Cell type-specific differences in chloride-regulatory mechanisms and GABA(A) receptor-mediated inhibition in rat substantia nigra

Abstract

The regulation of intracellular chloride has important roles in neuronal function, especially by setting the magnitude and direction of the Cl- flux gated by GABA(A) receptors. Previous studies have shown that GABA(A)-mediated inhibition is less effective in dopaminergic than in GABAergic neurons in substantia nigra. We studied whether this phenomenon may be related to a difference in Cl-regulatory mechanisms. Light-microscopic immunocytochemistry revealed that the potassium-chloride cotransporter 2 (KCC2) was localized only in the dendrites of nondopaminergic (primarily GABAergic) neurons in the substantia nigra, whereas the voltage-sensitive chloride channel 2 (ClC-2) was observed only in the dopaminergic neurons of the pars compacta. Electron-microscopic immunogold labeling confirmed that KCC2 is localized in the dendritic plasma membrane of GABAergic neurons close to inhibitory synapses. Confocal microscopy showed that ClC-2 was selectively expressed in the somatic and dendritic cell membranes of the dopaminergic neurons. Gramicidin-perforated-patch recordings revealed that the GABA(A) IPSP reversal potential was significantly less negative and had a much smaller hyperpolarizing driving force in dopaminergic than in GABAergic neurons. The GABA(A) reversal potential was significantly less negative in bicarbonate-free buffer in dopaminergic but not in GABAergic neurons. The present study suggests that KCC2 is responsible for maintaining the low intracellular Cl- concentration in nigral GABAergic neurons, whereas a sodium-dependent anion (Cl--HCO3-) exchanger and ClC-2 are likely to serve this role in dopaminergic neurons. The relatively low efficacy of GABAA-mediated inhibition in nigral dopaminergic neurons compared with nigral GABAergic neurons may be related to their lack of KCC2.

Figure 1.

Light micrographs demonstrate the localization of KCC2 in nondopaminergic cells of the rat SN. A, B, Low (A)- and high (B)-magnification micrographs illustrate the segregation of TH-immunolabeled neurons (brown) and KCC2-immunopositive dendrites (blue-black) in both the SNc (SNC) and SNr (SNR). Both somata and dendrites of the dopaminergic neurons are labeled for TH (arrows). KCC-2 was found only in the dendritic compartment of nondopaminergic cells (arrowheads). C, D, Double-immunofluorescent staining shows a mutually exclusive distribution pattern for TH and KCC2. It demonstrates that none of the TH-positive (green) dendrites or somata are outlined by KCC2 immunoreactivity (red). Scale bars, 25 μm.

Figure 2.

Conventional and epipolarization light micrographs show that, unlike dopaminergic cells, GABAergic neurons express KCC2 protein. A, Conventional light-microscopic image illustrates PV-immunopositive profiles in the rat SNr. PV exclusively labels a subset of GABAergic neurons in the SN. Black arrows indicate PV-immunopositive dendrites, whereas black arrowheads outline a PV-immunoreactive neuron with its labeled dendrite. A′, In the same section, epipolarization light microscopy illustrates KCC2-immunopositive dendrites. White arrowheads indicate a PV-immunopositive cell, of which only the dendrite but not the soma shows KCC2 immunoreactivity. White arrows show the PV-immunolabeled dendrites, which are also KCC2 immunoreactive. B, B′, Another example of conventional and epipolarization microscopy showing localization of KCC2 (B′) to PV⁺, nondopaminergic neurons (B). Scale bars, 20 μm. s, Soma.

Figure 3.

Electron-microscopic localization of KCC2 protein in the rat SN. A, B, Electron microscopy revealed that TH-immunopositive dendrites (DAB labeled) do not contain KCC2 protein (gold particles). Immunoreactivity for TH was found in dopaminergic dendrites, whereas KCC2 was localized in the dendritic membrane of nondopaminergic neurons. KCC2 immunolabeling (arrowheads) was often observed in the vicinity of symmetric synapses (arrows) at the postsynaptic site. Scale bars, 500 nm. b, Bouton; d, dendrite.

Figure 4.

