Early hyperactivity and precocious maturation of corticostriatal circuits in Shank3B(-/-) mice

Abstract

Some autistic individuals exhibit abnormal development of the caudate nucleus and associative cortical areas, suggesting potential dysfunction of cortico-basal ganglia (BG) circuits. Using optogenetic and electrophysiological approaches in mice, we identified a narrow postnatal period that is characterized by extensive glutamatergic synaptogenesis in striatal spiny projection neurons (SPNs) and a concomitant increase in corticostriatal circuit activity. SPNs during early development have high intrinsic excitability and respond strongly to cortical afferents despite sparse excitatory inputs. As a result, striatum and corticostriatal connectivity are highly sensitive to acute and chronic changes in cortical activity, suggesting that early imbalances in cortical function alter BG development. Indeed, a mouse model of autism with deletions in Shank3 (Shank3B(-/-)) shows early cortical hyperactivity, which triggers increased SPN excitatory synapse and corticostriatal hyperconnectivity. These results indicate that there is a tight functional coupling between cortex and striatum during early postnatal development and suggest a potential common circuit dysfunction that is caused by cortical hyperactivity.

Figure 1. Rapid development of striatal SPN excitatory input in mice after ~P10

(a) Whole cell voltage clamp recordings in SPNs of dorsomedial striatum in acute brain slices of Rbp4-Cre; ChR2-YFP^f/f mice and optogenetic fiber stimulation using whole field illumination. Scale bar, 1mm. (b) AMPAR oEPSCs recorded in SPNs under voltage clamp (V_h=−70 mV) at different postnatal days in response to brief pulses of 473 nm laser light (blue rectangle). (c) Mean oEPSC peak amplitude ± SEM recorded in SPNs at V_h=−70 mV from P6-P15 and (d) at V_h=−20 mV from P14-30. (e) Developmental progression of oEPSC amplitude values normalized to P30. (f) Representative traces of AMPAR (gray) and NMDAR (black) oEPSCs recorded in the same SPN of Rbp4-Cre; ChR2-YFP^f/wt at V_h=−70 mV and V_h=+40 mV, respectively. Red circle indicates time of NMDAR current amplitude analysis at 50ms post light stimulus (blue rectangle). (g) Mean AMPAR and (h) NMDAR oEPSC peak amplitude ± SEM in P10-11 and P14-15 SPNs. (i) Mean AMPAR to NMDAR ratio ± SEM for each SPN represented in (g–h). Scale bar, 1mm. (j) Average (solid line) ± SEM (dashed line) NMDAR oEPSCs from cells represented in (g–h). (k) Mean NMDAR oEPSC decay time constant (Tau) ± SEM of P10-11 and P14-15 SPNs. (l) Coronal brain slice of P12 mouse infected with AAV8-CAG-EGFP. Ctx- cortex; Str- striatum. Scale bar, 1mm. (m) Representative images of EGFP expressing SPN dendrites at different postnatal days. Scale bar, 10 μm. (n) Average dendritic spine density ± SEM from infected SPNs at P8-P24.

Figure 2. Correlated increase in cortical and striatal activity in vivo from P10 to P16

(a) Experimental diagram of in vivo recordings in a sagittal view of a mouse brain showing cortex (CTX) and striatum (STR). (b) Representative recordings of multi-unit activity in cortex (left) and striatum (right) at P10 and P14. (c) Median ± interquartile range of average FR of cortical units from P10-11 to P14-16. (d) Median AP burst frequency and (e) Intra-burst frequency ± interquartile range of cortical units shown in (c). (f) Median ± interquartile range of average FR of striatal units at different developmental time points. (g) Median AP burst frequency and (h) Intra-burst frequency ± interquartile range of striatal units shown in (f).

Figure 3. Corticostriatal coupling during early development

(a) Experimental diagram of in vivo recordings and extracranial optogenetic stimulation using 473nm laser (blue) in Rbp4-Cre;ChR2-YFP^f/f mice showing cortex (CTX) and striatum (STR). (b) Example raster plot (top) and 20 ms bin peri-stimulus time histogram (PSTH, bottom) of action potentials of a P11 striatal unit during optogenetic stimulation of cortex with a 10 Hz light pulse train (blue). Note the robust response to the first pulse of the train. (c) Raster plot (top) and 5 ms bin PSTH (bottom) of the unit shown in (b) in response to individual optogenetic pulses (blue). (d) PSTH (20 ms bin) representing firing rate of cortical neurons during ChR2-stimulation (blue) at P10-11 (black) and P14-16 (red). Shaded regions represent ± SEM (e) PSTH (5 ms bin) of units shown in (d) in response to the first pulse of the stimulation train. Shaded regions represent ± SEM (f) PSTH (20 ms bin) of firing rate of striatal neurons during cortical stimulation (blue) at P10-11 (black) and P14-16 (red). Shaded regions represent ± SEM (g) PSTH (5 ms bin) of striatal neurons in response to the first pulse of the stimulation train. Shaded regions represent ± SEM. Note the presence of secondary peaks indicative of burst firing in response to single light pulses.

