Novel subunit-specific tonic GABA currents and differential effects of ethanol in the central amygdala of CRF receptor-1 reporter mice

Abstract

The central nucleus of the amygdala (CeA) is an important integrative site for the reinforcing effects of drugs of abuse, such as ethanol. Activation of corticotropin-releasing factor type 1 (CRF1) receptors in the CeA plays a critical role in the development of ethanol dependence, but these neurons remain uncharacterized. Using CRF1:GFP reporter mice and a combined electrophysiological/immunohistochemical approach, we found that CRF1 neurons exhibit an α1 GABA(A) receptor subunit-mediated tonic conductance that is driven by action potential-dependent GABA release. In contrast, unlabeled CeA neurons displayed a δ subunit-mediated tonic conductance that is enhanced by ethanol. Ethanol increased the firing discharge of CRF1 neurons and decreased the firing discharge of unlabeled CeA neurons. Retrograde tracing studies indicate that CeA CRF1 neurons project into the bed nucleus of the stria terminalis. Together, these data demonstrate subunit-specific tonic signaling and provide mechanistic insight into the specific effects of ethanol on CeA microcircuitry.

Figure 1.

A, A 10× magnification photomicrograph of a coronal CeA slice indicating recording sites of CRF1⁺ neurons (*) and anatomical location (bregma −1.46 mm). Scale bar, 100 μm. B, A 60× magnification of a CRF1⁺ CeA neuron using fluorescent optics (left) and IR-DIC optics (right). Scale bar, 20 μm. C, A 25× magnification photomicrograph of a coronal CeA slice illustrating GFP expression in the CeA of a CRF1:GFP reporter mouse. Scale bar, 50 μm. D, Representative current-clamp recording illustrating spike characteristics of a low-threshold bursting CeA neuron (top trace), a late-spiking CeA neuron (middle trace), and a regular spiking CeA neuron (bottom trace) and the proportion of CRF1⁺ and CRF1⁻ neurons in each cell type. E, Representative voltage-clamp recording of sIPSCs and scaled average sIPSC (inset) from a CRF1⁺ neuron (top trace) and a CRF1⁻ neuron (bottom trace). F, Summary of membrane characteristics; *p < 0.05 comparing CRF1⁺ to CRF1⁻ by unpaired t test, n = 27 (CRF1⁺) and n = 21 (CRF1⁻). G, A biocytin-filled CRF1⁺ CeA neuron at 10× magnification (left), 25× magnification (middle), and a 60× magnification showing the primary dendrite indicated by the white box in the middle (right). Scale bars: left, 100 μm; middle, 50 μm; right, 5 μm. D, Dorsal; L, lateral; M, medial; V, ventral.

Figure 2.

A, Representative voltage-clamp recordings from a CRF1⁺ neuron during superfusion of GBZ (100 μm), followed by PTX (100 μm, top trace) and during superfusion of PTX followed by GBZ (bottom trace). Dashed lines indicate average holding current. B, Summary of the tonic current in CRF1⁺ neurons revealed by superfusion of GBZ (n = 10) and PTX (n = 9) and occlusion of tonic current by previous superfusion of either GBZ (n = 5) or PTX (n = 5); *p < 0.05 by one-sample t test, ^#p < 0.05 by unpaired t test. C, Representative voltage-clamp recordings from a CRF1⁻ neuron during superfusion of GBZ (100 μm, top trace) and during superfusion of nipecotic acid (NIP, 1 mm) followed by GBZ (bottom trace). Dashed lines indicate average holding current. D, Summary of the tonic current in CRF1⁻ neurons revealed by superfusion of GBZ alone (n = 7) and by superfusion of GBZ in the presence of NIP (n = 6). *p < 0.05 by one-sample t test, ^#p < 0.05 by unpaired t test.

Figure 3.

A, Representative voltage-clamp recordings from a CRF1⁺ neuron (top trace) and a CRF1⁻ neuron (bottom trace) during superfusion of TTX (1 μm). Dashed lines indicate average holding current. B, Summary of the tonic current revealed by TTX superfusion in CRF1⁺ neurons (n = 17) and CRF1⁻ neurons (n = 10); *p < 0.05 by one-sample t test, ^#p < 0.05 by unpaired t test. C, Summary of the change in IPSC frequency (% of Control) in CRF1⁺ neurons (n = 17) and CRF1⁻ neurons (n = 10); *p < 0.05 by unpaired t test, ^#p < 0.05 by unpaired t test. D, Summary of the correlation between the change in IPSC frequency and the magnitude of tonic current after TTX superfusion in CRF1⁺ neurons; slope = 9.0 ± 2.8, intercept = 5.1 ± 8.2, R² = 0.4329, p < 0.05, n = 16. E, Summary of the tonic current in CRF1⁺ neurons revealed by GBZ superfusion alone and compared with GBZ after TTX superfusion (n = 7); *p < 0.05 by one-sample t test, ^#p < 0.05 by unpaired t test.

