrodotoxin [TTX, 1 μM; n = 6 (LTB); n = 7 (RS); n = 6 (LS); n = 11 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison.

Figure 2

A. Representative voltage-clamp recordings from a Low Threshold Bursting (LTB, left panel) and Regular Spiking (RS, right panel) CeA neuron during focal application of zolpidem (Zolp, 100 nM) followed by focal application of bicuclline (BIC, 60 μM). Dashed lines indicate average holding current. B. Representative voltage-clamp recordings from a Late Spiking (LS, left panel) and Regular Spiking (RS, right panel) CeA neuron during focal application of zolpidem (Zolp, 100 nM) followed by focal application of bicuclline (BIC, 60 μM). Dashed lines indicate average holding current. C. Summary of the tonic current by cell type in CeA neurons revealed by focal application of zolpidem [n = 6 (LTB); n = 5 (RS); n = 5 (LS); n = 5 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. D. Summary of the tonic current by cell type in CeA neurons revealed by focal application of bicuculline after zolpidem [n = 6 (LTB); n = 5 (RS); n = 5 (LS); n = 5 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. E. Photomicrograph (60x) of α1 GABA_A receptor subunit expression (red fluorescence) and DAPI expression (blue fluorescence) in the CeA. Scale bar = 20 μm.

Figure 3

A. Representative voltage-clamp recordings from Low Threshold Bursting (LTB, left panel) and Regular Spiking (RS, right panel) CeA neurons during focal application of THIP (1 μm, top panel; 2 μm, middle panel; 5 μm, lower panel). B. Representative voltage-clamp recordings from Late Spiking (LS, left panel) and Regular Spiking (RS, right panel) CeA neurons during focal application of THIP (1 μm, top panel; 2 μm, middle panel; 5 μm, lower panel). C. Summary of the tonic current by cell type in CeA neurons revealed by focal application of 1 μm THIP [n = 6 (LTB); n = 4 (RS); n = 6 (LS); n = 5 (RS)]; 2 μm THIP [n = 6 (LTB); n = 6 (RS); n = 7 (LS); n = 6 (RS)]; 5 μm THIP [n = 5 (LTB); n = 7 (RS); n = 7 (LS); n = 8 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. D. Photomicrograph (60x) of δ GABA_A receptor subunit expression (red fluorescence) and DAPI expression (blue fluorescence) in the CeA. Scale bar = 20 μm.

Figure 4

A. Representative voltage-clamp recordings from a Low Threshold Bursting (LTB, left panel) and Regular Spiking (RS, right panel) CeA neuron during superfusion of nipecotic acid (NIP, 1 mM) followed by focal application of gabazine (GBZ, 100 μM). Dashed lines indicate average holding current. B. Representative voltage-clamp recordings from a Late Spiking (LS, left panel) and Regular Spiking (RS, right panel) CeA neuron during superfusion of nipecotic acid (NIP, 1 mM) followed by focal application of gabazine (GBZ, 100 μM). Dashed lines indicate average holding current. C. Summary of the tonic current by cell type in CeA neurons revealed by focal application of nipecotic acid [n = 7 (LTB); n = 5 (RS); n = 6 (LS); n = 5 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. D. Summary of the tonic current by cell type in CeA neurons revealed by focal application of gabazine after nipecotic acid [n = 7 (LTB); n = 5 (RS); n = 6 (LS); n = 5 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. E. Summary of the correlation between the magnitude of tonic current stimulated by focal application of THIP (5 μM) and the magnitude of tonic current produced by superfusion of nipecotic acid in Regular Spiking (RS) CeA neurons; slope = 2.7 ± 0.4, R² = 0.62, p<0.01, n = 10. F. Summary of the correlation between the magnitude of tonic current stimulated by focal application of THIP (5 μM) and the magnitude of tonic current blocked by focal application of gabazine in Regular Spiking (RS) CeA neurons; slope = 2.2 ± 0.2, R² = 0.78, p<0.001, n = 10.

