Molecular and Electrophysiological Characterization of GFP-Expressing CA1 Interneurons in GAD65-GFP Mice

Abstract

The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ∼20% of interneurons in this area. The majority of them expresses either reelin (70±2%) or vasoactive intestinal peptide (VIP; 15±2%), while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping) expression of neuropeptide Y (42±3%), cholecystokinin (25±3%), calbindin (20±2%) or calretinin (20±2%). Most of these subtypes (with the exception of calretinin-expressing interneurons) target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials.

Precision of Inhibition: Dendritic Inhibition by Individual GABAergic Synapses on Hippocampal Pyramidal Cells Is Confined in Space and Time

“…The firing profiles are consistent with previous reports for VIP-or Reelin-positive CGE-derived interneurons (Miyoshi et al, 2010;Wierenga et al, 2010) and consist of $50% delayed and (moderately) fast-spiking (20/21 dNSF3), $20% delayed and regular-spiking (9/10 LS1/2), and $30% non-delayed, mostly regular-spiking interneurons (5/15 bNA2) ( Figures 1D and 1E). Reversal potentials were well within the physiological range, as confirmed by gramicidin perforatedpatch recordings ( Figure 1F, À69.6 ± 1.5 mV versus 71.4 ± 1.2 mV at 24 C and À74.1 ± 2.2 mV versus À75.3 ± 2.1 mV at 34 C in whole-cell versus perforated, mean ± SEM, differences not significant, n = 9/11/7/5), and stable during wholecell recordings of the cells ( Figures 1G and S1).…”

“…Hippocampal slices (350 mm thick) were prepared from postnatal day 4-6 GAD65-GFP (Wierenga et al, 2010;Ló pez-Bendito et al, 2004) or C57Bl/6 mice and maintained in culture for 2-6 weeks following standard protocols (Stoppini et al, 1991, medium glucose reduced to 5.7 mg/ml).…”

“…Left: connected INs (n = 49); right: subset of INs included in our dataset (n = 12), excluding, e.g., cells with no contacts detected or with IPSC rundown. (E) Representative firing pattern of the three groups delayed fast/delayed regular/non-delayed (Wierenga et al, 2010). (F) Representative IV curves for perforated-patch and whole-cell recordings.…”

“…paired patch-clamp recordings from GABAergic interneurons and nearby pyramidal cells in organotypic hippocampal slice cultures prepared from GAD65-GFP mice (Ló pez-Bendito et al, 2004). Here we took advantage of the strong GFP expression in a subset of mostly dendrite-targeting GABAergic interneurons (Wierenga et al, 2010). A GFP-positive interneuron with its soma in stratum radiatum/oriens or at the border to stratum pyramidale and a nearby pyramidal cell were patched simultaneously ( Figure 1A).…”

Immunofluorescently labeling glutamic acid decarboxylase 65 coupled with confocal imaging for identifying GABAergic somata in the rat dentate gyrus—A comparison with labeling glutamic acid decarboxylase 67

“…However, the uses of colchicine are limited because colchicine is neurotoxic (Sutula et al, 1983) and it has to be given hours or days before sacrificing the animals. Over the past decade, transgenic animals knocking in GAD 67 -GFP or GAD 65 -GFP have been favored for the identification of GABAergic cells (Tamamaki et al, 2003;Wierenga et al, 2010). Using transgenic animals has numerous advantages over immunohistochemistry or in situ hybridization.…”