Figure 4.

A, Representative voltage-clamp recordings from a CRF1⁺ neuron during focal application of 2 μm THIP (top trace) and 5 μm THIP (bottom trace). B, Representative voltage-clamp recordings from a CRF1⁻ neuron before and after focal application of 2 μm THIP (top trace) and 5 μm THIP (bottom trace). C, Summary of the tonic current elicited by THIP (1–10 μm) in CRF1⁺ and CRF1⁻ neurons; *p < 0.05 by unpaired t test, n = 4, 6, 8, and 3 (CRF1⁺) and n = 3, 5, 5, and 3 (CRF1⁻) for 1, 2, 5, and 10 μm THIP, respectively. D, Representative voltage-clamp recordings from a CRF1⁺ neuron (left trace) and a CRF1⁻ neuron (right trace) during focal application of EtOH (44 mm). E, Summary of the tonic current stimulated by EtOH (44 mm); *p < 0.05 by unpaired t test, n = 5 (CRF1⁺) and n = 5 (CRF1⁻). F, Summary of the change in sIPSC frequency and amplitude produced by EtOH (44 mm); *p < 0.05 by one-sample t test, n = 5 (CRF1⁺) and n = 5 (CRF1⁻). G, Photomicrograph (60×) of GFP expression (green fluorescence) and total number of CeA neurons that displayed GFP expression (119). Arrow indicates a GFP⁺, CRF1-containing neuron. H, Photomicrograph (60×) of δ GABA_A receptor subunit expression (red punctate fluorescence) and total number of CeA neurons that displayed δ subunit expression (112). Arrow indicates a CRF1⁻ neuron expressing the δ subunit. I, Photomicrograph (60×) of merged GFP and δ subunit expression (green plus red fluorescence) and total number of CeA neurons that displayed both GFP and δ subunit expression (27). Arrows indicate the same CRF1⁺ and CRF1⁻ neurons as in G and H. Box indicates a CRF1⁺ neuron that expresses the δ subunit. Scale bar, 20 μm.

Figure 5.

A, Representative voltage-clamp recordings from a CRF1⁺ neuron (top trace) and a CRF1⁻ neuron (bottom trace) during superfusion of zolpidem (100 nm) followed by bicuculline (60 μm). Dashed lines indicate average holding current. B, Summary of the tonic current produced by zolpidem superfusion in CRF1⁺ neurons (n = 6) and CRF1⁻ neurons (n = 6); *p < 0.05 by unpaired t test. C, Summary of the tonic current inhibited by subsequent bicuculline superfusion in CRF1⁺ (n = 6) and CRF1⁻ neurons (n = 6); *p < 0.05 by unpaired t test. D, Photomicrograph (60×) of GFP expression (green fluorescence) and total number of CeA neurons quantified as displaying GFP expression (121). Arrows indicate CRF1⁺ neurons (top and middle arrows) and a CRF1⁻ neuron (bottom arrow). E, Photomicrograph (60×) of α1 GABA_A receptor subunit expression (red fluorescence) and total number of CeA neurons that displayed α1 subunit expression (111). Arrows are the same as in D showing α1 subunit expression (top and bottom arrow) and lack of α1 subunit expression (middle arrow). F, Photomicrograph (60×) of merged GFP and α1 subunit expression (green and red fluorescence) and total number of CeA neurons that displayed both GFP and α1 subunit expression (94). Arrows are the same as in D and E. Scale bar, 20 μm.

Figure 6.

A, Representative cell-attached recording from a CRF1⁺ neuron before (left trace) and during (right trace) superfusion of EtOH in physiological conditions (EtOH 44 mm, aCSF only). B, Representative cell-attached recording from a CRF1⁻ neuron before (left trace) and during (right trace) superfusion of EtOH in physiological conditions (EtOH 44 mm, aCSF only). C, Summary of the change in event frequency (% of Control) in CRF1⁺ neurons (n = 6) and CRF⁻ neurons (n = 6) before and during EtOH superfusion in physiological condition (aCSF only); *p < 0.05 by one-sample t test, ^#p < 0.05 by unpaired t test. D, Representative cell-attached recording from a CRF1⁺ neuron before (left trace) and during (right trace) superfusion of EtOH with pharmacological isolation of GABA_A receptor transmission (DNQX, AP-5, CGP). E, Representative cell-attached recording from a CRF1⁻ neuron before (left trace) and during (right trace) superfusion of EtOH with pharmacological isolation of GABA_A receptor transmission (DNQX, AP-5, CGP). F, Summary of the change in event frequency (% of Control) in CRF1⁺ neurons (n = 5) and CRF⁻ neurons (n = 5) before and during EtOH superfusion with pharmacological isolation of GABA_A receptor transmission (DNQX, AP-5, CGP); *p < 0.05 by one-sample t test, ^#p < 0.05 by unpaired t test.

Figure 7.