Figure 5

A. Representative voltage-clamp recordings from a Low Threshold Bursting (LTB, left panel) and Regular Spiking (RS, right panel) CeA neuron during focal application of ethanol (EtOH, 44 mM). B. Representative voltage-clamp recordings from a Late Spiking (LS, left panel) and Regular Spiking (RS, right panel) CeA neuron during focal application of ethanol (EtOH, 44 mM). C. Summary of the tonic current by cell type in CeA neurons produced by focal application of ethanol [n = 6 (LTB); n = 6 (RS); n = 6 (LS); n = 5 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. D. Summary of the change in sIPSC frequency by cell type in CeA neurons produced by focal application of ethanol [n = 6 (LTB); n = 6 (RS); n = 6 (LS); n = 5 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison.

Figure 6

A. Representative cell-attached recording from a Low Threshold Bursting (LTB, left panels) and a Regular Spiking (RS, right panels) CeA neuron before (left trace) and during (right trace) superfusion of ethanol (EtOH 44 mM). B. Representative cell-attached recording from a Late Spiking (LS, left panels) and a Regular Spiking (RS, right panels) CeA neuron before (left trace) and during (right trace) superfusion of ethanol (EtOH 44 mM). C. Summary of the change in event frequency (% of Control) by cell type in CeA neurons produced by superfusion of ethanol [n = 6 (LTB); n = 4 (RS); n = 3 (LS); n = 6 (RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison.

Figure 7

A. Representative voltage-clamp recording of a Low Threshold Bursting (LTB) CeA neuron from a chronic intermittent ethanol (CIE) exposed rat during focal application of gabazine (GBZ, 100 μM). Dashed lines indicate average holding current. B. Representative voltage-clamp recording of a Late Spiking (LS) CeA neuron from a CIE exposed rat during focal application of gabazine (GBZ, 100 μM). Dashed lines indicate average holding current. C. Summary of the tonic current in LTB and RS CeA neurons from naïve and CIE exposed rats revealed by focal application of GBZ [n = 13 (naïve LTB), n = 10 (CIE LTB); n = 5 (naïve RS), n = 5 (CIE RS); n = 6 (naïve LS), n = 5 (CIE LS); n = 7 (naïve RS), n = 6 (CIE RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. D. Summary of the tonic current by cell type in CeA neurons from naïve and CIE exposed rats revealed by superfusion of tetrodotoxin [TTX, 1 μM; n = 6 (naïve LTB), n = 6 (CIE LTB); n = 7 (naïve RS), n = 4 (CIE RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. E. Summary of the tonic current by cell type in CeA neurons from naïve and CIE exposed rats revealed by focal application of 5 μm THIP [n = 5 (naïve LTB), n = 12 (CIE LTB); n = 7 (naïve RS), n = 9 (CIE RS); n = 7 (naïve LS), n = 6 (CIE LS); n = 9 (naïve RS), n = 6 (CIE RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison. F. Summary of the tonic current by cell type in CeA neurons from naïve and CIE exposed rats produced by focal application of ethanol (EtOH 44 nM) [n = 6 (naïve LTB), n = 12 (CIE LTB); n = 6 (naïve RS), n = 8 (CIE RS); n = 6 (naïve LS), n = 4 (CIE LS); n = 5 (naïve RS), n= 6 (CIE RS)] *p<0.05 by one-way ANOVA with Bonferroni post hoc comparison.

Figure 8

Schematic illustrating local cell type-specific phasic and tonic GABA_A receptor transmission in CeA neurons from naïve (left panel) and chronic intermittent ethanol (CIE, right panel) rats. In naïve rats (left panel), Low Threshold Bursting (LTB) and some Regular Spiking (RS) CeA neurons possess an ongoing tonic conductance that is mediated by α1 subunit-containing tonic GABA_A receptors located in or near the synaptic cleft. Late Spiking (LS) and a separate population of RS neurons do not possess an ongoing tonic conductance but do possess the potential for tonic conductance that is mediated by extrasynaptic δ subunit-containing tonic GABA_A receptors that can be stimulated by elevations in ambient GABA or acute ethanol. Dashed lines indicate a proposed synaptic connection between LS/RS and LTB/RS neurons that may explain the differential effects of acute ethanol on CeA neuron firing. In CIE rats (right panel), the tonic conductance observed in LTB/RS CeA neurons from naïve rats is lost resulting in disinhibition and increased synaptic output (GABA release). In contrast, an ongoing tonic conductance is now present in LS/RS CeA neurons resulting in decreased synaptic output (GABA release).