An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size

Abstract

Quantal neurotransmitter release at excitatory synapses depends on glutamate import into synaptic vesicles by vesicular glutamate transporters (VGLUTs). Of the three known transporters, VGLUT1 and VGLUT2 are expressed prominently in the adult brain, but during the first two weeks of postnatal development, VGLUT2 expression predominates. Targeted deletion of VGLUT1 in mice causes lethality in the third postnatal week. Glutamatergic neurotransmission is drastically reduced in neurons from VGLUT1-deficient mice, with a specific reduction in quantal size. The remaining activity correlates with the expression of VGLUT2. This reduction in glutamatergic neurotransmission can be rescued and enhanced with overexpression of VGLUT1. These results show that the expression level of VGLUTs determines the amount of glutamate that is loaded into vesicles and released and thereby regulates the efficacy of neurotransmission.

Fig. 1.

Targeting of VGLUT1. (A) Targeting vector and VGLUT1 locus before and after homologous recombination. The horizontal arrow indicates the start codon. Coding exons 1–5 were replaced by a synaptobrevin 2-enhanced yellow fluorescent protein neomycin resistance (syb2-EYFP-neo) cassette, the positions of AseI restriction sites and 5′ Southern probe are marked. (B) Genomic Southern blot with 5′ probe after AseI digest of stem-cell DNA. The replacement of 6.2 kb of the VGLUT1 locus with the 2.5-kb synaptobrevin 2-enhanced yellow fluorescent protein neomycin resistance cassette results in a shift of the 8.4-kb WT band to 15.6 kb. (C) Genomic PCR with 280 bp for WT and 357 bp for targeted locus.

Fig. 2.

Protein and morphological analyses. (A) Parasagittal Nissl-stained brain sections of a P17 -/- animal and a +/- littermate. (B) Western blot of +/+ and -/- synaptosomes. VGAT, vesicular GABA transporter. SNAP-25, synaptosomal-associated protein 25. GluR, glutamate receptors. (C and D) Immunofluorescence analysis of VGLUT1 (green) and VGLUT2 (red) in P17 +/+ and -/- cerebellum. (C) VGLUT1^+/+ staining is found throughout parallel fibers in the molecular layer (ML). VGLUT2 staining is weak, except in climbing fibers. PL, purkinje layer. GL, granular layer. (D) VGLUT2 staining alone in +/+, for comparison with VGLUT2 staining (E) in -/- cerebellum. (Scale bar, 40 μm.)

Fig. 3.

VGLUT1 and VGLUT2 expression in autaptic neurons. (A) VGLUT1^+/+ neuron with strong staining for VGLUT1 (green) and VGLUT2 (red). Colocalization is shown in yellow. (Scale bar: 25 μm.) (B) VGLUT1^-/- neuron with small EPSC that is weakly positive for VGLUT2 (red), apparent at higher magnification in D. Synapses are stained for synapsin (green). (Scale bar, 25 μm.) (C) High magnification of VGLUT1 (green) and VGLUT2 (red) colocalization (yellow) in the +/+ neuron from A. (Scale bar, 5 μm.) (D) High magnification of -/- synapses labeled for synapsin (green). Some synapses are also positive for VGLUT2 (red). Clearly VGLUT2-positive synapses are marked with arrows, and colocalization with synapsin is shown in yellow. (Scale bar, 5 μm.)

Fig. 4.

EPSC amplitudes and release probability. (A) Sample traces from WT and small response KO cells and distribution of EPSC amplitudes from WT (gray) and KO neurons. (Inset) Mean amplitudes of WT, KO, and KO neurons rescued with a VGLUT1 Semliki Forest virus. (B) Release probabilities calculated from the ratio of EPSC and readily releasable pool size. (C) EPSC amplitudes during a 10-Hz stimulation, normalized to the size of the first response.

Fig. 5.

mEPSC frequency and amplitude. (A) Sample traces of mEPSC recordings in WT and KO neurons in the presence and absence of kynurenic acid. A KO mEPSC is magnified, and distribution of mEPSC amplitudes in WT (gray) and KO neurons is shown. (Inset) Cumulative representation of mEPSC distribution. (B) mEPSC frequency, silent KO neurons not included. (C) Mean mEPSC amplitudes with KO neurons sorted according to evoked EPSC size. (D) Rescue of KO cells with VGLUT1 (dark gray) shifts mEPSC amplitudes to larger values. (E) VGLUT1 overexpression in WT cells (dark gray) shifts mEPSC amplitudes to sizes larger than WT (gray) amplitudes.

Fig. 6.

FM1-43 staining and destaining. The fluorescence difference before and after specific destaining is shown. Synaptic FM1-43 uptake and release is indistinguishable between WT neurons (A), KO neurons with small EPSCs (B), and silent KO neurons (C). (D) Mean EPSC sizes of imaged WT and KO neurons. (E) Mean integrated FM1-43 fluorescence differences in WT cells and KO cells with small evoked responses and no response. (Scale bar, 20 μm.)