Input-specific GABAergic signaling to newborn neurons in adult dentate gyrus

Abstract

Adult neurogenesis is the multistage process of generating neurons from adult neural stem cells. Accumulating evidence indicates that GABAergic depolarization is an important regulator of this process. Here, we examined GABAergic signaling to newly generated granule cells (GCs) of the adult mouse dentate gyrus. We show that the first synaptic currents in newborn GCs are generated by activation of GABA(A) receptors by GABA with a spatiotemporal profile suggestive of transmitter spillover. However, the GABAergic response is not attributable to spillover from surrounding perisomatic synapses. Rather, our results suggest that slow synaptic responses in newborn GCs are generated by dedicated inputs that produce a relatively low concentration of GABA at postsynaptic receptors, similar to slow IPSCs in mature GCs. This form of synaptic signaling drives robust phasic depolarization of newborn GCs when the interneuron network is synchronously active, revealing a potential mechanism that translates hippocampal activity into regulation of adult neurogenesis via synaptic release of GABA.

Figure 1.

Initial synaptic currents in adult generated neurons are slow. a, POMC-EGFP-labeled GCs illustrate the immature morphology of newborn GCs. The white line approximates the outer edge of the molecular layer. b, The mature morphology of unlabeled neighboring GCs is illustrated by biocytin fills during recording in acute slices. Scale bars: a, b, 50 μm. c, Representation of cell morphologies and location of focal stimulating electrode. Green, Newborn GC; gray, mature GC. GCL, Granule cell layer; IML, inner molecular layer; MML, middle molecular layer; OML, outer molecular layer. d, Synaptic stimulation evoked PSCs in newborn cells with slow rise and decay kinetics, whereas stimulation at the same location evoked IPSCs in neighboring mature granule cells that are larger and faster. In all figures, stimulus artifacts are blanked for clarity. Glutamate receptor antagonists were used to isolate monosynaptic GABAergic input (see Materials and Methods).

Figure 2.

Manipulating the GABA transient reveals different synaptic GABA profiles in newborn GCs and mature GCs. a, The GABA transporter inhibitor NO711 enhanced the amplitude and prolonged the decay of slow PSCs in newborn GCs (left), whereas in mature GCs (right), NO711 only modestly prolonged the decay of fast IPSCs. The prolongation of the weighted decay in mature GCs was mediated entirely by an increase in the amplitude of the slow component (slow component from 106 ± 5 to 178 ± 21 ms in NO711; p < 0.05). b, Dextran increased the amplitude of PSCs in newborn cells (left) but did not affect the amplitude of GABA_A-IPSCs in mature cells (right).

Figure 3.

Synaptic GABA concentrations differ between fast and slow responses. a, The low-affinity GABA_A antagonist TPMPA (200 μm) produced a greater block in newborn GCs than in mature GCs (top). However, subsequent addition of the high-affinity antagonist gabazine (100 nm) blocked synaptic currents to a similar extent (bottom). Traces were normalized to the peak amplitude in each control condition. b, Histograms illustrating the enhanced sensitivity of slow PSCs to TPMPA. GABA_A,slow IPSCs in mature GCs evoked by stimulation of the MML (mature slow) also showed enhanced sensitivity to TPMPA. Error bars indicate SEM. *p < 0.05.

Figure 4.

Reducing spillover does not affect the kinetics of PSCs in newborn GCs. a, Reducing extracellular [Ca²⁺] decreased the amplitude of IPSCs in mature GCs by ∼60% (left) and increased the paired-pulse ratio (right), as expected for a reduction in Pr. Reduced GABA release was accompanied by a speeding of the IPSC decay (right). b, In newborn GCs, reduced extracellular [Ca²⁺] decreased the amplitude to the same extent (left) and increased the paired-pulse ratio of slow PSCs (right). However, the decay of slow PSCs was not altered.

Figure 5.

Coincident spontaneous currents in newborn and mature GCs. a, Example traces from a simultaneously recorded newborn (green) and mature (black) GC. The inset shows the expanded region overlaid. b, Left, GABAergic synaptic events in the newborn GC were detected and aligned, and are shown above the corresponding traces in the mature GC. Right, Fast events in the mature GC were detected and aligned, and are shown below the corresponding traces in the newborn GC. For clarity, 10 traces are shown for each group. c, Averages of all the detected events and the corresponding traces for the cell pair shown in b. Spontaneous events were aligned by the detected events in the newborn GC (left, average of 76 events), fast events in the mature GC (middle, average of 3325 events), or slow events in the mature GC (right, average of 392 events). No correlated event was evident in the newborn GC when fast events in the mature GC were averaged, whereas correlated events were present when slow events were averaged in both the newborn and mature GC. The amplitude of correlated sIPSCs in mature GCs was relatively large despite a majority of “failures” because individual correlated sIPSCs had large amplitudes. Similar results were obtained in each cell pair (n = 5) (Table 1). Calibration: 20 pA for currents in newborn GC; 40 pA for mature GC.

Figure 6.

Presynaptic GABA_B receptors differentially contribute to GABAergic PSCs in newborn and mature GCs. a, Left, Examples of synaptic currents in newborn and mature GCs that are normalized to the peak of the first response and overlaid to illustrate the difference in the PPR. Right, At each extracellular Ca²⁺ concentration tested, the PPR (300 ms interstimulus interval) in newborn GCs was significantly lower than in mature GCs (p < 0.05, ANOVA with Student–Newman–Keuls test). The numbers in parentheses represent number of cells. b, Application of the GABA_B receptor antagonist CGP55845 (2 μm) increased both the amplitude (left) and paired-pulse ratio (right) of slow PSCs in newborn GCs. c, CGP55845 did not affect the amplitude or paired-pulse ratio of fast IPSCs in mature GCs. Error bars indicate SEM. *p < 0.05.

Figure 7.

4-AP induces rhythmic activity in interneurons presynaptic to newborn GCs. a, The K⁺ channel blocker 4-AP (100 μm) increased spontaneous synaptic activity and triggered large, rhythmic inward currents in mature GCs (middle). The large events were reduced by picrotoxin (100 μm), but rhythmic activity was still present (bottom). These experiments were done in the absence of glutamate receptor antagonists. b, 4-AP also induced large, rhythmic inward currents in newborn GCs at the same frequency as in mature GCs (middle). All spontaneous activity was blocked by picrotoxin (bottom). c, Representative examples of individual events induced by 4-AP on an expanded timescale. i, A large event from the mature GC shown in a. ii, A large event from the newborn GC shown in b. Note that 4-AP events in newborn GCs are faster than those in mature GCs. iii, An individual spontaneous event in 4-AP from the newborn GC shown in b.

Figure 8.

4-AP-induced network activity generates robust depolarization of newborn GCs. a, Representative example of a current-clamp recording from a newborn GC in 4-AP. Spontaneous phasic depolarizations were observed in all cells (n = 6). b, Comparison of 4-AP-induced events in the same newborn GC in current-clamp (top) and voltage-clamp recording mode (bottom).