Differential synaptic integration of interneurons in the outer and inner molecular layers of the developing dentate gyrus

Abstract

The dentate gyrus (DG) undergoes continued reorganization and lamination during early postnatal development. Interneurons with anatomically identified synaptic contacts migrate from the outer to the inner regions of the molecular layer (ML) of the DG. By using the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein transgenic mouse, we were able to target and physiologically characterize Dlx2(+) developing ML interneurons. We investigated whether synapses on migrating ML interneurons were functional and defined properties of synaptic inputs onto interneurons that were located in the outer ML (OML) or inner ML (IML). Consistent with ongoing maturation, IML interneurons displayed lower input resistances and more hyperpolarized resting membrane potentials than OML interneurons. Both OML and IML interneurons received a direct excitatory monosynaptic input from the entorhinal cortex via the perforant paths, but this input was differentially sensitive to activation of presynaptic group II and III metabotropic glutamate receptors. Furthermore, only IML interneurons also received significant synaptic input from the CA3/hilar region, especially under conditions of experimentally induced disinhibition. These changes are attributed to a significant reorganization of dendritic fields. GABA(A) receptor-mediated innervation of OML and IML interneurons also displayed significant differences in miniature IPSC amplitude, frequency, and decay kinetics. Finally, cell-attached recordings indicated that GABA(A) receptor activation was depolarizing in OML interneurons but predominantly shunting in IML interneurons. Our data provide evidence that developing ML interneurons receive functional glutamatergic and GABAergic inputs and undergo significant changes in synaptic integration during migration from the OML to the IML.

Figure 1.

The CNP-EGFP mouse permits identification of developing GABAergic interneurons in the early postnatal ML. A, Bright EGFP⁺, small, irregularly shaped cells (arrowheads) and fainter EGFP⁺, larger round/ovoid-shaped cells (arrows) are present throughout the ML. B, The bright EGFP⁺ cells correspond to NG2⁺ CNP gene-expressing progenitors (arrowhead), whereas faint EGFP⁺ cells are NeuN⁺ neurons (arrow). C, Stacked confocal images with corresponding orthogonal reconstruction demonstrate that these neurons are developing interneurons, based on GAD65/67 and Dlx2 expression. D, E, Eliciting action potential firing in response to varying current injections demonstrates a fast-spiking phenotype. F, No statistical difference was noted between the firing frequency in the first and last 200 ms epochs of a 1000 ms depolarizing 300 pA current injection. No statistical differences in any of the measured parameters were noted between OML and IML EGFP⁺ neurons, and hence data were pooled. hf, Hippocampal fissure. Scale bars, 50 μm.

Figure 2.

OML and IML interneurons are differentially integrated into the excitatory hippocampal circuitry as a consequence of dendritic field reorganization. A, EPSCs in OML and IML interneurons were evoked by subicular (perforant paths; filled circle) or CA3 pyramidal layer (cross) stimulation. In all experiments, an incision was made between the CA1 and CA3. All blue and red traces denote evoked responses from OML and IML, respectively. B, Subicular stimulation elicits a monosynaptic EPSC response in OML interneurons but results in a “flurry” of asynchronous EPSCs in IML interneurons. C, Addition of 10 μm GBZ has no effect on the evoked EPSC response in OML interneurons but results in a large barrage of EPSCs in IML interneurons. Traces on the right are 3–5 min after addition of GBZ. D, Similar results are attained after preincubation of slices with 50 μm picrotoxin (picro). C, D, Arrows highlight the presence of initial EPSC that is temporally very close to the stimulus. E, OML interneurons display an NMDA receptor-mediated outward EPSC, even after the application of 10 μm CNQX, indicating that the response is monosynaptic. In IML interneurons, an outward NMDA receptor-mediated response is observed that is temporally coincident with the initial inward AMPA receptor-mediated EPSC (arrowhead and arrow). This is followed by a barrage of AMPA receptor-mediated EPSCs with corresponding NMDA receptor currents. After CNQX application, only the NMDA receptor response (arrowhead) that was temporally coincident with the initial AMPA receptor response remained, indicating that this was indeed monosynaptic. F, CA3 stimulation in the presence of GBZ elicited no response in OML interneurons, but an EPSC profile similar to that seen after perforant path stimulation (in absence and presence of GBZ) was noted in IML interneurons. G, H, Biocytin filling of OML and IML interneurons shows distinct nonoverlapping dendritic fields. EC, Entorhinal cortex; hf, hippocampal fissure; hil, hilar region; sub, subiculum. Scale bar, 50 μm.

Figure 3.

Selective inhibition of monosynaptic perforant path transmission by group II and III mGlus in IML and OML developing interneurons, respectively. All blue and red traces denote evoked EPSC responses from OML and IML, respectively. A, B, DCG-IV inhibits AMPA receptor-mediated monosynaptic EPSC peak responses after perforant path stimulation in IML but not OML interneurons. C, D, l-AP4 inhibits the AMPA receptor-mediated monosynaptic EPSC peak responses after perforant path stimulation in OML but not IML interneurons. E, Bar graph illustrating differential inhibition by group II (open bars) and group III (filled bars) mGlu activation on the perforant path evoked EPSCs in OML and IML interneurons. F, In IML interneurons, DCG-IV inhibits the charge transferred by the monosynaptic response in a quantitatively similar manner to that noted with EPSC peak measurements (red filled circles in F vs red filled circles in B). DCG-IV totally abolishes the charge transferred by the EPSC barrage (A; F, open red circles). Data are means ± SEM from five to six OML and IML interneurons.

Figure 4.

Differences in GABA_A receptor-mediated innervation of developing OML and IML interneurons. All blue and red traces denote evoked EPSC responses from OML and IML, respectively. A, Representative traces illustrating outward mIPSC events in OML and IML interneurons plus an ensemble average of all mIPSC events. B, C, Representative frequency distribution histograms of mIPSC amplitude from an OML and IML interneuron. The presence of larger-amplitude mIPSCs (>50 pA) in the OML interneuron (see A, asterisks) are never observed in IML interneurons. D, mIPSC decay kinetics differ between OML and IML interneurons. In OML interneurons, a double-exponential is statistically better than a single-exponential fit (black lines depict the exponential fit superimposed on the blue EPSC decay), as assessed by r² coefficient values (D2). In contrast, a single-exponential fit adequately describes the mIPSC decay in IML interneurons (D2). Average data for mIPSC amplitude, frequency, and decay values are summarized in supplemental Table 2 (available at www.jneurosci.org as supplemental material). E, Using a cell attached protocol (see supplemental methods, available at www.jneurosci.org as supplemental material), activation of GABA_A receptors by muscimol depolarizes OML interneurons (top; blue vs black trace) but has minimal effect in IML interneurons (bottom; red vs black trace). F, Summary graph depicting the average effect in OML and IML interneurons. Data were pooled according to three different criteria based on the range of developmental ages at which the experiments were performed. Data are means ± SEM from four to eight OML and IML interneurons. amp, Amplitude; No., number; exp, exponential; ns, not significant.