Figure 1.
Characterization and in vivo localization/expression of CaMKII inhibitory (EAC3I) peptide in EAC3I mice.
(A) Schematic of breeding strategy for production of EAC3I mice. CaMKIIalpha promoter drives expression of tTA which binds to the tetO promoter and drives expression of EAC3I peptide fused to EGFP. (B) (Top) Brain slice of EAC3I under brightfield (left) and EGFP epifluorescence (right). (Bottom) Brain slice of EAC3I as above following 3 weeks of doxycycline feeding (200 mg/kg). Note lack of EAC3I-EGFP expression. (C) Sagittal section of EAC3I mouse brain showing restricted endogenous EAC3I-EGFP expression (green) and nissl stain (red, neuronal marker). Scale bar 500 µm. (D) Coronal image of mosaic expression of endogenous EAC3I-EGFP expression (green) in dorsal lateral striatum with a NeuN stain (blue, neuronal marker). Note little to no expression of EAC3I peptide in cortex. CC = corpus callosum, Scale bar 100 µm. (E) 63× image of unstained EAC3I-EGFP expressing MSN (green = endogenous EGFP signal). Note expression in soma, dendrites and dendritic spines (arrows). Scale bar 10 µm. (F) Images of globus pallidus (GP) (left) and substantia nigra pars reticulata (SNR) (right) showing MSN axon terminals (green) and nissl stain (red). GP and SNR cell somas (red, nissl stain) are devoid of EGFP signal. CP = cerebral peduncle. Scale bars 20 µm. (G) (left) DIC image of patch pipette on a MSN in whole cell mode, (right) epifluorescence image of left panel confirming EAC3I-EGFP expression. Scale bar 20 µm. (H) Overlaid coronal images showing EAC3I-EGFP MSN axon terminal field expression in globus pallidus (left) and substania nigra pars reticulata (right) confirming indirect and direct pathway expression, respectively. Scale bar 0.5 mm.
Figure 2.
In vivo expression of EAC3I decreases s/mEPSC frequency in dorsal lateral striatum MSNs.
(A) Representative sEPSC traces for NON EGFP (black) and EGFP (CaMKII-inhibited, green). Scale bars 50 ms and 10 pA. (B) Representative mEPSC traces for NON EGFP (black) and EGFP (CaMKII-inhibited, green). Scale bars 50 ms and 10 pA. (C) Representative sEPSC traces for NON EGFP (black) and EGFP (CaMKII-inhibited, green) MSNs from animals that were fed DOX from birth to six weeks and then removed to allow EAC3I transgene expression. Recordings were made 4–5 weeks following DOX removal at a similar age to previous. The scale bars are 50 ms and 10 pA. (D) (Left) Average sEPSC frequencies from EAC3I-containing MSNs compared to controls, (right) Cumulative probability distributions of sEPSC inter-event intervals (Wt: n = 7, p = 0.0001; tTA: n = 5, p = 0.0001; Non EGFP: n = 9, p = 0.0009; versus EGFP: n = 13). (E) (left) Average mEPSC frequency from EAC3I MSNs compared to controls, (right) cumulative probability distributions of mEPSC frequency (Wt: n = 5, p = 0.0022; tTA: n = 6, p<0.0001; Non EGFP: n = 6, p = 0.0019; versus EGFP: n = 6). (F) (Left) Average sEPSC frequencies from EAC3I-containing MSNs versus controls, (right) cumulative probability distributions of sEPSC inter-event intervals (Wt: n = 8, p = 0.99; tTA: n = 7, p = 0.99; Non EGFP: n = 8, p = 0.99; versus EGFP: n = 10). (G) (left) Average sEPSC amplitudes, (right) cumulative probability distributions of sEPSC amplitude (Wt: n = 7, p = 0.91; tTA: n = 5, p = 0.26; Non EGFP: n = 9, p = 0.99; versus EGFP: n = 13). (H) (left) Average mEPSC amplitude, (right) cumulative probability distributions of mEPSC amplitude (Wt: n = 5, p = 0.90; tTA: n = 6, p = 0.71; Non EGFP: n = 6, p = 0.96; versus EGFP: n = 6). (I) (left) Average sEPSC amplitudes, (right) cumulative probability distributions of sEPSC amplitude from animals that were fed DOX from birth to six weeks and then removed to allow EAC3I transgene expression (Wt: n = 8, p = 0.99; tTA: n = 7, p = 0.34; Non EGFP: n = 8, p = 0.99; versus EGFP: n = 10). * P<0.05; ** P<0.01; *** P<0.001; error bars represent SEM, N.S. = not significant. Note: All neurons in panels A-I are held at −70 mV during recordings.
Figure 3.
Dorsal lateral striatum MSN CaMKII inhibition reduces excitatory transmission independently of changes in release probability.
