a. Cre expression driven by Drd1a (D1-Cre, top) and Drd2 (D2-Cre, bottom) BACs was visualized via activation of TdTomato in a reporter mouse. Red fluorescence reveals expression throughout striatum and in axons in expected target nuclei of direct and indirect pathway MSNs (SNr and GP, respectively). Green fluorescence reflects expression of a GAD67-GFP that reports GABAergic neurons. As seen in the red channel, there is diffuse cortical expression of Cre in the D1-Cre mice; however, this occurs in non-GABAergic neurons as noted by the lack of overlap with GFP fluorescence (see Supplementary Figure 2 for complete anlaysis). Scale bar: 500 µm.
b. AAV DIO-ChR2-mCherry injected into the striatum of a mouse carrying D2-Cre and D2-GFP transgenes shows ChR2-mCherry labeling in GFP⁺ cells, indicating pathway specific conditional expression of the virally encoded protein. ChR2-mCherry-expressing somata are marked with an asterisk and represent over 2/3 of the GFP⁺ MSNs in the area of dense infection. ChR2-mCherry was never observed in D2-GFP⁻ MSNs in these mice. Scale bar: 10 µm.
c. Voltage-clamp recordings from ChR2-mCherry⁻ MSNs demonstrate GABAergic synaptic currents evoked by 2 ms-long pulses of 473 nm light that stimulates neighboring ChR2-mCherry⁺ MSNs. Example currents from MSNs in D1-Cre (left) and D2-Cre (right) mice that were either homozygous (gray, Slc32a1^f/f) or heterozygous (black, Slc32a1^f/+) for the Slc32a1 conditional allele are shown. GABAergic currents are inward due to high intracellular Cl⁻ concentration. Insets, graphs of average peak current amplitudes evoked in animals of each genotype. * indicates p<0.05 for comparison of Slc32a1^f/+ and Slc32a1^f/f data. Error bars: SEM

a. Schematic of experimental design and hypotheses for changes in excitatory synapse number induced by extensive and bilateral expression of hM4D in direct or indirect pathway MSNs and subsequent injections of cno or saline. Widespread inhibition of direct pathway MSN firing with cno is expected to decrease excitatory synapse number, whether circuit level or cell-intrinsic mechanisms dictate corticostriatal synaptogenesis. The converse is expected for widespread inhibition of indirect pathway MSNs.
b. left, hM4D-mCherry⁺ MSNs in tissue densely infected with hM4D-encoding AAV (mCherry, red; DAPI, blue). Scale bar: 20 µm. right, Summary data demonstrate that in vivo manipulation of neuronal activity in direct or indirect pathway MSNs in the time window of excitatory synaptogenesis led to opposing changes in excitatory synapse number. Decreased mEPSC frequency was observed in cno-treated D1-Cre mice, whereas mEPSC frequency was enhanced in cno-treated D2-Cre mice, compared to respective saline-injected controls. * indicates p<0.05 for the comparison of same pathway MSNs from saline and cno-injected mice.
c. left top, 2PLSM images of a dendrite from a direct pathway MSN in a saline-injected mouse and a less spiny dendrite from a cno-injected sibling. Scale bar: 2 µm. left bottom, Images showing an example of increased spine density in indirect pathway MSNs of cno-treated animals compared to saline controls. right, Summaries of spine density in cno- and saline-treated animals demonstrating the opposite sign changes resulting from inhibition of the direct or indirect pathway. * indicates p<0.05 for the comparison of same pathway MSNs from saline and cno-injected mice.
d. Schematic of experimental design and hypotheses for possible changes in excitatory synapse number induced by sparse and unilateral expression of hM4D in direct or indirect pathway MSNs and subsequent injections of cno. Manipulation of activity in a small subset of neurons is not expected to engage circuit-wide mechanisms regulating synapse numbers. In contrast, unknown cell-intrinsic mechanisms could regulate the number of synapses formed onto the manipulated neurons, compared to same pathway uninfected MSNs.
e. left, Confocal image showing an hM4D-mCherry expressing MSN in a sparse injection configuration (mCherry, red; DAPI, blue). Scale bar: 20 µm. right, With sparse activity manipulations, no differences in mEPSC frequency were observed in either direct or indirect pathway infected, compared to uninfected, MSNs.
f. left top, 2PLSM images of a dendrite from control or neighboring hM4D-expressing direct pathway MSN. Scale bar: 2 µm. left bottom, Images of spiny dendrites from control or neighboring hM4D-expressing indirect pathway MSN. right, Summary graph shows that inhibiting activity of a sparse subset of MSNs does not alter dendritic spine density in manipulated direct or indirect pathway neurons. Error bars: SEM

