Figure 4. PVALB neocortical interneuron enhancers display distinct subcortical expression patterns.

(A) Twenty putative PVALB enhancers from snATAC-seq data cloned into AAV vectors. Seven of the 20 (35%) exhibited low or high specificity for PVALB cells in mouse retro-orbital assay (indicated with green boxes).

(B–G) mFISH in L2/L3 of VISp demonstrating positive labeling of Pvalb⁺ cells (arrows) by each of the indicated enhancer-AAV vectors. eHGT_023h and eHGT_064h also label non-Pvalb⁺ cells (asterisks). Percentages indicate the mean ± SD of SYFP2 labeling specificity for Pvalb⁺ cells across three independent mice.

(H–M) scRNA-seq in VISp confirming the PVALB transcriptomic cell subclass identity of enhancer-AAV vector-labeled cells. Bar graph shows the percentage of single cells that map to a transcriptomic cell type within that subclass. In contrast, the percentages given in the text are the percentage of cells recovered that expressed the indicated gene. Note that although only 65% of the eHGT_079h-marked cell types mapped to the PVALB subclass, 94% of the eHGT_079h-marked cells expressed Pvalb mRNA. This is because several SST subclass cell types also express Pvalb mRNA.

(N) Pvalb mRNA expression pattern (Allen Institute public in situ hybridization data) with multiple sites of expression throughout mouse brain.

(O–T) Labeling of both neocortical PVALB cells and various subcortical brain regions by PVALB-specific enhancers. These subcortical brain regions are also seen in the endogenous Pvalb mRNA expression pattern. Two enhancers (eHGT_079h and 140h) show exceptional specificity to neocortical PVALB cells. CTX, cerebral cortex; HPF, hippocampal formation; MOB, main olfactory bulb; MB, midbrain nuclei; MY, medulla nuclei; P, pons; IC, inferior colliculus; CBX, cerebellar cortex; CBN, cerebellar nuclei.

(U and V) Subcortical labeling by eHGT_023h in Purkinje cells (U) and eHGT_082h in CBN (V). eHGT_023h-labeled Purkinje cells are Pvalb⁺Gad1⁺, and eHGT_082h-labeled CBN cells are either Pvalb⁺Gad1⁺ or Pvalb⁺Gad1⁻.