(A) (Upper trace, black) NON EGFP (EAC3I-lacking) MSN PPR (50 ms ISI) example trace baseline (average of 20 sweeps, 0.05 Hz). (Middle trace, black) EAC3I-lacking MSN PPR (50 ms ISI) example trace post 10 µM Baclofen wash in (average of 20 sweeps, 0.05 Hz). (Bottom trace, black) EAC3I-lacking MSN PPR (50 ms ISI) example trace 20 minutes post wash out of drug (average of 50 sweeps, 0.05 Hz). (B) Same as in (A), but for an EGFP (EAC3I-containing) MSN. (C) Average PPR recorded by paired-pulse stimulation eliciting EPSCs with two different interstimulus intervals (40 and 60 ms) for EAC3I-containing MSNs versus all controls. (Wt: n = 9; tTA: n = 6; NON EGFP: n = 9; EGFP: n = 10; 40 ISI p = 0.38, 60 ISI p = 0.27). (D) Baclofen increases the CV of EPSCs. Coefficient of variation (CV = SD/Mean) change of EPSCs from before, during and after application of baclofen for EAC3I-containing and EAC3I-lacking MSNs (NON EGFP: n = 6; EGFP: n = 5; p = 0.95). (E) Baclofen increases the PPR. PPR (50 ms ISI) for EAC3I-containing and EAC3I-lacking MSNs before, during and after application of baclofen (NON EGFP: n = 6; EGFP: n = 5; p = 0.48). (F) Baclofen decreases sEPSC frequency. Plot of normalized sEPSC frequency post 10 µM Baclofen compared to baseline for EAC3I-containing and EAC3I-lacking MSNs (NON EGFP: n = 6; EGFP: n = 5; p = 0.96). Note: All MSNs held at −70 mV during recordings.
Figure 4.
Dorsal lateral striatum MSN CaMKII inhibition does not alter the level of glutamate at the cleft.
(A) Normalized average NMDAR-mediated EPSC Charge (AUC) measured with 0.1 Hz stimulation in the presence of 10 µM Mk-801 over time for EAC3I-containing and EAC3I-lacking MSNs. (B) The rate of NMDA-mediated EPSC decay is best fit with a single exponential decay function. The time constant (tau) in seconds was not significantly different between groups (NON EGFP: n = 6; EGFP: n = 5; p = 0.65). All neurons held at -70 mV to relieve NMDAR-dependent voltage blockade by magnesium.
Figure 5.
CaMKII inhibition does not alter dendritic spine density, but decreases dendritic length and complexity.
Rendering and quantification of a confocal image of a Lucifer yellow filled dendritic segment (80–100 µm from the cell soma) from a MSN from the dorsal lateral striatum. (A) (top) Confocal image of EAC3I-lacking MSN (NON EGFP) and the Imaris dendrite and spine model overlaid from segment above. (below) Same as above, but for an EAC3I-containg MSN segment. Scale bars 1.5 µm. Fluorescent signal (green) pertains to Lucifer yellow fill. (B) Average dendritic spine density (number of spines/10 µm) scatter plot for each neuron. NON EGFP n = 17, EGFP n = 16; p = 0.93. (C) Neuronal reconstructions of representative EAC3I-lacking (NONEGFP) and EAC3I-containing (EGFP) dorsal striatal MSNs. Scale bar 50 µm. (D) Average total dendritic length in EAC3I-lacking (black) and EAC3I-containing (green) MSNs. (E) Sholl analysis of dendritic complexity in EAC3I-lacking (black) and EAC3I-containing MSNs (green). **p<0.01, *p<0.05; error bars represent SEM.
Figure 6.
GluA1KO mice mimic the EAC3I mice decrease in sEPSC frequency.
(A) Example traces of sEPSCs collected from dorsal lateral striatum MSNs in Wt (top) and GluA1KO (bottom). Scale bars 0.5 sec, 30 pA (Control: n = 15; GluA1KO: n = 18; p = 0.013). (B) (left) Average sEPSC frequency in GluA1KO versus controls. (right) Cumulative probability graph of inter-event interval. (C) (left) Average sEPSC amplitude in GluA1KO versus controls. (right) Cumulative probability graph of amplitude (Control: n = 15; GluA1KO: n = 18; p = 0.72). * P<0.05 ; error bars represent SEM. All MSNs were held at −70 mV.
Figure 7.
CaMKII inhibition enhances MSN intrinsic excitability.
(A) Traces of EAC3I-lacking (NON EGFP, black) and EAC3I-containing MSNs (EGFP, green) sEPSPs recorded at −85 mV. Scale bars 0.6 mV, 200 ms. (B) Traces of EAC3I-lacking (black) and EAC3I-containing MSNs (green) with 20 pA hyperpolarizing and depolarizing current injections (−120 pA to +100 pA above AP threshold, 20 pA steps). Scale bars 200 ms, 20 mV. (C) Average sEPSP frequency in EAC3I-containing MSNs versus controls (Wt: n = 4; tTA: n = 4; NON EGFP: n = 9; versus EGFP: n = 6; p = 0.015). (D) Average sEPSP amplitude (current clamped at −85 mV) in EAC3I-containing MSNs versus controls (Wt: n = 4; tTA: n = 4; NON EGFP: n = 9; versus EGFP: n = 6; p = 0.037). (E) Resting membrane potential (RMP) (mV) of EAC3I-containing and control MSNs (Wt: n = 18; tTA: n = 15; NON EGFP: n = 22; versus EGFP: n = 24; p = 0.0043). (F) Input resistance of EAC3I-containing and control MSNs (Wt: n = 18; tTA: n = 15; NON EGFP: n = 22; versus EGFP: n = 24; p = 0.0002). (G) Rheobase current injection or current injection to reach 1st AP in EAC3I-containing and control MSNs (Wt: n = 18; tTA: n = 15; NON EGFP: n = 22; versus EGFP: n = 24; p<0.0001). (H) Firing frequency (Hz) after 4 sweeps (20 pA steps) following threshold firing in EAC3I-containing and control MSNs (Wt: n = 18; tTA: n = 15; NON EGFP: n = 22; versus EGFP: n = 24; p = 0.0023). * P<0.05; ** P<0.01; *** P<0.001; error bars represent SEM.