Perineuronal Nets in the Dorsomedial Striatum Contribute to Behavioral Dysfunction in Mouse Models of Excessive Repetitive Behavior

Abstract

Background: Excessive repetitive behavior is a debilitating symptom of several neuropsychiatric disorders. Parvalbumin-positive inhibitory interneurons in the dorsal striatum have been linked to repetitive behavior, and a sizable portion of these cells are surrounded by perineuronal nets (PNNs), specialized extracellular matrix structures. Although PNNs have been associated with plasticity and neuropsychiatric disease, no previous studies have investigated their involvement in excessive repetitive behavior.

Methods: We used histochemistry and confocal imaging to investigate PNNs surrounding parvalbumin-positive cells in the dorsal striatum of 4 mouse models of excessive repetitive behavior (BTBR, Cntnap2, Shank3, prenatal valproate treatment). We then investigated one of these models, the BTBR mouse, in detail, with DiI labeling, in vivo and in vitro recordings, and behavioral analyses. We next degraded PNNs in the dorsomedial striatum (DMS) using the enzyme chondroitinase ABC and assessed dendritic spine density, electrophysiology, and repetitive behavior.

Results: We found a greater percentage of parvalbumin-positive interneurons with PNNs in the DMS of all 4 mouse models of excessive repetitive behavior compared with control mice. In BTBR mice, we found fewer dendritic spines on medium spiny neurons (targets of parvalbumin-positive interneurons) and differences in neuronal oscillations as well as inhibitory postsynaptic potentials compared with control mice. Reduction of DMS PNNs in BTBR mice altered dendritic spine density and inhibitory responses and normalized repetitive behavior.

Conclusions: These findings suggest that cellular abnormalities in the DMS are associated with maladaptive repetitive behaviors and that manipulating PNNs can restore normal levels of repetitive behavior while altering DMS dendritic spines and inhibitory signaling.

Keywords: Dorsal striatum; Inhibitory signaling; Medium spiny neurons; Parvalbumin interneurons; Perineuronal nets; Repetitive behavior.

Figure 1

Mouse models with excessive repetitive behaviors have more DMS PV+ interneurons with PNNs. (A–C) Histological analysis of WFA+ PNNs and PV+ interneurons revealed an increase in DMS percent colocalization compared with control mice in BTBR mice (p = .0204) (A), Cntnap2^−/− mice (p = .0195) and Shank3^+/−ΔC mice (p = .0041) (B), and VPA-treated mice (p = .0445) (C). No differences were found in the DLS (A–C). (D) Representative images of PV+ interneurons (magenta) and WFA+ PNNs (green) in the DMS. White triangles indicate PV+WFA+ cells. Scale bar = 50 μm. (E–G) No differences were found in WFA+ PNN density in DMS of BTBR mice (E), Cntnap2^−/− and Shank3^+/−ΔC mice (F), or VPA mice (G). DLS PNN+ density was higher in BTBR mice compared with control mice (p = .0186) (E), but not in Cntnap2^−/− and Shank3^+/−ΔC mice (F) or VPA mice (G). PV+ density was lower in the DMS of BTBR (p = .0187) and VPA (p = .0379) mice compared with respective control mice, but not in Cntnap2^−/− or Shank3^+/−ΔC mice. (H–J) No differences were detected in the DLS of BTBR mice (H), Cntnap2^−/− and Shank3^+/−ΔC mice (I), or VPA-treated mice (J). Data are represented as mean ± SEM. See also Figure S1. See Table S1 for complete statistics. ∗p < .05. DLS, dorsolateral striatum; DMS, dorsomedial striatum; PNN, perineuronal net; PV, parvalbumin; VPA, valproic acid; WFA, Wisteria floribunda agglutinin.

Figure 2

Reducing PNNs in DMS rescues excessive repetitive behaviors and normalizes grooming and digging behaviors in BTBR mice. (A) Mice were habituated to an enclosed open field box twice for 5 minutes a day before behavioral testing. The next day mice were tested in both open field grooming and thick bedding digging paradigm (order counterbalanced). (B) BTBR mice showed elevated time spent grooming compared with control mice (p = .0293) and longer time spent grooming per bout (p = .0202). (C) BTBR mice showed greater time spent digging compared with control mice (p < .0001) and longer time spent digging per bout (p < .0001). (D) Experiment timeline of behavior pre-injection, stereotaxic surgery, behavior postinjection, and tissue collection for histology. (E) Mean change (Δ) in time spent grooming from pre- to postinjection per mouse. Negative values depict a decrease in time spent, and positive values depict an increase in time spent, where means near zero (dashed line) represent no change in behavior from pre- to postinjection. BTBR-chABC mice show decreases in grooming (Δ−91.44 ± 25.43 seconds), a significant change compared with the control group (p = .0036). (F, I) Graphical representation of the mean bout duration from pre- to postinjection. (G) 10 dpi of chABC in the DMS normalized BTBR grooming bout duration (3.73 ± 0.36 seconds), comparable to C57BL/6 mice (3.11 ± 0.39 seconds), while 10 dpi of pnase in BTBR mice maintained prolonged grooming bouts (8.00 ± 0.66 seconds) (p = .0014). (H) Mean Δ in time spent digging from pre- to postinjection shows a decrease in BTBR-chABC mice (Δ−62.36 ± 32.88 seconds), a significant change compared with control mice (p = .0295). (J) 10 dpi of chABC in the DMS of BTBR mice normalized digging bout duration (4.81 ± 0.74 seconds), comparable to C57BL/6 mice (4.22 ± 0.54 seconds). (K) Histological analysis of DMS WFA+ labeled PNNs confirms reduction in the percent of PV+ interneurons positive for PNNs in BTBR-chABC mice. BTBR-pnase mice show an increase in percent of PV+ interneurons positive for PNNs compared with C57BL/6-pnase mice (p = .023), (L) with no differences in overall WFA+ cell density. (M) PV+ density was lower in BTBR mice compared with control mice in both chABC (p = .0009) and pnase (p = .0028) treatment groups. Data are represented as mean ± SEM. See also Figure S2 and S3. See Table S2 for complete statistics. ∗p < .05; #p < .075. chABC, chondroitinase ABC; DMS, dorsomedial striatum; dpi, days post injection; pnase, penicillinase; PNN, perineuronal net; PV, parvalbumin; WFA, Wisteria floribunda agglutinin.

