GABAC receptor-mediated inhibition is altered but not eliminated in the superior colliculus of GABAC rho1 knockout mice

Abstract

GABA(C) receptors (GABA(C)Rs) are widely expressed in the mammalian subcortical visual system, particularly in the retina and superior colliculus (SC). GABA(C)Rs are composed of specific rho1-3 subunits the expression of which varies among visual structures. Thus rho1 subunits are most abundant in retina, and their loss eliminates GABA(C)R expression and function. In the SC, rho2 subunit expression may be equal to or stronger than rho1 subunit expression; however, results across studies vary considerably. To more directly assess the expression of GABA(C)R subunits, we characterized inhibition in the SC of wild-type (WT) and GABA(C) rho1 Null mice that lack expression of GABA(C) rho1 subunits. We used whole cell patch-clamp recordings and evaluated GABA(C)R-mediated modulation of electrically evoked post synaptic currents using either agonists or antagonists in WT mice. In GABA(C) rho1 Null stratum griseum superficiale (SGS) cells, inhibitory postsynaptic currents were shorter in duration and their excitatory postsynaptic currents (EPSCs) were longer, indicating that a slow GABA(C)R-mediated inhibitory component was reduced in each case. In contrast to retina, GABA(C)R-mediated currents in the SC were altered but not eliminated in GABA(C) rho1 Null mice. In the majority of SC cells in GABA(C) rho1 Null mice, GABA(C)R activation could still be induced to alter EPSC peak amplitudes in putative interneurons and in many projection neurons. These results, compared with previously published data, indicate a fundamental difference between retina and SC in the control of GABA(C)R expression and subunit composition.

FIG. 1.

Muscimol at low concentration (0.5 μM) selectively activates GABA_C receptors (GABA_CRs) in wild-type (WT) mouse stratum griseum superficiale (SGS) cells. Inhibitory effect of muscimol on postsynaptic currents in 2 WT mouse SGS cells (A, B and C, D). In both cells, bath application of 0.5 μM muscimol reduced excitatory postsynaptic current (EPSC) amplitudes (A and C). Although 10 μM bicuculline, a GABA_AR antagonist, enhanced postsynaptic responses when applied alone, it had no effect on the muscimol induced reduction (B and D), indicating that muscimol at this concentration selectively activates GABA_CRs. Similar muscimol effects were found in 65% of the recorded WT SGS cells. Current traces in this and in the following figures represent responses averaged from five consecutive stimulus applications. Stimulus artifacts have been truncated for clarity.

FIG. 2.

Postsynaptic excitation is enhanced by muscimol in WT mouse SGS cells. In these 3 cells, bath application of 0.5 μM muscimol either increased EPSC amplitudes (A), decreased IPSC amplitudes (B), or both increased EPSC and decreased inhibitory PSC (IPSC) amplitudes (C). Again, 10 μM bicuculline did not alter the effects induced by 0.5 μM muscimol (A).

FIG. 3.

cis-Aminocrotonic acid (CACA) selectively activates GABA_CRs and reduces postsynaptic responses in WT mouse SGS cells. In these 2 cells, 50 μM CACA reduced EPSC amplitudes (A and C) and also IPSC amplitudes (A), which leads to a prolonged EPSC. In both cases, 10 μM bicuculline, which strongly enhanced EPSCs when applied alone, could not block the CACA effects (B and D).

FIG. 4.

GABA_CRs contribute to postsynaptic inhibition in WT mouse SGS cells. Postsynaptic responses, at −30 mV holding potential, to stratum opticum (SO) stimulation in 2 SGS cells in the presence of 5 μM [3-[[(3,4-dichlorphenyl)methyl]amino]propyl](diethoxymethyl) phosphinic acid (CGP 52432), CGP +50 μM 1,2,5,6-tetrahydropyridine-4-yl phosphinic acid (TPMPA), and GCP + TPMPA +20 μM bicuculline to block GABA_ARs, GABA_BRs and GABA_CRs, respectively. While TPMPA application reduced the IPSC amplitudes, a complete block of synaptic inhibition only appeared in the presence of all antagonists.

FIG. 5.

Synaptic responses to SO stimulation of SGS neurons in WT and GABA_CR ρ1 Null mice differ. A: at a holding potential of −50 mV, EPSCs from GABA_CR ρ1 Null cells on average have longer durations than EPSCs from WT cells. B: a complete blockade of GABAergic inhibition (GABAR block), using antagonists to all 3 GABAR types, produces no change in EPSCs durations in WT or GABA_CR ρ1 Null cells. C: at a holding potential of −30 mV, IPSCs from WT cells have longer durations than IPSCs from GABA_CR ρ1 Null cells in control solution. D: antagonism of GABA_CRs in WT cells by application of TPMPA leads to IPSCs that are considerably shorter in duration than IPSCs in GABA_CR ρ1 Null cells. E: when the GABA_CR contribution to the IPSC is eliminated by TPMPA in both WT and GABA_CR ρ1 Null cells, synaptic responses are similar in their temporal profile. Synaptic responses (A–E) were averaged from 6 cells and normalized to equal EPSC or IPSC amplitudes.

FIG. 6.

Functional GABA_CRs are present in GABA_CR ρ1 Null SGS cells. Inhibitory effect of muscimol on postsynaptic currents in 2 GABA_CR ρ1 Null SGS cells. As in WT cells, bath application of 0.5 μM muscimol reduced EPSC amplitudes (A and C), and this reduction was not altered by 10 μM bicuculline (B and D). Similar muscimol effects were observed in 67% of GABA_C ρ1 Null cells.

FIG. 7.

In GABA_CR ρ1 Null, as in WT SGS cells, muscimol enhanced postsynaptic excitation. In the 3 cells shown, bath application of 0.5 μM muscimol either increased EPSC amplitudes (A), decreased IPSC amplitudes (B), or both increased EPSC and decreased IPSC amplitudes (C).

FIG. 8.

Postsynaptic responses are reduced by CACA in GABA_CR ρ1 Null SGS cells. In these 2 cells, 50 μM CACA reduced EPSC amplitudes (A and C) and also IPSC amplitudes (A). In both cases 10 μM bicuculline, which strongly enhances EPSCs when applied alone, could not block the effects of CACA (B and D). This indicates that CACA effects are mediated by GABA_CRs in SGS cells.

FIG. 9.

Quantitative comparison of muscimol-induced reductions of postsynaptic response amplitudes. Average reductions of EPSC and IPSC amplitudes (indicated as percentage of control amplitude) in the presence of either 0.5 μM muscimol or 50 μM CACA alone or of both agonists in the presence of 10 μM bicuculline, were not significantly different between WT and GABA_CR ρ1 Null SGS cells. However, a statistically significant difference for IPSC amplitude reductions was observed in response to bicuculline application. Error bars indicate SEs.

FIG. 10.

Schematic diagram of the proposed synaptic arrangement in SGS. In SGS, both projection neurons (PN) and local interneurons (IN) receive excitatory input from retinal afferents mediated by ionotropic GluRs. Interneurons release GABA onto projection neurons where it acts mainly through GABA_ARs (red) but also through GABA_CRs (yellow). GABA_CRs also serve as autoreceptors in GABAergic interneurons. In addition to excitatory input, interneurons also receive inhibitory input through GABA_ARs and GABA_CRs.