Recruitment of early postnatal parvalbumin-positive hippocampal interneurons by GABAergic excitation

Abstract

GABAergic synaptic inputs targeting cortical principal cells undergo marked changes in their functional properties from depolarizing at early postnatal life to hyperpolarizing at mature stages. In contrast, the nature of GABA(A) receptor-mediated signaling in interneurons during maturation of neuronal networks is controversial. By using gramicidin perforated-patch and whole-cell recordings from LIM homeobox 6 (Lhx6)-positive dentate gyrus perisomatic-targeting parvalbumin-expressing interneurons (PV-INs), we show that signaling at first formed GABAergic synapses at postnatal day 3 (P3) is excitatory and switches to shunting during the course of the first to second postnatal week. GABAergic synaptic inputs at P3-P6 reliably evoke action potentials in 65% of Lhx6-EGFP-expressing perisomatic-targeting cells and boost spike induction upon conjoint activation of glutamatergic fibers. Thus, GABAergic inputs change their functional role during maturation. They facilitate the recruitment of perisomatic-targeting INs in early postnatal circuits when network connectivity and synaptic glutamate receptor-mediated excitation are low and control spike timing at later stages when connectivity and glutamate-mediated drive are high.

Figure 1.

Lhx6-EGFP is reliably expressed in fast-spiking perisomatic-targeting PV⁺ cells. A, Confocal image stack from a horizontal section (50 μm) through the DG of a juvenile Lhx6-EGFP mouse. Note the high density of Lhx6-EGFP-positive somata at the gcl–hilus border (arrows). Inset, Confocal image demonstrating colocalization of EGFP and Lhx6 detected with a primary antibody and visualized using a secondary antibody conjugated to Cy3. Scale bar, 20 μm. B, Left, A single Lhx6-EGFP-positive neuron was filled with biocytin during whole-cell recording and visualized subsequently. The dense axonal arborizations in the gcl (arrows) identify this neuron as a perisomatic-targeting cell. The selected somatic area (red box) is shown on the right at higher magnification. Inset, Perisomatic-targeting cells had a typical fast-spiking non-adapting discharge pattern (600 pA, 1 s; V_hold −70 mV). Right, Confocal image stack of the same cell stained against parvalbumin, a characteristic marker for perisomatic-targeting cells. Scale bar, 10 μm. ml, Molecular layer.

Figure 2.

First formed GABAergic synapses on perisomatic-targeting cells are depolarizing. A, Plot summarizing the onset of functional GABAergic (blue) and glutamatergic (red) synaptic transmission on Lhx6-EGFP-expressing perisomatic-targeting cells. Inset, Average IPSC (30 single traces; in the presence of 20 μm CNQX) and EPSC (in the presence of 5 μm SR95531) during extracellular stimulation in the granule cell layer. IPSCs and EPSCs were blocked after bath application of the GABA_A receptor antagonist SR95531 (5 μm) and the AMPA/kainate receptor antagonist CNQX (20 μm; gray), respectively. B, Gramicidin perforated-patch recordings were used to measure the reversal potential of synaptically evoked IPSPs (E_syn) in perisomatic-targeting cells. Alexa Fluor 488 was added to the pipette solution to monitor stability of the perforated-patch. Left, Epifluorescence is restricted to the recording pipette. Right, Soma of the recorded cell is labeled after spontaneous break through. C, Representative GABA_A receptor-mediated IPSPs (average from 6–13 traces) evoked in an early postnatal (orange) and a juvenile (black) perisomatic-targeting cell at different holding potentials (V_hold). Bottom right, IPSP peak amplitudes plotted against V_hold to reveal E_syn in an early postnatal and a juvenile perisomatic-targeting cell. Data were fitted with a polynomial function. D, Summary plot of E_syn over postnatal age. Gray line indicates a linear fit. E, Action potential threshold (V_thr, dashed line) was similar in early postnatal (orange) and juvenile (black) perisomatic-targeting cells. F, Plot of resting membrane potential (V_rest) and V_thr over age. Gray lines correspond to linear functions fitted to the data. G, Superposition of three lines representing linear fits to E_syn, V_thr, and V_rest in perisomatic-targeting cells at P3–P21 emphasizing the developmental change of GABAergic signaling from excitatory (red area; E_syn ≥ V_thr) to shunting (blue area; V_rest ≤ E_syn < V_thr).

Figure 3.

Depolarizing GABAergic signaling facilitates action potential generation in early postnatal perisomatic-targeting cells. A, Top, Schematic illustration of the experimental configuration. Bottom, Two examples are shown for extracellular activation of presynaptic GABAergic fibers (blue) in the presence of 4 mm kynurenic acid, of glutamatergic fibers (red) in the presence of 5 μm SR95531, or both GABAergic and glutamatergic fibers (black) in P3–P6 perisomatic-targeting cells (V_hold = V_rest). Two traces are superimposed. Recordings were performed with a pipette solution mimicking the native internal Cl⁻ concentration at P3–P6 (“early postnatal E_syn” solution) estimated from perforated-patch recordings (Fig. 2). B, Bar graph summarizing the percentage of perisomatic-targeting cells discharging upon stimulation of pharmacologically isolated GABAergic, glutamatergic, or both types of presynaptic inputs (values above bars represent number of cells). C, Extracellular stimulation intensity was chosen to evoke action potentials in early postnatal perisomatic-targeting cells with ∼50% reliability (V_hold = V_rest; 2 superimposed traces). Middle, Blocking GABA_A-mediated signaling with 5 μm SR95531 obstructs action potential generation. Right, Blocking effect on spike induction is reversed after washout of SR95531. D, Summary of the effect of SR95531 on discharge probability in early postnatal (P3–P6) perisomatic-targeting cells. Circles connected by lines represent single experiments (control 53 ± 5%, 5 cells; SR95531 4 ± 3%, 5 cells; wash 55 ± 19%, 3 cells). E, Left, Example of action potential initiation during recording with “early postnatal (P3–P6) E_syn” pipette solution as in C. Middle, Repatching the same cell with “juvenile (P18–P21) E_syn” pipette solution that induces shunting inhibition abolishes action potential generation. Right, Re-repatching the neuron with the initial P3–P6 E_syn pipette solution restores spiking. F, Bar graph summarizing discharge-probability in the experiments shown in E. *p < 0.05; **p < 0.01.