Figure 3

BTBR mice exhibit altered neuronal oscillations, inhibitory transmission, and dendritic spine density in the DMS. (A) All mice were implanted unilaterally with an electrode array targeting the DMS. Targeting was confirmed histologically with GFAP and DAPI labeling. Example local field potential traces of a C57BL/6 (top, gray) and a BTBR (bottom, green) mouse. Scale bar = 200 mV (y-axis) by 0.2 second (x-axis). (B–D) Normalized local field potential power spectrum data for grooming behavior during the pre-event period (−1 to 0 second prior to event start) (B), event period (0 to 1 second) (C), and end of event period (0 to 1 second prior to event end) (D). (E–G) Normalized local field potential power spectrum data for digging behavior during the pre-event period (E), event period (F), and end of event period (G). Gray bars indicate significant differences in power (A.U.) across frequency (Hz): light gray (p ≤ .01); dark gray (p ≤ .001). (H) Example depiction and traces of whole-cell, mIPSC recordings from putative MSNs of a C57BL/6 (top, gray) and a BTBR (bottom, green) mouse. Scale bar = 40 pA (y-axis) by 2 seconds (x-axis). (I) mIPSC frequency (Hz) analysis of C57BL/6 (gray) and BTBR (green) mice data from DMS slice preparations yields no differences in mean frequencies. (J) Significant effect in amplitude (pA) of mIPSC events shows increased mean amplitudes in BTBR mice compared with C57BL/6 mice (p = .0335). (K) Bar graph of mean frequency by amplitude depicts the increased frequency of events at amplitudes >60 pA in BTBR mice. (L) Representative images of MSN cell (left panel) and dendritic segments (DiI, red) in the DMS from a control (top right panel) and BTBR (bottom right panel) mouse. Scale bar = 5 μm. (M) Spine density differences between C57BL/6 and BTBR mice were observed in the DMS (p = .0048), but not in the DLS. Data are represented as mean ± SEM. See Table S3 for complete statistics. ∗p < .05. Amp., amplitude; A.U., arbitrary units; DLS, dorsolateral striatum; DMS, dorsomedial striatum; Freq, frequency; GFAP, glial fibrillary acid protein; mIPSC, miniature inhibitory postsynaptic current; MSN, medium spiny neuron.

Figure 4

Reducing perineuronal nets alters inhibitory synaptic strength at DMS MSNs and normalizes dendritic spine density. (A) Mice were injected bilaterally with chABC in the DMS and lived in their home cage for 10 dpi, and subsequently tissue was collected for slice electrophysiology. (B) Example traces of whole-cell, mIPSC recordings from putative MSNs of a C57BL/6 (left, dark gray) and a BTBR (right, dark green) mouse, 10 dpi of chABC. Scale bar = 40 pA (y-axis) by 2 seconds (x-axis). (C) mIPSC frequency (Hz) analysis of C57BL/6 and BTBR mice10 dpi of chABC data from DMS slice preparations shows different effects of chABC on C57BL/6 and BTBR mice (p = .00126). (D) Significant effect in amplitude (pA) of mIPSC events shows increased mean amplitudes in BTBR mice compared with C57BL/6 mice (p = .0146). (E) Bar graph of mean frequency by amplitude depicts the overall increased frequency of events across amplitudes >20 pA in BTBR mice. (F) Representative images of MSN dendritic segments (DiI, red) in the DMS from C57BL/6 mice (top panels), 10 dpi of pnase (top left panel) and 10 dpi of chABC (top right panel) and BTBR mice (bottom panels), 10 dpi of pnase (bottom left panel) and 10 dpi of chABC (bottom right panel). Scale bar = 5 μm. (G) Comparison of DMS MSN spine density yields no differences across all groups. Data are represented as mean ± SEM. See Table S4 for complete statistics. ∗p < .05. Amp. amplitude; chABC, chondroitinase ABC; DMS, dorsomedial striatum; dpi, days post injection; Freq, frequency; mIPSC, miniature inhibitory postsynaptic current; MSN, medium spiny neuron; pnase, penicillinase.