Fig 2: Expression levels of neurotrophins, neurotrophin-receptors and functional neuronal markers. Overall brain gene expression levels of A neurotrophins and B neurotrophin-receptors; C gene expression levels of glutamate transporters (EAAT2, VGLUT1) and GABAa1 subunit; D expression of glutamate decarboxylases GAD65 and GAD67 and F western blot analysis; E expression levels of GABA transporters (GAT-1, GAT-2, GAT-3, VGAT); G protein levels of beta-III-tubulin (TUBB3). Bar charts represent mean values + SEM obtained in two independent experiments and analyzed together, n = 4–5 per experiment, *p < 0.05, **p < 0.01 (Mann–Whitney U-test); Control (black bars) and PACAP-treated (white bars). Western blot membranes show two representative samples of each group, and bar charts represent mean values + SEM, n = 4

Fig 3: tPA-Cer cells are positive for immunocytochemical markers of O-LM inhibitory interneurons. Dorsal hippocampal sections from tPABAC-Cer transgenic mice were probed for previously confirmed immunocytochemical markers of O-LM inhibitory interneurons. While O-LM interneurons are known for expressing SST (A), other morphologically distinct interneuron subgroups have been shown to stain positive for the neuropeptide. O-LM interneurons, however, have been shown to be strongly decorated with the metabotropic glutamate receptor 1a (mGluR1a), and they have been shown to express the calcium-binding protein calbindin. High-resolution, confocal images (63×) of 10-µm-thick z-stack maximum intensity projections demonstrate that tPA-Cer-positive cells bodies in SO/alveus coexpress both mGluR1a (B) and calbindin (C). Sections were also stained for GAD65, confirming the GABAergic nature of the tPA-Cer-expressing cells (D). In agreement with this, O-LM immunocytochemical profile was the observation of axonal-like projections in SR, as axons of O-LM interneurons extend to stratum lacunosum moleculare (E). Pictures are representative images from SO/alveus of stainings from at least four transgenic mice (n = 4–8) per interneuronal marker. mGluR1a, metabotropic glutamate receptor 1a; GAD65, glutamic acid decarboxylase 65. Scale bars: 5 µm (A–E).

Fig 4: DZP downregulates gephyrin independent of ?2 surface levels. (A) Cortical neurons were treated for 24 h with vehicle or 1 µM DZP, then immunostained for surface ?2 GABAAR (green), followed by permeabilization and immunostaining for (P)Ser270 gephyrin (red), and GAD65 (blue). Panels below show enlargements of GABAAR synapses on dendrites. (B) Dendrite surface synaptic and extrasynaptic ?2 levels are not significantly altered by DZP. Synaptic phospho-gephyrin was enhanced in response to DZP (n = 69–74 neurons; 4 independent cultures). (C) Neurons were treated as in (A) followed by antibody staining for total gephyrin (green) and (P)Ser270 gephyrin (red). Panels below show enlargements of dendrite region. (D) The dendritic pool of gephyrin was decreased, while (P)Ser270 gephyrin levels were augmented, resulting in a dramatic increase in the ratio of phosphorylated gephyrin to total gephyrin (n = 52–59 neurons; 3 independent cultures). Int., fluorescence intensity. Image scale bars: main panels = 5 µm, enlargements = 1 µm. *p = 0.05, **p < 0.01, ***p < 0.001, Student’s t-test; error bars ± s.e.m.

Fig 5: Specific elimination of a5-GABAARs from interneurons versus pyramidal neurons. A) Genetic strategies used to eliminate a5-GABAARs from interneurons (top) or pyramidal neurons (bottom). For a5-i-KO mice, Cre recombinase was driven by the interneuron-specific GAD2 promoter to target the floxed Gabra5 gene. For a5-pyr-KO mice, Cre was driven by the pyramidal neuron-specific CaMKIIa promoter. B) Western blot analysis of a5-GABAAR (55 kDa) expression in male and female WT, p-WT, a5-i-KO, and a5-gl-KO mice. Expression levels were normalized to ß-actin (42 kDa) and quantified by densitometry. Note that receptor levels were unchanged in p-WT and a5-i-KO mice versus WT mice, consistent with maintained expression in pyramidal neurons. C) Immunohistochemical analysis of a5-GABAAR expression levels in interneurons, in WT, a5-gl-KO, a5-i-KO, and a5-pyr-KO mice (ordered from top to bottom). Significant loss of a5-GABAAR staining from interneurons (GAD2-positive cells) was seen in a5-gl-KO and a5-i-KO but not a5-pyr-KO mice. (D) Quantification of punctate a5-GABAAR staining within all identified interneurons (GAD2+/DAPI+ neurons) in WT, a5-gl-KO, a5-i-KO, and a5-pyr-KO mice (N = 3 for each genotype). Expression levels were significantly reduced in a5-gl-KO and a5-i-KO mice but not in a5-pyr-KO mice.