To determine if there were fewer synapses after retinal lesions, we measured the density of boutons colocalizing with one or both of the synaptic markers in lesioned (72 hr after complete lesion) and control animals. We found that the fraction of GFP-labeled boutons with both synaptic markers did not change after lesioning (85% ± 0.02%), but that the overall GABAergic synapse density did decrease (Figure 6B), confirming the results observed with chronic structural imaging (Figures

4C and 4E). Having established that inhibitory synapse density decreases following retinal lesions, we next determined if the bouton loss actually reflected a loss of functional GABAergic synapses in the cortical circuit. Layer 2/3 GABAergic cells are known to target both layer 2/3 pyramidal cell somata and the dendrites of layer 5 pyramidal cells located in layer 2/3 (Chen et al., 2011, Kätzel et al., 2011 and Silberberg signaling pathway et al., 2005). As structural changes in excitatory pathways associated with functional circuit

plasticity in adult visual cortex occur preferentially on layer 5, but not layer 2/3 cells (Hofer et al., 2009), we determined whether there was a reduction in functional inhibition onto layer 5 neurons. Therefore, we measured miniature inhibitory postsynaptic currents (mIPSCs) in layer 5 Sotrastaurin clinical trial pyramidal cells in acute slices of visual cortex 48 hr after complete retinal lesions. We found that mIPSC frequency was decreased (Figures 6C and 6D), consistent with a reduction in the number of GABAergic synapses onto these cells. The amplitude of the mIPSCs was unchanged 48 hr after lesions, suggesting no postsynaptic Olopatadine receptor changes had occurred (Figures 6C and 6E). Together, these data indicate that following retinal lesions, there is a rapid decrease in the number of inhibitory synapses in the affected cortical region. Finally, we compared the time course of changes in inhibitory neuron bouton and spine density after retinal lesions. Inside the LPZ

in mice with focal lesions (Figure 7A), spine density (dashed line) was significantly decreased within 6 hr after the lesion, preceding the decrease in bouton density (solid line), which was significant only 24 hr after the lesion. The observation that changes of synaptic input structures (i.e., spines) of the interneurons precede changes in synaptic output structures (i.e., boutons) could possibly reflect a causal relation. In contrast, in animals with complete lesions (Figure 7B), spine and bouton density decrease over the same time course, 48 hr after the lesion. Together these data suggest that the exact timing of these structural changes may depend on the nature of the input loss. We have used chronic two-photon imaging to monitor structural plasticity of inhibitory neurons in adult mouse visual cortex. We observe that a subset of inhibitory neurons, many of which are NPY positive, carry dendritic spines.

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