Abstract
Homeostatic scaling of glutamatergic and GABAergic transmission is triggered by prolonged alterations in synaptic neuronal activity. We have previously described a presynaptic mechanism for synaptic homeostasis and plasticity that involves scaling the level of vesicular glutamate (VGLUT1) and gamma-aminobutyric acid (GABA) (VGAT) transporter biosynthesis. These molecular determinants of vesicle filling and quantal size are regulated by neuronal activity in an opposite manner and bi-directionally. Here, we report that a striking induction of VGLUT2 mRNA and synaptic protein is triggered by a prolonged increase in glutamatergic synaptic activity in mature neocortical neuronal networks in vitro together with two determinants of inhibitory synaptic strength, the neuronal activity-regulated pentraxin (Narp), and glutamate decarboxylase (GAD65). Activity-dependent induction of VGLUT2 and Narp exhibits a similar intermediate-early gene response that is blocked by actinomycin D and tetrodotoxin, by inhibitors of ionotropic glutamate receptors and L-type voltage-gated calcium channels, and is dependent on downstream signaling via calmodulin, calcium/calmodulin-dependent protein kinase (CaMK) and extracellular signal-regulated kinase 1/2 (ERK1/2). The co-induction of VGLUT2 and Narp triggered by prolonged gamma-aminobutyric acid type A receptor blockade is independent of brain-derived nerve growth factor and TrkB receptor signaling. VGLUT2 protein induction occurs on a subset of cortically derived synaptic vesicles in excitatory synapses on somata and dendritic processes of multipolar GABAergic interneurons, recognized sites for the clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptors by Narp. We propose that VGLUT2 and Narp induction by excitation-transcription coupling leads to increased glutamatergic transmission at synapses on GABAergic inhibitory feedback neurons as part of a coordinated program of Ca(2+)-signal transcription involved in mechanisms of homeostatic plasticity after prolonged hyperactivity.
Highlights
Whereas decreased synaptic strength occurs at most excitatory synapses after prolonged neuronal hyperactivity [5], increased glutamatergic synaptic strength has been reported at GABAergic bipolar interneurons [19, 20] providing a mechanism for inhibitory feedback [21]
We conclude that the increased synaptic glutamate receptor activation and the resulting depolarization of neurons after prolonged disinhibition by gabazine initiates a coordinated program of actionpotential-dependent gene transcription for these three established molecular determinants of excitatory or inhibitory strength; VGLUT2 or neuronal activity-regulated pentraxin (Narp) and GAD65
Our results indicate that CaM, calmodulin-dependent protein kinases (CaMK), and extracellular signal-regulated kinase 1/2 (ERK1/2) are important components in Ca2ϩ signal transcription involved in the coordinate induction of VGLUT2 and Narp triggered by prolonged endogenous increase in glutamatergic synaptic activity
Summary
Primary Neuronal Cultures and PC12 Cell Transfection— Primary neuronal cultures were prepared as described [12, 49] with minor modifications. After 3 washes for 10 min with PBS, species-specific and highly cross-adsorbed secondary antibodies coupled to Alexa 488, 594, or 647 (Molecular Probes, Eugene, OR) diluted 1:200 in blocking buffer were applied for 1 h at room temperature and followed by three PBS washes. For analysis of the time-course of VGLUT2 expression after gabazine treatment, vesicle membranes from post-35,000 ϫ g supernatants were pelleted by high speed centrifugation (100,000 ϫ g, 30 min) and subjected to SDS-PAGE electrophoresis and Western blotting. Coupled beads were washed 5 times in PBS and blocked for 10 min with PBS containing 2% glycine, 2% lysine (w/v) followed by a final PBS wash. Control beads were beads coupled with nonspecific rabbit IgGs. Beads were separated from unbound proteins in the supernatant by brief centrifugation (30 s) and washed 5 times with cold PBS with or without 1% Nonidet P-40. VGLUT1, VGLUT2, and synaptophysin were detected using their respective primary antibodies (guinea pig anti-VGLUT1 or VGLUT2, 1/4000, and mouse anti-p38 from Sigma, 1/5000) and horseradish peroxidase-conjugated antiguinea pig or mouse IgG secondary antibodies (Sigma, 1/8000) followed by enhanced chemiluminescence (West Pico, Pierce) and exposure to film (Hyperfilm ECL, Amersham Biosciences)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
