Presynaptic GABA(B) receptors decrease neurotransmitter release in vestibular nuclei neurons during vestibular compensation

Abstract

Unilateral damage to the peripheral vestibular receptors precipitates a debilitating syndrome of oculomotor and balance deficits at rest, which extensively normalize during the first week after the lesion due to vestibular compensation. In vivo studies suggest that GABA(B) receptor activation facilitates recovery. However, the presynaptic or postsynaptic sites of action of GABA(B) receptors in vestibular nuclei neurons after lesions have not been determined. Accordingly, here presynaptic and postsynaptic GABA(B) receptor activity in principal cells of the tangential nucleus, a major avian vestibular nucleus, was investigated using patch-clamp recordings correlated with immunolabeling and confocal imaging of the GABA(B) receptor subunit-2 (GABA(B)R2) in controls and operated chickens shortly after unilateral vestibular ganglionectomy (UVG). Baclofen, a GABA(B) agonist, generated no postsynaptic currents in principal cells in controls, which correlated with weak GABA(B)R2 immunolabeling on principal cell surfaces. However, baclofen decreased miniature excitatory postsynaptic current (mEPSC) and GABAergic miniature inhibitory postsynaptic current (mIPSC) events in principal cells in controls, compensating and uncompensated chickens three days after UVG, indicating the presence of functional GABA(B) receptors on presynaptic terminals. Baclofen decreased GABAergic mIPSC frequency to the greatest extent in principal cells on the intact side of compensating chickens, with concurrent increases in GABA(B)R2 pixel brightness and percentage overlap in synaptotagmin 2-labeled terminals. In uncompensated chickens, baclofen decreased mEPSC frequency to the greatest extent in principal cells on the intact side, with concurrent increases in GABA(B)R2 pixel brightness and percentage overlap in synaptotagmin 1-labeled terminals. Altogether, these results revealed changes in presynaptic GABA(B) receptor function and expression which differed in compensating and uncompensated chickens shortly after UVG. This work supports an important role for GABA(B) autoreceptor-mediated inhibition in vestibular nuclei neurons on the intact side during early stages of vestibular compensation, and a role for GABA(B) heteroreceptor-mediated inhibition of glutamatergic terminals on the intact side in the failure to recover function.

Fig.1

Confocal images of positive and negative controls for antibody specificity performed on chicken cerebellum (A-D) and/or the tangential nucleus (E). A: Confocal tile image of positive control for guinea pig anti-GABA_BR2 primary antibody showing positive immunolabeling for cerebellar Purkinje cell bodies (arrow) and dendrites in the molecular layer (M). Scale bar, 20 μm. B_1: Positive control for primary antibody to Syt1 which labeled many terminals in the cerebellar molecular layer (M). B₂: On plotting the line scan from B_1, regions of increased pixel brightness were detected along the line, representing high signal/noise ratio that corresponded to Syt1-labeled terminals. Length of line scan in B_1, 200 μm. C_1: Positive control for primary antibody to Syt2 which labeled many terminals surrounding the cerebellar Purkinje cell bodies (arrow). C₂: On plotting the line scan from C1 regions of increased pixel brightness were observed along the line, representing high signal/noise ratio that corresponded to Syt2-labeled terminals. Length of line scan in C1 250 μm. D: Negative control for GABA_BR2 immunolabeling was performed by omitting guinea pig anti-GABA_BR2 primary antibody. D_1: Confocal image of cerebellar Purkinje cells showed no GABA_BR2 signal when captured using the same parameters applied to take experimental images in the study. D₂: Confocal image brightness was boosted so that a line could be drawn through the Purkinje cell bodies. D3: Line scan through the Purkinje cell bodies in D₂ indicated low GABA_BR2 signal/noise ratio. Length of the line scan in D₁ and D₂ was 200 μm. E: Confocal image demonstrating a cross-over control experiment in which guinea pig anti-GABA_BR2 primary antibody was reacted with secondary anti-mouse IgG, used here to reveal Syt1, Syt2, and MAP2. No GABA_BR2 immunolabeling of principal cell bodies in the tangential nucleus was detected. E₁: Image of a principal cell body lacking GABA_BR2 signal when captured using the same parameters to take experimental images in this study. E₂: Image brightness was boosted to detect the principal cell body so that a line could be drawn through the soma. E₃: Line scan through the principal cell body in E₂ revealed low signal/noise ratio for GABA_BR2 immunolabeling. Length of the line scans in E₁ and E₂ was 100 μm.

