Cholinergic interneurons are preferentially reduced in 3-week-old Isl1 conditional knock-outs. A–H, Light micrographs of striatal interneuron marker expression demonstrating a marked reduction in the cholinergic interneuron marker ChAT in Isl1 conditional knock-outs (B, arrowheads) compared with controls (A, arrowheads; average number of ChAT⁺ interneurons ± SD: controls, 266 ± 25, n = 5; Isl1^fl/fl; Six3-cre, 36 ± 20, n = 5; p < 0.0002, Student’s t test). NPY⁺ interneurons were not significantly different between Isl1 conditional knock-outs (D) and controls (C; average number of NPY⁺ interneurons ± SD:controls,232 ± 24, n = 5; Isl1^fl/fl; Six3-cre, 230 ± 33, n = 4; p ≤ 0.93, Student’s t test). Parvalbumin-positive interneurons (E, F) also showed no significant difference between groups (average number of parvalbumin-positive interneurons ± SD: controls, 264 ± 80, n = 5; Isl1^fl/fl; Six3-cre, 206 ± 46, n = 5; p ≤ 0.20, Student’s t test). Similarly, calretinin-positive interneurons (G,H) were not significantly different (average number of calretinin-positive interneurons ± SD: controls, 15 ± 3, n = 5; Isl1^fl/fl; Six3-cre, 18 ± 6, n = 5; p ≤ 0.35, Student’s t test). NC, Neocortex. Scale bar, 250 µm.

Restricted reduction of cortically projecting cholinergic neurons of the nucleus basalis in the Isl1 conditional knock-out. A–D, Light micrographs of anti-ChAT staining in the nucleus basalis (NB) of control and Isl1 conditional knock-outs at 3 weeks of age demonstrating a loss of the majority of ChAT⁺ cells in the NB. E, F, Light micrographs of acetylcholinesterase staining at the neocortical surface in coronal sections of control (E) and Isl1 conditional knock-out (F) brains showing reduced acetylcholinesterase staining in the cortex. G–J, Light micrographs of anti-ChAT staining of additional forebrain cholinergic populations. Anti-ChAT expression in the MS and vertical limb of the diagonal band (DBv) is not markedly reduced in conditional knock-outs (H) compared with controls (G). Expression of ChAT⁺ interneurons in the nucleus accumbens shell (NaS), however, is reduced (compare G, H). Anti-ChAT staining of the MCPO and adjacent SI is not substantially reduced in Isl1 conditional knock-outs (J) compared with controls (I). Additional acetylcholinesterase staining reveals no clear disruption in cholinergic innervation to the hippocampus in conditional knock-outs (L) versus controls (K). GP, Globus pallidus; NC, neocortex; DG, dentate gyrus. Scale bars: (in J) A, B, G–J, 250 µm; (in L) E, F, K, L, 20 µm.

Subsets of cholinergic neurons fail to differentiate in the Isl1 conditional knock-out telencephalon at P0. A, B, Light micrographs of anti-ChAT staining at P0 demonstrating its reduction in the nucleus basalis (NB) and caudate–putamen (CP) in knock-outs (B, arrowheads) versus controls (A, arrowheads). Ngfr mRNA expression is considerably reduced in the NB of P0 knock-outs (D, arrowheads) versus controls (C, arrowheads), whereas expression in the MCPO of knock-outs (F , arrowheads) is not clearly disrupted compared with controls (E, arrowheads). Anti-TrkA immunofluorescence shows a reduction in TrkA⁺ neurons in the CP of knock-outs (H, arrowhead) compared with controls (G, arrowheads) at P0, and a similar reduction in TrkA⁺ neurons in the NB of knock-outs (J, arrowheads) compared with controls (I, arrowheads). Lhx8 mRNA expression reveals no clear reduction of Lhx8 expression in the MS, but a decrease in CP expression levels in knock-outs (L, arrowheads) versus controls (K , arrowheads). Higher magnification of Lhx8 mRNA expression in the CP demonstrates the presence of sustained Lhx8 expression despite the lower expression levels in knock-outs (N, arrowheads) compared with controls (M, arrowheads). Lhx8 mRNA is also maintained in the globus pallidus (GP) and, putatively, the ansa lenticularis (AL) in conditional knock-outs (P, arrowheads) as in controls (O, arrowheads). Lhx6 mRNA expression is not clearly disrupted, both in the neocortex (NC) (asterisks) and CP (arrowheads) in conditional knock-outs (R, arrowheads) versus controls (Q, arrowheads). HP, Hippocampus. Scale bars: (in B) A, B, 250 µm; (in F) C–F, 100 µm; (in J) G–J, 50 µm; (in R) K, L, O–R, 500 µm.

Isl1-lacZ expression in restricted forebrain cholinergic neurons. A–I, Light micrographs of X-gal-stained tissue from 3-week-old Isl1^lacZ/+ mice double labeled for cholinergic and striatal markers demonstrating colocalization of Isl1-lacZ expression with the cholinergic marker ChAT in the caudate–putamen (CP) (A), olfactory tubercle (OT) (B), and nucleus basalis (NB) (C), with varying colocalization in the MCPO (D) and MS (E). Colocalization was not encountered in DARPP-32⁺ triatal projection neurons (F), or other interneuron subtypes in the CP, includingNPY⁺ (G), parvalbumin-positive (H), or calretinin-positive (I) interneurons.J–O, Light micrographs of X-gal-stained tissue from 3-week-old Six3-cre; R26R mice demonstrating colocalization of the recombination pattern of Six3-cre with ChAT in the CP (J), NB (K), and MS (L). Colocalization was also demonstrated in NPY⁺ (M) and parvalbumin-positive (N) but not calretinin-positive (O) interneurons. P–Q, Confocal micrographs of Isl1 and pH3-immunolabeling at E13.5 demonstrating exclusion of Isl1 from pH3⁺ mitotic cells in the LGE subventricular zone (SVZ) as well as the AEP/POa. Six3-cre-mediated recombination of floxed Isl1 resulted in depletion of Isl1 immunolabeling in the AEP/POa (Q, arrowheads), but residual expression in the LGE (Q, brackets). MGE, Medial ganglionic eminence. Scale bars: (inO) A–O, 20 µm; (in Q) P, Q, 100 µm.