Shank1 regulates excitatory synaptic transmission in mouse hippocampal parvalbumin-expressing inhibitory interneurons - PubMed (original) (raw)

. 2015 Apr;41(8):1025-35.

doi: 10.1111/ejn.12877. Epub 2015 Mar 25.

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Shank1 regulates excitatory synaptic transmission in mouse hippocampal parvalbumin-expressing inhibitory interneurons

Wenjie Mao et al. Eur J Neurosci. 2015 Apr.

Abstract

The Shank genes (SHANK1, 2, 3) encode scaffold proteins highly enriched in postsynaptic densities where they regulate synaptic structure in spiny neurons. Mutations in human Shank genes are linked to autism spectrum disorder and schizophrenia. Shank1 mutant mice exhibit intriguing cognitive phenotypes reminiscent of individuals with autism spectrum disorder. However, the molecular mechanisms leading to the human pathophysiological phenotypes and mouse behaviors have not been elucidated. In this study it is shown that Shank1 protein is highly localized in parvalbumin-expressing (PV+) fast-spiking inhibitory interneurons in the hippocampus. Importantly, a lack of Shank1 in hippocampal CA1 PV+ neurons reduced excitatory synaptic inputs and inhibitory synaptic outputs to pyramidal neurons. Furthermore, it is demonstrated that hippocampal CA1 pyramidal neurons in Shank1 mutant mice exhibit a shift in the excitatory and inhibitory balance (E-I balance), a pathophysiological hallmark of autism spectrum disorder. The mutant mice also exhibit lower expression of gephyrin (a scaffold component of inhibitory synapses), supporting the dysregulation of E-I balance in the hippocampus. These results suggest that Shank1 scaffold in PV+ interneurons regulates excitatory synaptic strength and participates in the maintenance of E-I balance in excitatory neurons.

Keywords: GABAergic synapse; excitatory synapse; hippocampus; interneurons; synaptic transmission.

© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

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Figures

Figure 1

Figure 1. Shank1 is highly expressed in Parvalbumin-expressing interneurons

(A, B) Confocal images of double-labeled immunofluorescence staining against parvalbumin (PV, red) and Shank1 (green) from hippocampal CA1 pyramidal cell layer of wild-type (+/+) (left) and Shank1−/− (−/−) (right) mice. Note that the immunoreactivity of Shank1 in Shank1−/− tissue is minimal, confirming the specificity of the antibody. Enlargements of the indicated rectangular fields are shown at bottom. (B) Relative proportion of hippocampal CA1 PV+ neurons as a fraction of neurons decorated by Shank1 signals (left), and that of Shank1-decorated neurons as a fraction of PV+ neurons (right). Numbers of neurons obtained from 4 wild-type mice were: Shank1-decorated PV+ neurons, 137; PV+ and Shank1 non-decorated neurons, 14; PV negative and Shank1 positive decorated neurons, 22. Note that the majority of PV+ neurons were immunopositive for Shank1. (C) Confocal images of triple-labeled immunofluorescence staining against PV (red), Shank1 (green) and VGluT1 (red) from proximal region of hippocampal CA1 stratum pyramidale (SP) of a wild-type mouse. Note that the dense VGluT1 signals (arrow heads) were localized close to Shank1 puncta in PV+ neurons. (D, E) Confocal images of double-labeled immunofluorescence staining against calbindin (Calb, D, red), calretinin (Calr, E, red) and Shank1 (green) from hippocampal CA1 stratum pyramidale (SP) and radiatum (SR) regions of wild-type (+/+) mice. (F) Relative expression of SHANK genes (SHANK1, SHANK2 and SHANK3) in PV+ and pyramidal (Py) neurons in adult mouse hippocampus. The expression level was normalized to that of GAPDH. Approximately 1000 PV+ and pyramidal neurons in hippocampal CA1 pyramidal cell layer per mouse were cut from one PV-RFP mouse (N = 6–8 mice). Scale bars: 10 μm.

Figure 2

Figure 2. Altered expression of postsynaptic proteins in Shank1-deficient PV+ neurons

Triple- or double-labeled immunofluorescence staining for PV and synaptic proteins in hippocampal primary cultures (14 DIV) prepared from wild-type (+/+) or Shank1−/− (−/−) mice. (A) PV+ interneurons in wild-type and Shank1−/− cultures were triple-stained for PV, Shank1 and PSD-95. (B) Double-immunostaining for pan-Shank, PSD-95, GKAP, Homer, GluA1, or Bassoon, and PV. (C) Quantification of puncta density per 10 μm dendrite length (left) and puncta area (right) for the indicated proteins. Shank1−/− dendrites show a significant reduction in pan-Shank, PSD-95, GKAP and GluA1 (n = 10 cells from 3 mice, 3 independent cultures) signals. The error bars show standard error. Scale bars, 10 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001, student t-test.