A, Representative cell-attached recording from a CRF1⁺ neuron in control conditions (left trace), during superfusion of GBZ (100 μm; middle trace), and during superfusion of EtOH in the presence of gabazine (GBZ + EtOH, right trace). All recordings were performed with pharmacological isolation of GABA_A receptor transmission (DNQX, AP-5, CGP). B, Representative cell-attached recording from a CRF1⁻ neuron in control conditions (left trace), during superfusion of GBZ (100 μm; middle trace), and during superfusion of EtOH in the presence of gabazine (GBZ + EtOH, right trace). All recordings were performed with pharmacological isolation of GABA_A receptor transmission (DNQX, AP-5, CGP). C, Summary of the change in event frequency (% of Control) in CRF1⁺ neurons (n = 5) and CRF1⁻ neurons (n = 5) before and during GBZ (100 μm) superfusion. *p < 0.05 by one-sample t test, ^#p < 0.05 by unpaired t test. D, Summary of the change in event frequency (% of Control) in CRF1⁺ neurons (n = 5) and CRF1⁻ neurons (n = 5) before and during superfusion of EtOH in the presence of GBZ (GBZ + EtOH). *p < 0.05 by one-sample t test, ^#p < 0.05 by unpaired t test.

Figure 8.

A, Photomicrograph (10×) of a CeA slice during a paired recording. B, Photomicrographs (60×) of synaptically coupled CRF1⁻ (left) and CRF1⁺ (right) CeA neurons. C, Representative traces of an action potential elicited from a CRF1⁻ neuron (top trace) and superimposed (gray traces) and averaged (black trace) IPSCs from a synaptically coupled CRF1⁺ neuron (bottom trace). D, Representative traces of an action potential elicited from a CRF1⁻ neuron (top trace) and superimposed (gray traces) and averaged (black trace) IPSCs from a synaptically coupled CRF1⁺ neuron (bottom trace) recorded using a low Ca²⁺ buffered intracellular solution.

Figure 9.

A, Photomicrograph (20×) of microsphere injection site into the dlBNST dorsal and lateral to the anterior commissure (ac). Scale bar, 100 μm. B, Photomicrograph (25×) of retrogradely transported red microspheres (red punctate fluorescence) and CRF1⁺ neurons (green fluorescence) in the CeA of the same brain shown in A. Scale bar, 50 μm. C, Photomicrograph (180×) of the CRF1⁺ CeA neuron with red microsphere labeling indicated by the white box in B. Scale bar, 10 μm. D, Photomicrograph of the microsphere injection site into the dlBNST (left). Scale bar, 100 μm. Photomicrographs (60×) of a CRF1⁺ CeA neuron from the same brain in IR-DIC (top right), red fluorescence (middle right), and green fluorescence (bottom right). Scale bar, 10 μm. E, Photomicrograph of the microsphere injection site into the dlBNST (left). Scale bar, 100 μm. Photomicrographs (60×) of a CRF1⁻ CeA neuron from the same brain in IR-DIC (top right) and red fluorescence (bottom right). Scale bar, 10 μm. F, Representative cell-attached recording from a CRF1⁺ neuron containing microspheres before (left trace) and during (right trace) superfusion of EtOH with pharmacological isolation of GABA_A receptor transmission (EtOH 44 mm; DNQX, AP-5, CGP). G, Summary of the event frequency in CRF1⁺ neurons containing microspheres during control and during EtOH superfusion. *p < 0.05 by unpaired t test. H, Representative cell-attached recording from a CRF1⁻ neuron containing microspheres before (left trace) and during (right trace) superfusion of EtOH with pharmacological isolation of GABA_A receptor transmission (EtOH 44 mm; DNQX, AP-5, CGP). I, Summary of the event frequency in CRF1⁻ neurons containing microspheres during control and during EtOH superfusion. *p < 0.05 by unpaired t test.

Figure 10.

Proposed microcircuitry of the CeA illustrating local phasic and tonic GABA_A receptor transmission in CRF1⁺ and CRF1⁻ neurons. Phasic GABA_A receptor transmission is depicted on both CRF1⁺ and CRF1⁻ neurons, although CRF1⁺ neurons are depicted with more baseline GABA activity. The CRF1⁺ neuron possesses α1 subunit-containing tonic GABA_A receptors located in or near the synaptic cleft to reflect the dependence on action-potential-dependent GABA release and projects out of the CeA into the BNST. The CRF1⁻ neuron possesses δ subunit-containing GABA_A receptors located outside the synaptic cleft to reflect activation by ambient GABA. We hypothesize that EtOH acts on this CeA circuitry by enhancing tonic conductance in CRF1⁻ neurons through actions at δ-containing GABA_A receptors, resulting in decreased firing of this subpopulation. The ongoing tonic inhibition of CRF1⁺ neurons is subsequently diminished, resulting in disinhibition and increased firing in this subpopulation. This increased firing then results in increased GABA release in target brain regions, such as the BNST.