a. left, 2PLSM image of a direct pathway Slc32a1^f/+ MSN filled with Alexa Fluor 594 through the recording pipette during whole-cell voltage-clamp analysis of mEPSCs. right, At a higher magnification, dendritic spines are visible. Examples of a dendrite from a control direct pathway MSN (top) and of a less spiny dendrite from a direct pathway MSN of a Slc32a1 null sibling (bottom) are shown. Scale bars: 20 µm, 2 µm.
b. As in (a), but illustrating the increase in dendritic spine density seen with silencing of indirect pathway MSNs.
c. Examples and summary of frequencies of mEPSCs in direct pathway Slc32a1 null mice and their heterozygous sibling controls. Preventing GABA release from direct pathway MSNs reduced mEPSCs frequency, compared to direct pathway MSNs in Slc32a1^f/+ sibling controls (top). Summary graph illustrates the decrease in direct and indirect pathway MSNs that were paralleled by differences in the density of dendritic spines. * indicates p<0.05 for comparison of Slc32a1^f/+ and Slc32a1^f/f data.
d. As in (c) for indirect pathway mutants indicating that preventing GABA release from indirect pathway MSNs increased mEPSC frequency and dendritic spine density in indirect and direct pathway MSNs. * indicates p<0.05 for comparison of Slc32a1^f/+ and Slc32a1^f/f data. Error bars: SEM

a. Focal release of glutamate is sufficient to trigger de novo spinogenesis in MSNs. 2PLSM image of a tdTomato (red) and GFP (green) expressing MSNs in a D2-GFP; tdTomato^f/f mouse sparsely injected with Cre-mCherry encoding AAV at P0. Imaging was performed in an acute slice of striatum at P10. Scale bar: 20 µm. right, higher magnification image of a dendrite before (top) and after (bottom) triggering new spine growth. Scale bar: 2 µm. The stimulation protocol consisted of 40 uncaging pulses directed at the indicated spot (arrow) with 15 mW of 720 nm light measured at the objective back aperture.
b. Summary graph demonstrating ~50% success rate in generating new spines with glutamate uncaging in direct and indirect pathway MSNs at P8-11.
c. left, Cre expression driven by an Rbp4 BAC (Rbp4-Cre) targets Cre to corticostriatal projection neurons. Red fluorescence from a TdTomato reporter allele is present in deep layer cortical neurons and densely labels axons throughout striatum. Scale bar: 500 µm. middle, AAV DIO hM4D-mCherry injected into the cortex of a mouse carrying Rbp4-Cre and D2-GFP transgenes shows strong red fluorescence in deep layer somata in cortex and green fluorescence in striatum. Scale bar: 500 µm. right, Red channel, higher magnification view of boxed area in the center panel shows extensive hM4D-mCherry labeling of corticostriatal axons.
d and e. In vivo inhibition of Rbp4-Cre neurons expressing hM4D during the window of excitatory synaptogenesis leads to a decrease in excitatory synapse number for both direct and indirect pathway MSNs. d. 2PLSM images of a dendrite from a direct pathway MSN in a saline-injected mouse and a less spiny dendrite from a cno-injected littermate. Scale bar: 2 µm. e. Summary data showing a decrease in direct and indirect pathway MSN mEPSC frequency (left) and spine density (right) for cno-treated mice versus saline treated littermates.
f – h. hM4D/cno-dependent decreases in MSN excitatory synapse number persist into early adulthood. f. Timeline for experiments. g. 2PLSM images of dendrites from indirect pathway MSNs from sibling mice in their early adulthood (P25- 28) after treatment with cno or saline during the time window for excitatory synaptogenesis (P8-15). h. Summary data showing that decreases in mEPSC frequency (left) and spine density (right) persist into early adulthood. Error bars: SEM