Figure 1.
Layer 5 neurons in primary visual cortex project to dorsomedial striatum.
A–F. Representative serial coronal sections from the brain of a Rbp4-Cre mouse virally injected in the primary visual cortex (V1) with an AAV encoding Cre-dependent EGFP to label the cell bodies and axonal processes of neurons in layer 5 (A). EGFP-labeled axons leave V1 in through the external capsule (B) and innervate various structures including several layers of the superior colliculus (SC; B) and dorsal lateral geniculate nucleus (DLG; C). V1 axons enter the posterior tail of the striatum (caudate/putamen, CPu; D) and course throughout the length of the striatum (E) before innervating the anterior dorsomedial quadrant (F). Left, entire hemisphere stained with DAPI to highlight different brain structures overlaid with the EGFP fluorescence. Middle, detailed view of regions outlined in white in the corresponding left panel. Right, corresponding images from Paxinos Mouse Brain Atlas highlighting the putative structures where EGFP fluorescence is detected. Similar results were observed eight different mouse brains. M1; primary motor cortex. S1BF; primary somatosensory cortex barrel field.
Figure 2.
Retrograde labeling of V1 neurons innervating the DMS.
A. Coronal striatal section showing the site of red retrobead (RRB) injection into the dorsomedial striatum (DMS). The section is counterstained with DAPI. B. Coronal brain section of V1 containing RRBs retrogradedly transported from the dorsomedial striatum. Secondary visual cortex (V2ML and V2L) and secondary auditory cortex (AuD), but not Au1 (primary auditory cortex) were also labeled. As expected, retrobeads were also detected in putative dopaminergic neurons within the substantia nigra pars compacta (SNc). C. Detailed view of V1 showing the relative distribution of retrobead-labeled cells bodies across cortical layers. D. Distribution of retrobead-labeled neurons in V1 across cortical layers. The majority (81%) of labeled cells are situated within layer 5, while only 19% distribute to layers 2/3. E. Representative coronal section from a Rbp4-Cre;ZsGreen1 brain showing EGFP fluorescence concentrated in the cell bodies of Cre-containing cortical neurons in layer 5. The section is counterstained with DAPI. F. Detailed view of V1 in a Rbp4-Cre;ZsGreen1 brain injected with RRBs in the DMS illustrating the extensive overlap between retrobead- and Cre-containing (EGFP-positive) cells in upper layer 5. G. Percentage of all retrobead-labeled layer 5 neurons that also contain Cre (RRB/EGFP+; 65%, shown in yellow) vs. cells that are only RRB+ (35%, red). H. Coronal brain section showing red retrobead labeled cells in thalamus. ILT: Intralaminar thalamic nuclei. Scale: 1 mm for panels A, B, E and H; 100 µm for panels C and F.
Figure 3.
Stimulation of V1 axons engages glutamatergic and GABAergic synaptic transmission onto SPNs.
A. Representative high magnification view of the dorsomedial striatum in a Rbp4-Cre; Drd2-EGFP mouse virally injected in V1 with an AAV encoding Cre-dependent ChR2-mCherry. Axonal projections from V1 (red) are clearly seen within the DMS. The section is stained with DAPI (blue) to label cell bodies. iSPNs are easily identified using EGFP fluorescence (green). A presumptive dSPN (DAPI+; EGFP−) is also shown. B. Cell-attached recording from a SPN showing that a 1 ms 473 nm light flash (blue bar) reliably evokes a single action potential. Three consecutive extracellular waveforms are overlaid. C. As in B for another SPN recorded in the whole-cell current-clamp configuration. D. Whole-cell voltage-clamp traces from a SPN upon optogenetic stimulation (1 ms, blue bar) of ChR2-expressing V1 axons. EPSCs (black) were recorded at ECl = −70 mV, while IPSCs (red) were recorded at 0 mV (the reversal potential for ionotropic glutamate receptor mediated currents). Dashed gray lines mark the onset of both currents and highlight the delayed onset of IPSCs relative to EPSCs. Both EPSC and IPSC were eliminated after bath application of the glutamate receptor antagonists NBQX and CPP (both at 10 µM; gray and pink lines, respectively), confirming the disynaptic origin of IPSCs. E. Mean (± s.e.m) peak EPSC amplitude in dSPNs (blue) and iSPNs (green). F. Mean (± s.e.m) latency from flash onset to current onset of EPSCs (black) and IPSCs (red). G. Mean (± s.e.m) EPSC (black) and IPSC (red) amplitudes. Asterisk represents statistical significance, P<0.05 vs. baseline amplitude. Data in E–G represent mean ± s.e.m. Number of recordings are indicated in parentheses.