Figure 4. Hyperexcitability of SPNs during early development

(a) Example membrane responses to discreet current injection steps in SPNs of dorsomedial striatum. (b) Mean resting membrane potential ± SEM and (c) Mean spike threshold potential ± SEM of SPNs recorded at different postnatal periods. (d) Mean ± SEM current-voltage (I–V) relationship in SPNs across development. Dashed lines represent linear fits to voltage steps to 10, 25 and 50 pA whose slopes were used to determine the input resistance. (e) Current-firing rate (I–F) plot of SPNs across development. Error bars represent ± SEM (f) Mean SPN rheobase current ± SEM from P10-11 to P16-17.

Figure 5. Precocious maturation of striatal glutamatergic inputs in Shank3B^−/− SPNs

(a) Representative mEPSC recordings in SPNs of dorsomedial striatum of WT and Shank3B^−/− mice at P14. (b) Cumulative distribution of amplitude and (c) inter-event interval of mEPSCs recorded from SPNs of WT and Shank3B^−/− littermates at P14. (d) Representative mEPSC recordings of WT and Shank3B^−/− SPNs at P60. (e) Cumulative distribution of amplitude and (f) inter-event interval of mEPSCs recorded from WT and Shank3B^−/− litter mates at P60. (g) Mean mEPSC frequency and (h) amplitude ± SEM of SPNs from WT and Shank3B^−/− animals at different developmental time points. WT maturation is characterized by a continuous increase in mEPSC frequency throughout development whereas Shank3B^−/− show a precocious maturation followed by an arrest in later stages. (i) Experimental diagram depicting whole cell voltage clamp recordings in SPNs of dorsomedial striatum in acute brain slices of Shank3B^−/−;Rbp4-Cre;ChR2-YFP^f/wt mice and optogenetic fiber stimulation using whole field illumination. Scale bar, 1mm. (j) Representative average AMPAR and NMDAR oEPSCs from WT (black) and Shank3B^−/− (red) SPNs. Blue rectangle represents 5ms 473nm light stimulation. (k) Mean AMPAR and (h) NMDAR oEPSC peak amplitude ± SEM in P14 WT and KO SPNs. (l) Mean AMPAR to NMDAR ratio ± SEM for SPNs represented in (k).

Figure 6. Cortical hyperactivity in neonatal Shank3B^−/− mice

(a) Experimental diagram of in vivo recordings in a sagittal view of a mouse brain showing cortex (CTX) and striatum (STR). (b) Representative recordings of multi-unit activity in cortex and (c) striatum of WT and Shank3B^−/− animals at P13-14. (d) Median ± interquartile range of average FR of cortical units from WT and Shank3B^−/− mice. (e) Median frequency of AP bursts and (f) Intra-burst frequency ± interquartile range of cortical units shown in (d). (g) Median ± interquartile range of average FR of striatal units from WT and Shank3B^−/− mice. (h) Median frequency of AP bursts and (i) intra-burst firing rate ± interquartile range of cortical units shown in (e).

Figure 7. Elevated cortical activity during early development increases corticostriatal connectivity

(a) Silencing of cortical interneuron output was achieved by injecting Cre expressing adenovirus in the cortex of Slc32a1^f/f animals at P4. (b) Local field potential (LFP) recordings from cortex of control and AAV injected animals at P14 show epileptiform patterns of activity after VGAT deletion. (c) Spectrogram of LFPs shown in (b). Scale bar, 1 min. Color scale represents normalized power. (d) Example mEPSC recordings in SPNs of dorsomedial striatum of control and AAV-Cre injected animals. (e) Cumulative distribution of mEPSC amplitude and (f) mEPSC inter-event interval values for the total pool of mEPSCs recorded from control (black) and Cre injected (red) litter mates at P12-14. (g) Cell average mEPSC frequency and (h) amplitude ± SEM of SPNs from control and Cre injected animals. (i) Schematic showing optogenetic cortical stimulation using extracranial implant of a low mass LED (blue) in Rbp4-Cre; ChR2^f/f animals (top panel) and subsequent oEPSC measurements in SPNs in the ipsilateral (stimulated) and contralateral (control) hemispheres (bottom panel). (j) Example AMPAR oEPSCs recorded in SPNs located in dorsomedial striatum of the stimulated (ipsi, red) or opposite (contra, black) hemisphere in response to 5 ms pulses of 473 nm laser light (blue rectangle). (k) Mean oEPSC amplitude ± SEM of control (contra) and stimulated (ipsi) SPNs. (l) Pair wise comparison of average oEPSC amplitude in animals recorded in (k)

Figure 8. Early increase in corticostriatal drive in Shank3B^−/− mice is due to cortical hyperactivity

(a) Schematic representing bilateral injection of AAV8-DI-hM4D_i into cortex of Shank3B^−/−; Rbp4-Cre mice at P1-2 and bi-daily administration of CNO for 3 days before mEPSC recordings at P13-14. (b) Coronal brain slice of P13 Shank3B^−/−;Rbp4-Cre mouse infected with AAV8-DI-hM4D_i-mCherry. Ctx- cortex, Str- striatum. Scale bar, 1 mm. (c) Example mEPSC recordings in SPNs of dorsomedial striatum of saline or CNO injected animals. (d) Cell average mEPSC frequency and (e) amplitude ± SEM of SPNs of saline or CNO injected animals.