Parvalbumin-immunoreactive neurons express KCC2 in the rat SN. A, B, Electron micrographs taken from two consecutive sections demonstrate the presence of KCC2 (gold particles) in the PV-immunopositive (DAB-labeled) dendrites. Gold particles show that KCC2 is often localized near to the symmetric synapses (arrows). Arrowheads indicate gold particles. Scale bar: (in A) A, B, 500 nm.

Figure 5.

Light-microscopic illustration of the distribution of ClC-2-immunolabeled neurons in the rat SN. A, Low-magnification light micrograph shows the presence of ClC-2-immunopositive cells in the SNc (SNC). ClC-2-immunopositive cells were visualized by immunoperoxidase reaction (DAB; black end product). The SNr (SNR) did not display ClC-2 immunoreactivity. B, High-magnification micrograph demonstrates that ClC-2 is present not only in the perikarya (arrowheads) but also in distal dendrites of the SNc neurons (arrows). Scale bars: A, 100 μm; B, 25 μm.

Figure 6.

Confocal microscopic images of double-immunostained sections show that TH-immunoreactive dopaminergic neurons express ClC-2 in the SNc. A, Immunofluorescence staining against TH visualizes the dopaminergic neurons in the SNc (green). Both perikarya and dendrites display TH immunoreactivity. B, In the same section, ClC-2-immunopositive cells were identified on the basis of the presence of a red fluorescent signal. Similarly, immunolabeling was observed selectively in perikarya and proximal dendrites of dopaminergic neurons. Scale bars, 25 μm.

Figure 7.

Identification of principal dopaminergic and GABAergic neurons in substantia nigra from current-clamp recordings. A1, A2, Dopaminergic neurons exhibit slow pacemaker-like firing with broad action potentials exceeding 2.5 msec in duration and a large-amplitude, long-duration spike afterhyperpolarization. A3, Dopaminergic neurons exhibit a prominent time-dependent sag in the voltage deflection in response to hyperpolarizing current injection because of a slowly activating I_h. A4, I-V plots both before (filled circles) and during (open circles) activation of I_h, calculated at the times indicated by the filled and open arrows in A3 above. The I-V relationship was best fit by linear regression before activation of I_h but showed a pronounced inward rectification after its activation, which was best fit by a fourth-power polynomial. B1, B2, In contrast to dopaminergic neurons, pars reticulata principal GABAergic neurons fire spontaneously at higher rates, have a shorter-duration action potential of ∼1 msec in duration, and do not exhibit a prominent large-amplitude spike afterhyperpolarization. B3, B4, Principal GABAergic neurons lack I_h, and the I-V plot, calculated at the time indicated by the arrow, is best fit by linear regression (B4). In both cases, the hyperpolarizing current was applied from rest. Traces in A3 and B3 are the digital averages of four consecutive single sweeps.

Figure 8.

Pharmacologically isolated GABA_A receptor-mediated IPSPs in response to stimulation of the SNr in vitro. A, Representative current-clamp recordings of a dopaminergic neuron at various membrane potentials show an IPSP in response to stimulation of the SNr. Inset is a plot of the IPSP amplitude versus the membrane potential. The plot of the regression line revealed that the reversal potential of the IPSP was -62.9 mV. B, Representative current-clamp recordings of IPSP in GABAergic SNr neuron shows significantly more hyperpolarized reversal potential of -75.6 mV. C, D, In bicarbonate ion-free slice buffer, the GABA_A receptor-mediated IPSP reversal potential in a representative dopaminergic neuron (C) becomes significantly less hyperpolarized at -49.3 mV, whereas there is no significant change in a nondopaminergic neuron in which the IPSP reverses at -67.7 mV (D). Each of the traces is an average of four sweeps.

Figure 9.

A summary of values for the GABA_A IPSP reversal potential (E_IPSP-A) and the GABA_A IPSP driving force (DF_IPSP-A) in GABAergic and dopaminergic (DA) cells in standard (CTRL) and bicarbonate-free (HEPES) solution. E_IPSP-A is significantly less hyperpolarized in dopaminergic than in GABAergic neurons under standard conditions. In the absence of bicarbonate, the E_IPSP-A is significantly more positive, and the polarity of DF_IPSP-A is reversed in the dopaminergic neurons. In contrast, there are no significant changes in these parameters in the GABAergic neurons.