Figure 3

Figure 3. Shank1 regulates excitatory synaptic transmission in Parvalbumin-expressing basket cells

(A, D) Top, Consecutive sample sEPSC (A) and mEPSC (D) traces of PV+ basket cells in wild-type (black traces) and Shank1−/− (grey traces) mice. Bottom, Averaged and normalized sample sEPSC (A) and mEPSC (D) traces in PV+ basket cells. (B, C, E and F) Summary of the frequency and amplitude (B and E), and kinetics (C and F) of sEPSCs and mEPSCs in wild-type (+/+) and Shank1−/− (−/−) animals. The averaged EPSC frequency and amplitude for each cell were superimposed as circles while the bar graphs indicate the means ± SEM. Number of cells: sEPSC: wild-type, 16 cells from 7 mice; Shank1−/−, 19 / 11; mEPSC: wild-type, 10 cells from 5 mice; Shank1−/−, 10 / 4. (G) Left, Sample traces from CA1 fast-spiking PV+ basket cells in wild-type and Shank1−/− hippocampal slices showing spikes elicited by current injections of 300 and 900 pA for 400 ms. Right, Summary graph of the frequency of action potentials in wild-type and Shank1−/− animals. Input-output relationship [number of spikes elicited versus amount of current injection (400 ms duration)] was plotted for wild-type and Shank1−/− animals. Neurons were held at the indicated resting membrane potentials. Number of cells: wild-type, 10 cells from 7 mice; Shank1−/−, 11 / 6.

Figure 4

Figure 4. Shank1 deficit causes reduced basal firing rate in PV+ neurons and PV neuron-mediated inhibitory synaptic output onto CA1 pyramidal neurons

(A) Sample traces of PV+ neurons in wild-type (black traces) and Shank1−/− (gray traces) mice. (B) Summary graph of the firing rates measured by cell-attached recordings of PV+ neurons in wild-type and Shank1−/− animals. Number of cells: wild-type, 7 cells from 7 mice; Shank1−/−, 6 / 5. (C–D) Effect of Shank1 deficiency on unitary inhibitory synaptic transmission between hippocampal CA1 PV+ and pyramidal neurons. (C) Left, superimposed fluorescent and Nomarski images. Averaged sample unitary IPSC (uIPSC) traces by one (middle) and double (right) presynaptically applied depolarization commands. (D) Summary of uIPSC amplitude (left), connectivity (middle) and paired-pulse ratio (right). Number in each bar represents the number of synaptically connected cell pairs (left and right), total number of cell pairs tested (middle).

Figure 5

Figure 5. Shank1 deficit causes reduced E-I ratio by reducing inhibitory synaptic function and gephyrin expression

(A) Shank1−/− mice display reduced E-I ratio compared with wild-type mice. Left, Sample traces (average of 10 consecutive responses) mediated by GABAAR (upward) and AMPAR (downward) from wild-type (+/+) or Shank1−/− (−/−) hippocampal slices. Stimulus artifacts were truncated. Calibration, 200 and 50 pA, 20 msec. Right, Summary graph of E-I ratio of wild-type (total n = 20 cells from 6 mice) and Shank1−/− mice (n = 24 / 6, student t-test) (see text for definition). (B) Shank1−/− mice reduce inhibitory synaptic transmission. Left, Sample traces of mIPSC events (top) and the average traces of mIPSC traces (bottom) obtained from wild-type (+/+) or Shank1−/− (−/−) hippocampal slices. The averaged traces scaled to match the amplitude and aligned at the onset of response are shown at the bottom right (norm). Note that the time course of events is the same. Calibration, 40 pA, 500 msec (top), 5 pA, 20 msec (bottom). Right, Summary graphs of the frequency (left) and amplitude (right) of mIPSCs in wild-type (+/+) and Shank1−/− (−/−) animals. Number of cells: wild-type, total 9 cells from 3 mice; Shank1−/−, 9 / 3. (C, D, E) Shank1−/− mice exhibit reduced expression of gephyrin. (C) Left, Immunoblot analysis of membrane (P2) fractions from individual wild-type (+/+) and Shank1−/− (−/−) mice for the indicated proteins. Right, Quantitation of various proteins in membrane fractions. Syn: Synaptophysin. (D) Confocal images of gephyrin puncta in hippocampal CA1 stratum pyramidale (SP) and radiatum (SR) areas from wild-type and Shank1−/− mice. Left, double-labeled staining for gephyrin and DAPI. Right, the gephyrin images were deconvoluted and quantified after thresholding of fluorescence intensity (grey images).

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