fm1-43 and biocytin

Synaptic scaling is a form of homeostatic plasticity that scales synaptic strengths up or down to compensate for prolonged changes in activity. It has been controversial whether this plasticity is expressed presynaptically, postsynaptically, or both. Here we describe in detail the homeostatic changes that take place at excitatory synapses in visual cortical cultures after 1 or 2 d of activity blockade. After 7-10 d in vitro, activity blockade significantly increased postsynaptic accumulation of synaptic AMPA receptors via proportional increases in glutamate receptor 1 (GluR1) and GluR2. Time-lapse imaging of enhanced green fluorescent protein-tagged AMPA receptors revealed that receptor accumulation increased progressively over 2 d of activity blockade and affected the entire population of imaged synapses. The strength of synaptic connections between pyramidal neurons was more than doubled after activity blockade without affecting short-term depression or the coefficient of variation of the postsynaptic responses. Furthermore, uptake of the fluorescent styryl dye FM1-43 (N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl] pyridinium dibromide) by presynaptic terminals was not different at control and activity-blocked synapses. In addition to the increased accumulation of postsynaptic AMPA receptors, boosting of dendritic AMPA currents by sodium channels was increased by activity blockade. These data indicate that, at young neocortical synapses, synaptic scaling has a predominantly postsynaptic locus and functions as a gain control mechanism to regulate neuronal activity without affecting the dynamics of synaptic transmission.

Topics: Action Potentials; Animals; Animals, Newborn; Cells, Cultured; Diagnostic Imaging; Disks Large Homolog 4 Protein; Electric Stimulation; Homeostasis; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Lysine; Membrane Proteins; Neocortex; Neuronal Plasticity; Neurons; Patch-Clamp Techniques; Potassium Chloride; Pyridinium Compounds; Quaternary Ammonium Compounds; Rats; Rats, Long-Evans; Receptors, AMPA; Synapses; Synapsins; tau Proteins; Tetrodotoxin; Time Factors; Transfection

Competitive synaptic remodeling is an important feature of developmental plasticity, but the molecular mechanisms remain largely unknown. Calcium/calmodulin-dependent protein kinase II (CaMKII) can induce postsynaptic changes in synaptic strength. We show that postsynaptic CaMKII also generates structural synaptic rearrangements between cultured cortical neurons. Postsynaptic expression of activated CaMKII (T286D) increased the strength of transmission between pairs of pyramidal neuron by a factor of 4, through a modest increase in quantal amplitude and a larger increase in the number of synaptic contacts. Concurrently, T286D reduced overall excitatory synaptic density and increased the proportion of unconnected pairs. This suggests that connectivity from some synaptic partners was increased while other partners were eliminated. The enhancement of connectivity required activity and NMDA receptor activation, while the elimination did not. These data suggest that postsynaptic activation of CaMKII induces a structural remodeling of presynaptic inputs that favors the retention of active presynaptic partners.

Topics: Alanine; Anesthetics, Local; Animals; Animals, Newborn; Aspartic Acid; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Count; Cell Surface Extensions; Cerebral Cortex; Dizocilpine Maleate; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fluorescent Dyes; Gene Expression Regulation, Enzymologic; Green Fluorescent Proteins; Immunohistochemistry; Luminescent Proteins; Lysine; Mutation; Nerve Tissue Proteins; Neuronal Plasticity; Neurons; Organ Culture Techniques; Pyridinium Compounds; Quaternary Ammonium Compounds; Rats; Rats, Long-Evans; Synapses; Synapsins; Synaptic Transmission; Tetrodotoxin; Threonine; Transfection; Valine