Evidence that GABA rho subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

Abstract

Ionotropic gamma-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for rho subunit-containing GABA(C) over other GABA receptors. Exogenous application of the GABA(C)-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABA(C) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABA(A) receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABA(A)/GABA(C) pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone-Purkinje cell (IN-PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that rho subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABA(A) alpha1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that rho subunits can form complexes with GABA(A) receptor alpha1 subunits in the cerebellar cortex. Overall, these data suggest that rho subunits contribute to functional ionotropic receptors that mediate a component of phasic inhibitory GABAergic transmission at IN-PC synapses in the cerebellum.

Figure 1. Ionotropic GABA receptors display an unusual pharmacological profile

A, example raw data traces of GABA- and CACA-evoked inward currents. Holding potential (V_H = −70 mV). Inward mIPSCs, due to spontaneous, action potential-independent release of endogenous GABA, are also evident. Note also current desensitization induced by GABA. B, example raw data traces showing reversible TPMPA-induced reduction of GABA- (a) and CACA- (b) evoked current, and summary data (c; mean ± s.e.m. with number of replicants (n) in parentheses). TPMPA produced a larger block of CACA- than GABA-evoked currents (*P < 0.01); V_H = −70 mV. C, example raw data traces showing bicuculline-induced abolition of CACA-evoked current; V_H = −70 mV.

Figure 2. Focal application of ionotropic GABA receptor agonists evoke membrane currents with distinct profiles

A, agonists were focally applied to distinct sites, as approximately indicated by arrows on the left-hand schematic panel, namely directly onto the Purkinje cell (PC) soma (a), ∼30–60 μm away from the soma (b), and at the edge of the inner-third of the molecular layer (ML) (c); the granular layer (GL) is also shown for reference. Representative raw data traces for 500 μm CACA and 500 μm GABA are shown for corresponding positions (a–c); responses were normalized to peak agonist effect at the cell soma. CACA responses typically showed a sharp attenuation in amplitude with increasing distance from the soma, whilst GABA responses showed a far less pronounced attenuation. Note also that CACA-evoked currents had a slower onset and much less desensitization in comparison to those evoked by equimolar GABA. B, summary data for effects of 500 μm CACA (n = 5) and 500 μm GABA (n = 7) showing differences in degree of attenuation of current amplitude with increasing distance from the soma, *P < 0.05, unpaired t test assuming unequal variance.

Figure 3. TPMPA reduces synaptic transmission in Purkinje cells

A, example raw data traces showing the inhibition of mean mIPSC amplitude and frequency by TPMPA; V_H = −70 mV. B, TPMPA-induced reduction in mIPSC amplitude as shown by a pooled cumulative frequency plot (n = 7) for mIPSC amplitude (a; each replicant P < 0.05, KS test), and summary of normalized data (b; *P < 0.01, **P < 0.001, unpaired t test assuming unequal variance). C, TPMPA-induced reduction in mIPSC frequency as shown by a pooled cumulative frequency plot (n = 7 cells) for mIPSC inter-event intervals (a; each replicant P < 0.05, KS test), and summary of normalized data (b; *P < 0.01; **P < 0.001, unpaired t test assuming unequal variance).

Figure 4. TPMPA has both pre- and postsynaptic actions

A, mIPSC amplitude distribution histograms in control (a) and TPMPA-treated cells (b). Values are mean ± s.e.m. with number of replicants (n) in parentheses. Bin width = 2 pA. TPMPA caused a reduction in larger events, but no shift in the peak response. B, 10–90% rise time distribution histograms in control (a) and TPMPA-treated cells (b). Bin width = 0.05 ms. Data are best described by the sum of two Gaussian components fitted to grouped data, shown for clarity in c: sum of two Gaussians fitted to control, TPMPA and wash data. TPMPA reduced the number of events in both fast and slow rise-time populations.

Figure 5. TPMPA has no effect on a NO-711/SNAP-5114-induced tonic GABA current in Purkinje cells

A, example raw data trace of NO-711/SNAP-5114-induced increase in membrane holding current, lack of effect of TPMPA and subsequent block of NO-711/SNAP-5114-induced current by bicuculline methiodide (BMI). Note also the increase in baseline noise induced by NO-711/SNAP-5114 and subsequent reduction in noise by BMI (but not TPMPA); V_H = −70 mV. B, summary data for effects on holding current. V_H = −70 mV, *P < 0.01. C, representative all-point histograms of 500 ms event-free recording under different conditions. NO-711/SNAP-5114 caused a broadening in the distribution of baseline noise (shaded region) compared to control cells; whilst TPMPA was without effect, BMI narrowed the noise distribution. Mean values of single Gaussian fits to distributions from 6 cells were 4.2 ± 0.3 pA (control); 7.8 ± 0.6 pA (NO-711/SNAP-5114; P < 0.01 versus control); 8.7 ± 0.7 pA (TPMPA; P = 0.10 versus NO-711/SNAP-5114) and 4.5 ± 0.5 pA (BMI; P < 0.01 versus TPMPA).

Figure 6. ρ subunit expression in mouse cerebellum, and comparison with α1 subunit expression

Confocal images showing ρ_(1&2) subunit (green; A) and α1 subunit (green) together with calbindin D_28K (red) immunoreactivity (B) in the mouse cerebellar cortex (> 3 weeks). A, strong ρ_(1&2) subunit immunostaining can be observed in the somatic and somatodendritic regions of Purkinje cells (PC) while moderate staining is observed in the molecular layer (ML). B, strong, uniform α1 subunit immunostaining can be observed in the somatic, proximal and distal dendritic regions of PCs and in the ML. The mean normalized fluorescence intensities (NFI) of ρ_(1&2) and α1 subunit immunoreactivity in somatic, proximal dendritic and distal dendritic locations (see arrows) of cerebellar PCs are also shown (n = 4 separate slices each).

Figure 7. ρ subunits co-immunoprecipitate with α1 subunits in cerebellum lysates

Protein complexes precipitated from cerebellum lysates using anti-ρ_(1&2) and probed with the α1 antibody resulted in a band at the molecular weight appropriate to the α1 subunit. No bands were detected when the ρ_(1&2) antibody was omitted. The data are representative of n = 3 separate experiments. IP, immunoprecipitation antibody; IB, immunoblot antibody.