Ca2+ / Calmodulin-dependent kinase II (CaMKII) plays a central role in long-term potentiation (LTP), which underlies some forms of learning and memory. Here we monitored the spatiotemporal dynamics of CaMKII activation in individual dendritic spines during LTP using 2-photon fluorescence lifetime imaging microscopy in combination with 2-photon glutamate uncaging. Induction of LTP and associated spine enlargement in single spines triggered transient (∼ 1 min) CaMKII activation restricted to the stimulated spines. CaMKII in spines was specifically activated by NMDA receptors and L-type voltage sensitive calcium channels, presumably via nanodomain Ca2+ near the channels, in response to glutamate uncaging and depolarization, respectively. The high degree of compartmentalization and channel specificity of CaMKII signalling allow stimuli-specific spatiotemporal patterns of CaMKII signalling and may be important for synapse-specificity of synaptic plasticity.

NMDA receptors are key regulators of synaptic plasticity, and their hypofunction is thought to contribute to the pathophysiology of CNS disorders. Furthermore, NMDA receptors participate in the formation, maintenance, and elimination of synapses. The consequences of NMDA receptor hypofunction on synapse biology were explored in a genetic mouse model, in which the levels of NMDA receptors are reduced to 10% of normal levels (i.e., NR1-knockdown mice). In these mice, synapse number is reduced in an agedependent manner; reductions are observed at the postpubertal age of 6 wk, but normal at 2 wk of age. Efforts to uncover the biochemical underpinnings of this phenomenon reveal synapsespecific reductions in 14-3-3ε protein and in Disrupted in Schizophrenia-1 (DISC1), two schizophrenia susceptibility factors that have been implicated in the regulation of spine density. Subchronic administration of MK-801, an NMDA receptor antagonist, produces similar synaptic reductions in both spine density and DISC1, indicating that synaptic levels of DISC1 are regulated by NMDA receptor function. The synaptic reduction of DISC1 and 14-3-3ε is developmentally correlated with the age-dependent decrease in striatal spine density.glutamate | neurodevelopmental A defining feature of neurons is their ability to alter the number and strength of synaptic connections with experience. At the cellular level, changes in synapse number, or postsynaptic spine density, occur with learning and memory formation (1) or exposure to psychoactive drugs (2), and in neurodevelopmental diseases including schizophrenia (3, 4), fragile-X mental retardation (5), and Rett syndrome (6). At the molecular level, NMDA-type glutamate receptors have long been appreciated for their role in the formation and maintenance of glutamatergic synapses (7), and as mediators of synaptic plasticity (8). Several studies have shown a positive correlation between NMDA receptor activity and spine density (9-12), with notable exceptions (13,14). However, the molecular mechanisms by which NMDA receptors regulate spine density remain to be fully elucidated. In the case of disease states such as schizophrenia, a fuller understanding of this molecular machinery may point to new therapeutic strategies.The striatum represents an ideal brain region in which to further explore the biochemical mechanisms by which NMDA receptors regulate spine density, because the vast majority of neurons (95%) within this brain structure are medium spiny neurons (MSNs), which have densely spinous dendrites, upon which glutamate and dopamine afferents converge (15,16). This neuronal homogeneity allows for ex vivo biochemical preparation of synaptic proteins from a nearly homogenous neuronal substrate. MSNs are thought to be a principal site of action of antipsychotic drugs because they express the highest levels of D2 dopamine receptors (17). Furthermore, they participate in many of the cognitive and limbic behaviors that are altered in schizophrenia (17, 18).We hypothesized that reduced NMDA receptor funct...

We use phase-sensitive detection of spectral hole refilling to demonstrate strong novel intrinsic nonlinear signatures of neuronal activation in hippocampal brain slices. The ability to gain access to this fundamentally new intrinsic contrast with modest power levels suggests a new approach to in vivo neural imaging. We expect that we can extrapolate our method to high spatial and temporal resolution in deep tissue while retaining the noninvasive character.

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Forebrain neurons deprived of activity become hyperactive when activity is restored. Rebound activity has been linked to spontaneous seizures in vivo following prolonged activity blockade. Here we measured the time course of rebound activity and the contributing circuit mechanisms using calcium imaging, synaptic staining and whole cell patch clamp in organotypic slice cultures of mouse neocortex. Calcium imaging revealed hypersynchronous activity increasing in intensity with longer periods of deprivation. While activity partially recovered three days after slices were released from five days of deprivation, they were less able to recover after ten days of deprivation. However, even after the longer period of deprivation, activity patterns eventually returned to baseline levels. The degree of deprivation-induced rebound was age-dependent, with the greatest effects occurring when silencing began in the second week. Pharmacological blockade of NMDA receptors indicated that hypersynchronous rebound activity did not require Hebbian plasticity evoked. In single neuron recordings, input resistance roughly doubled with a concomitant increase in intrinsic excitability. Synaptic imaging of pre- and postsynaptic proteins revealed dramatic reductions in the number of presumptive synapses with a larger effect on inhibitory than seen in excitatory synapses. Putative excitatory synapses colocalizing PSD-95 and Bassoon declined by 39% and 56% following five and ten days of deprivation, but presumptive inhibitory synapses colocalizing gephyrin and VGAT declined by 55% and 73% respectively. The results suggest that with prolonged deprivation, a progressive reduction in synapse number is accompanied by a shift in the balance between excitation and inhibition and increased cellular excitability.