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. 2005 Jul 20;25(29):6775-86.
doi: 10.1523/JNEUROSCI.1135-05.2005.

Neurogliaform neurons form a novel inhibitory network in the hippocampal CA1 area

Christopher J Price  1 , Bruno Cauli, Endre R Kovacs, Akos Kulik, Bertrand Lambolez, Ryuichi Shigemoto, Marco Capogna
Affiliations

Neurogliaform neurons form a novel inhibitory network in the hippocampal CA1 area

Christopher J Price et al. J Neurosci. .

Abstract

We studied neurogliaform neurons in the stratum lacunosum moleculare of the CA1 hippocampal area. These interneurons have short stellate dendrites and an extensive axonal arbor mainly located in the stratum lacunosum moleculare. Single-cell reverse transcription-PCR showed that these neurons were GABAergic and that the majority expressed mRNA for neuropeptide Y. Most neurogliaform neurons tested were immunoreactive for alpha-actinin-2, and many stratum lacunosum moleculare interneurons coexpressed alpha-actinin-2 and neuropeptide Y. Neurogliaform neurons received monosynaptic, DNQX-sensitive excitatory input from the perforant path, and 40 Hz stimulation of this input evoked EPSCs displaying either depression or initial facilitation, followed by depression. Paired recordings performed between neurogliaform neurons showed that 85% of pairs were electrically connected and 70% were also connected via GABAergic synapses. Injection of sine waveforms into neurons during paired recordings resulted in transmission of the waveforms through the electrical synapse. Unitary IPSCs recorded from neurogliaform pairs readily fatigued, had a slow decay, and had a strong depression of the synaptic response at a 5 Hz stimulation frequency that was antagonized by the GABA(B) antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP55845). The amplitude of the first IPSC during the 5 Hz stimulation was also increased by CGP55845, suggesting a tonic inhibition of synaptic transmission. A small unitary GABA(B)-mediated IPSC could also be detected, providing the first evidence for such a component between GABAergic interneurons. Electron microscopic localization of the GABA(B1) subunit at neurogliaform synapses revealed the protein in both presynaptic and postsynaptic membranes. Our data disclose a novel interneuronal network well suited for modulating the flow of information between the entorhinal cortex and CA1 hippocampus.

