In the adult hippocampus, chronic nerve growth factor deprivation shifts GABAergic signaling from the hyperpolarizing to the depolarizing direction

Abstract

GABA, the main inhibitory transmitter in adulthood, early in postnatal development exerts a depolarizing and excitatory action. This effect, which results from a high intracellular chloride concentration ([Cl(-)](i)), promotes neuronal growth and synaptogenesis. During the second postnatal week, the developmental regulated expression of the cation-chloride cotransporter KCC2 accounts for the shift of GABA from the depolarizing to the hyperpolarizing direction. Changes in chloride homeostasis associated with high [Cl(-)](i) have been found in several neurological disorders, including temporal lobe epilepsy. Here, we report that, in adult transgenic mice engineered to express recombinant neutralizing anti-nerve growth factor antibodies (AD11 mice), GABA became depolarizing and excitatory. AD11 mice exhibit a severe deficit of the cholinergic function associated with an age-dependent progressive neurodegenerative pathology resembling that observed in Alzheimer patients. Thus, in hippocampal slices obtained from 6-month-old AD11 (but not wild-type) mice, the GABA(A) agonist isoguvacine significantly increased the firing of CA1 principal cells and, at the network level, the frequency of multiunit activity recorded with extracellular electrodes. In addition, in AD11 mice, the reversal of GABA(A)-mediated postsynaptic currents and of GABA-evoked single-channel currents were positive with respect to the resting membrane potential as estimated in perforated patch and cell attached recordings, respectively. Real-time quantitative reverse transcription-PCR and immunocytochemical experiments revealed a reduced expression of mRNA encoding for Kcc2 and of the respective protein. This novel mechanism may represent a homeostatic response that counterbalances within the hippocampal network the Alzheimer-like neurodegenerative pathology found in AD11 mice.

Figure 1.

In AD11 mice, pressure application of the GABA_A receptor agonist isoguvacine increases the firing rate of hippocampal CA1 principal cells. Responses of CA1 pyramidal cells (recorded in cell-attached mode) to pressure application of isoguvacine (100 μm, arrows), in WT animals (top trace), in AD11 mice with high levels of NGF antibodies (group II, middle trace), and in AD11 mice with low levels of NGF antibodies (group I, bottom trace).

Figure 2.

Changes in the effects of isoguvacine on MUA recorded from WT and AD11 mice. A, A brief application of isoguvacine in the bath to hippocampal slices from WT animals reduced the frequency of MUA and eliminated higher-amplitude events in a reversible manner. B, In group II AD11 mice, isoguvacine increased the frequency of MUA, an effect that was blocked by bicuculline (10 μm). C, D, Amplitude and frequency probability distribution plots obtained from WT (C; n = 7) and AD11 (D; n = 6) mice in the absence (open circles) or presence (filled circles) of isoguvacine. Differences in amplitude (WT) and frequency (AD11) obtained in the presence of isoguvacine were significantly different from controls (p < 0.001, Wilcoxon's signed rank test).

Figure 3.

E_GPSCs is more positive in AD11 than in WT mice. A, Perforated patch recordings of cells in hippocampal slices from WT (white), AD11 (black), and AD11 exposed to bumetanide (gray). GPSCs were evoked by local stimulation of GABAergic interneurons in the presence of DNQX (20 μm) and d-AP-5 (50 μm). B, Synaptic currents (I_GPSCs) shown in A are plotted versus membrane potentials (V_m). C, Individual RMPs and E_GPSCs values in WT (n = 15; white), AD11 (n = 20; black), and AD11 exposed to bumetanide (n = 6; gray) slices. Larger symbols on the left and right refer to mean ± SEM values. D, Plot of the driving force for GABA_A(E_GABA–V_m) in individual experiments from WT (white), AD11 (black), and AD11 exposed to bumetanide (gray) slices. Larger symbols are mean ± SEM values. **p < 0.001.

Figure 4.

In AD11 mice, E_GABA is more positive than V_m. A, Cell-attached recordings of single NMDA channels obtained at two different pipette potentials in neurons from WT (open circles) and AD11 (filled circles) mice. B, Summary plot of single NMDA-mediated currents (I_NMDA) versus pipette potentials (V_p) in six WT (open circles) and five AD11 (filled circles) neurons. Currents through NMDA channels reversed at −75.3 mV in WT and at −76.2 mV in AD11. Assuming a reversal of NMDA currents equal to 0 mV, we estimated a mean resting membrane potential of −75 and −76 mV, respectively. C, Examples of cell-attached recordings of GABA-induced single-channel currents obtained at two different pipette potentials in neurons from WT (open circles) and AD11 (filled circles) mice. D, Summary plot of single GABA-mediated currents (I_GABA) versus pipette potentials (V_p) in eight WT (open circles) and 10 AD11 (filled circles) neurons. In WT mice, current reversed at −76 mV and in AD11 at −56 mV.

Figure 5.

mRNA expression analysis of nicotinic receptors and cation-chloride cotransporters, by real-time qRT-PCR. These experiments were performed in the basal forebrain and hippocampus of six mice, three AD11 mice and three age-matched control mice at 6 months of age, for selected genes: five subunits of the nicotinic cholinergic receptors (α3, α4, α7, β2, and β4) and the cation-chloride cotransporters Nkcc1 and Kcc2. Error bars are computed according to the SEM and error propagation. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

KCC2 protein is downregulated in the hippocampus of the AD11 mice. Immunohistochemistry of the CA1–CA2 region of the hippocampus with anti-KCC2 antibodies show a decreased labeling in AD11 mice (B), with respect to the hippocampus of age-matched control mice (A). Scale bar, 100 μm.