Fig. 2

A: Recording from a principal cell in H7 control during exposure to 10 μM baclofen for 90 seconds at a holding potential of −65 mV. No postsynaptic current was detected. B-J: High power (63X) confocal images of MAP2 (B, E, H) and GABA_BR2 (C, F, I) immunolabeling in principal cell bodies in the tangential nucleus from controls (B-D), lesion (E-G) and intact (H-J) sides of compensating chickens three days after UVG. Note the increased GABA_BR2 immunolabeling in MAP2-positive principal cell bodies on the intact side of compensating chickens (I, J) compared to controls (C, D). K: Mean pixel brightness of GABA_BR2 immunolabeling (gray scale 0-255) increased significantly in the principal cell body cytoplasm on the intact side of compensating chickens compared to controls. L: Mean pixel brightness of GABA_BR2 immunolabeling was weak in line scans drawn on the cell surface compared to that in the cytoplasm of MAP2-positive principal cell bodies from controls, and did not change in compensating chickens. K, L: Using two different approaches, GABA_BR2 immunolabeling increased in the principal cell body cytoplasm on the intact side of compensating chickens relative to controls (see Experimental Procedures). Scale bar (J), 20 μm.

Fig. 3

Decreased mEPSC frequency on exposure to 10 μM baclofen in principal cells from H7 controls (A) and on the intact side of uncompensated chickens three days after UVG (B). A₁ and A₂, and B₁ and B₂ were taken from the same cells, respectively. C: After baclofen exposure, mEPSC frequency decreased in principal cells in all experimental groups. D: After baclofen exposure, the percentage decrease in mEPSC frequency was significantly greater in principal cells on the intact side of uncompensated chickens compared to controls and the lesion side of compensating and uncompensated chickens three days after UVG.

Fig. 4

Decreased GABAergic mIPSC frequency on exposure to 10 μM baclofen in principal cells on the lesion (A) and intact (B) sides of compensating chickens three days after UVG. A₁ and A₂, B₁ and B₂ were taken from the same cells, respectively. C: After baclofen exposure, GABAergic mIPSC frequency decreased in principal cells in all experimental groups. D: After baclofen exposure, the percentage decrease in GABAergic mIPSC frequency was significantly greater n principal cells on the intact side of compensating chickens compared to the lesion side

Fig. 5

Decreased glycinergic mIPSC frequency on exposure to 10 μM baclofen in principal cells from H7 controls (A): A₁ and A₂ were taken from the same cell. B: After baclofen exposure, glycinergic mIPSC frequency decreased in principal cells from all experimental groups. C: After baclofen exposure, glycinergic mIPSC frequency decreased significantly only in principal cells on the intact side one and three days after UVG. D: After baclofen exposure, the percentage decrease in glycinergic mIPSC frequency was similar in principal cells from all experimental groups.

Fig. 6

(A-O) High power (63X) confocal images of Syt1 (A, D, G, J, M) and GABA_BR₂ (B, E, H, K, N) immunolabeling in the tangential nucleus from control (A-C), compensating (D-I) and uncompensated chickens (J-O) three days after UVG. Note the decrease in Syt1-labeled terminals on the lesion (J, L) and intact side (M, O) of uncompensated chickens. P, Q: enlarged images of the tangential nucleus from boxed-region in C and O, respectively. Arrows indicate overlap of Syt-1 and GABA_BR2. R: Mean Syt1-labeled terminal area in the tangential nucleus decreased significantly on both the lesion and intact side in uncompensated chickens. S: Mean GABA_BR₂ pixel brightness in Syt1-positive terminals of the tangential nucleus increased significantly on the lesion and intact sides from uncompensated chickens compared to controls and compensating chickens. T: Percentage overlap of Syt1-labeled terminal area and GABA_BR₂ increased significantly on the intact side in uncompensated chickens three days after UVG compared to controls. Scale bar in A-O: 30 μm. Scale bar in P and Q: 10 μm.

Fig. 7

(A-O) High power (63X) confocal images of Syt2 (A, D, G, J, M) and GABA_BR₂ (B, E, H, K, N) immunolabeling in the tangential nucleus from control (A-C), compensating (D-I) and uncompensated chickens (J-O) three days after UVG. P, Q: Enlarged images of the tangential nucleus from the boxed-in regions in C and O, respectively. Arrows point to the overlap of Syt-1 and GABA_BR2. R: Mean Syt2-labeled terminal area in the tangential nucleus decreased significantly in uncompensated chickens. S: Mean pixel brightness of GABA_BR₂ immunolabeling in Syt2-positive terminals in the tangential nucleus increased on the intact side of compensating chickens, and both the lesion and intact sides in uncompensated chickens. T: Percentage overlap of Syt2-labeled terminal area and GABA_BR₂ decreased significantly on the lesion side in compensating chickens compared to the intact side and controls. Scale bar in A-O: 30 μm. Scale bar in P and Q: 10 μm.