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Figures

Figure 1.
Figure 1.
Reconstruction of NG cells in the SLM of area CA1. A, Reconstruction of an NG cell (P18) illustrating dendrites (black) and axons (gray). Perforant pathway stimulation at 40 Hz elicited EPSCs with a facilitating/depressing pattern in this cell, like the pattern shown in Figure 4 A2. Note that the dendrites and axons cross the hippocampal fissure and extend both into the molecular layer of the dentate gyrus and the SLM of area CA1. B, Reconstruction of another NG cell (P13) illustrating dendrites (black) and axons (gray). Perforant pathway stimulation at 40 Hz evoked EPSCs with a depressing pattern in this cell, like the pattern shown in Figure 4 A1. The dendrites and axon are mostly restricted to the CA1 SLM. SLM, Stratum lacunosum moleculare; ML, molecular layer of the dentate gyrus. C1, Micrograph showing detail of dendrites from a different NG cell; an axonal process is also visible and is indicated by the arrowhead. C2, Micrograph illustrating the detail of axonal processes showing branching points and varicosities of another NG cell. Scale bars: A, 50 μm; B, 25 μm; C1, C2, 5 μm.
Figure 2.
Figure 2.
Molecular markers of NG cells in the SLM. A, Agarose gel electrophoresis of the multiplex single-cell RT-PCR product from the neuron in Figure 1 A using primers to test the GABAergic, calcium-binding protein, and neuropeptide phenotypes of the neuron. GAD 65 and 67, Glutamic acid decarboxylases 65 and 67; b-NOS, nitric oxide synthase; CaB, calbindin; PV, parvalbumin; CR, calretinin; NPY, neuropeptide Y; VIP, vasointestinal peptide; SOM, somatostatin; CCK, cholecystokinin; Dyn, dynorphin; Enk, enkephalin. B, Fluorescence images of a biocytin-filled NG cell, visualized with AMCA-conjugated avidin and double labeled with an antibody against α-actinin-2. Scale bar, 10 μm. C, Fluorescence images of neurons in the SLM of hippocampal area CA1 triple labeled for α-actinin-2, NPY, and b-NOS. Note that two neurons were α-actinin-2 and b-NOS double labeled, whereas only one was triple labeled for all three markers. Scale bar, 20 μm. D, Graphical representation of the cumulative percentage of SLM cells double or triple labeled for NPY, α-actinin-2, and b-NOS. One hundred percent represents the total number of cells labeled for each molecule, and this number is indicated above each column.
Figure 3.
Figure 3.
Electrophysiological characterization and perforant path-evoked excitability of NG cells. A, Current-clamp traces showing the results of hyperpolarizing current-clamp steps of 10, 20, and 30 pA demonstrate the low input resistance associated with NG cells, whereas a depolarizing current step (220 pA) to just beyond threshold shows the slow ramping of membrane voltage just before action potential initiation. Large depolarizing current steps, 700 and 500 pA, respectively, elicit weak accommodating spike trains (A1) and bursting spike trains (A2). B, Voltage-clamp recordings show the effect of stimulation of the perforant path from the entorhinal cortex. Short-latency, fast EPSCs were generated in NG cells and were abolished by bath application of 20 μm DNQX. The inset shows the position of stimulating and recording electrodes and the location of the cut made to excise area CA3. Traces are the average 10 sweeps. B1, Frequency histograms illustrating a unimodal distribution of EPSCs generated by perforant path stimulation (n = 60 events analyzed; n = 20 traces shown superimposed). B2, The distribution of onset times was clearly bimodal in a different neuron. The insets show voltage-clamp traces from which data in histograms were obtained (n = 84 events analyzed; n = 20 traces shown superimposed). C, NMDA receptor-dependent EPSC recorded at a holding potential of +40 mV in the presence of 20 μm DNQX. In the electrode solution, potassium gluconate was substituted with cesium methansulphonate. Traces are the average 10 sweeps. C1, The distribution of onset times of control EPSCs recorded at -70 mV (n = 85 events analyzed; n = 20 traces shown superimposed). C2, From the same neuron, the distribution of onset times for NMDA receptor-mediated EPSCs recorded at +40 mV and in the presence of 20 μm DNQX. Note that a similar range of onset times was present for the evoked responses recorded under either condition (n = 99 events analyzed; n = 20 traces shown superimposed).
Figure 4.
Figure 4.
Short-term plasticity of perforant path inputs onto NG cells. A1, Voltage-clamp trace of the response of an NG cell to 40 Hz stimulation with a train of EPSCs that depressed rapidly. A2, Recording from a second NG cell that showed initial facilitation, followed by depression after a 40 Hz stimulation. Associated with the facilitation was a slowly developing inward shift in baseline, likely reflecting the summation of EPSCs at high stimulus frequency. Each trace represents the average of 10 sweeps. A3, Summary graph illustrating the EPSC peak amplitudes during a 40 Hz stimulation normalized to the first EPSC (ANOVA; Bonferroni's post hoc values: *p < 0.05, **p < 0.01, and ***p < 0.001). Gray bars, Depressing pattern; black bars, facilitating/depressing pattern. B, Histograms comparing membrane time constant (tau; B1), input resistance (B2), and action potential (spike) half-width (B3) for NG cells showing either depressing (depress) or facilitating (fac)/depressing forms of short-term plasticity. C, Scatter dot plots illustrating the postnatal age for each cell displaying either depressing or facilitating/depressing short-term plasticity in response to a 40 Hz (C1) or 10 Hz (C2) stimulation. The line represents the mean for each group.
Figure 5.
Figure 5.
SC stimulation monosynaptically excites NG cells. A, SC stimulation evokes EPSCs with a short and constant latency in voltage-clamped NG cells. The left inset shows the differentiated traces illustrating onset latency; the right inset depicts the position of recording and stimulating electrodes. B, Under conditions favoring the recording of NMDA currents (20 μm DNQX; holding potential, +40 mV), monosynaptic EPSCs were also evoked in the same neuron. For each panel, 10 superimposed sweeps are shown. Onset latency distributions for both AMPA-mediated (C1) and NMDA-mediated (C2) EPSCs were similar, confirming that the glutamatergic input from the SCs onto this cell was monosynaptic.
Figure 6.
Figure 6.
Mixed electrical and chemical synapses between pairs of NG cells. A, Unitary IPSCs recorded in voltage clamp (bottom trace) evoked by an action current in the presynaptic neuron (top trace). The arrow points to the initial inward current flowing through the electrical synapse, which was then followed by an outward current with slow decay kinetics. B, Current-clamp recording showing the voltage deflection seen in the prejunctional neuron after injection of 250 pA of hyperpolarizing current (top) and the subsequent deflection seen in the postjunctional neuron (bottom). C, Current-clamp recording in which 190 pA of depolarizing current was injected into the prejunctional neuron to induce spiking activity (top). In the postjunctional neuron, smaller amplitude spikelets, followed by AHPs, were observed. D1, Results of a voltage-clamp experiment charting the time course of inhibition after application of 500 μm carbenoxolone. D2, Top traces superimposed show the currents evoked by hyperpolarizing voltage steps from -70 to -130 mV in the prejunctional cell before and after application of carbenoxolone. The middle and bottom traces are the postjunctional responses before (1) and after (2) carbenoxolone. Traces are the average of 10 individual sweeps.
Figure 7.
Figure 7.
Oscillations and synchronous behavior of electrically coupled NG cells. A, Current-clamp recordings of 7, 20, and 40 Hz sine waves injected into the prejunctional neuron and the resulting attenuated postjunctional potential. Each record is the average of 10 traces obtained at each frequency. The inset is an expanded trace for the first oscillation at 7 Hz to note the phase shift seen at all oscillation frequencies. B, The relationship between frequency of the injected sine wave and the coupling coefficient measured. C, The box plot shows the relationship between sine wave frequency and the measured phase shift (degrees). Median values are shown; the box represents the 25 and 75% intervals, whereas the minimum and maximum values are also indicated. D, Cross-correlogram for spiking activity between a pair of NG cells, both depolarized beyond threshold. The bin size used to construct cross-correlograms was 5 ms. E, The relationship between the coupling coefficient, measured with injection of hyperpolarizing current into the prejunctional neuron, and the cross-correlation value.
Figure 8.
Figure 8.
Involvement of the GABAB receptor in the unitary IPSCs observed in NG pairs. A, Voltage-clamp recordings (top trace, presynaptic action currents; bottom trace, postsynaptic unitary IPSCs) from a pair of NG cells showing the marked depression of the GABAergic IPSC seen in the postsynaptic neuron with a 5 Hz stimulation. B, Voltage-clamp recordings (top trace, presynaptic action currents; bottom traces, postsynaptic unitary IPSCs) from a different NG cell pair showing that 5 μm CGP55845 inhibits the depression seen with 5 Hz stimulation. C1, Histogram representing the amplitude of each of the four IPSCs recorded during each 5 Hz train under control and 5 μm CGP 55845 conditions normalized to the amplitude of the first control IPSC. Note the increase seen in the amplitude of the first pulse when GABAB receptors are inhibited, suggesting a tonic activation of these receptors. C2, Normalizing the IPSCs to the first IPSC in CGP55845 emphasizes that depression was reduced with GABAB antagonist treatment. D, Unitary IPSCs in a NG cell pair (top trace, presynaptic action current; bottom traces, postsynaptic unitary IPSCs). In the presence of the GABAA antagonist SR95531 (10 μm), a small residual IPSC was observed that was abolished with application of the GABAB antagonist CGP55845 (5 μm). Each trace represents the average of 10 IPSCs. E, Summary graph showing the mean and range of amplitudes for synaptically evoked GABAB responses between NG cell pairs.
Figure 9.
Figure 9.
Subcellular localization of the GABAB1 subunit in neurons of the CA1 SLM as revealed by the pre-embedding immunogold method. Aa-Ac, Electron micrographs of consecutive sections showing extrasynaptic localization of GABAB1 (double arrows) in an axon terminal of a biocytin-labeled (peroxidase reaction end product) NG cell (T). The bouton establishes symmetrical synaptic contact with a dendritic shaft of an unidentified neuron also labeled for GABAB1 (arrows). B, Electron micrograph showing the localization of the GABAB1 subunit at the extrasynaptic membrane (arrows) of the dendritic shaft of a biocytin-filled (peroxidase) NG cell receiving synapses from axon terminals making asymmetrical (T1, T3) and symmetrical (T2) synapses (arrowheads). The biocytin-filled bouton (T2) originates from a presynaptic NG cell. The inset in B demonstrates the symmetrical synaptic junction (arrowheads). C, Extrasynaptic distribution of the GABAB1 subunit (arrows) on a dendritic shaft of a non-NG interneuron of the SLM. Scale bars, 0.2 μm. D, Dendritic shaft.
Figure 10.
Figure 10.
Synapses between NG and non-NG cells of the CA1 SLM. A, Voltage-clamp recording showing the slow IPSC recorded in a non-NG cell after NG cell stimulation. B, Stimulation of a non-NG cell resulted in a fast IPSC being recorded in the NG cell. C, Stimulation of a non-NG cell at 5 Hz resulted in a train of nondepressing IPSCs being recorded in the NG cell. The top traces illustrate presynaptic action currents, and the bottom traces illustrate unitary postsynaptic IPSCs. Each trace is the average of 10 sweeps.
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