Prolonged activity-deprivation causes pre- and postsynaptic compensatory plasticity at neocortical excitatory synapses.

Homeostatic plasticity stabilizes firing rates of neurons, but the pressure to restore low activity rates can significantly alter synaptic and cellular properties. Most previous studies of homeostatic readjustment to complete activity silencing in rodent forebrain have examined changes after two days of deprivation, but it is known that longer periods of deprivation can produce adverse effects. To better understand the mechanisms underlying these effects and to address how presynaptic as well as postsynaptic compartments change during homeostatic plasticity, we subjected mouse cortical slice cultures to a more severe five-day deprivation paradigm. We developed and validated a computational framework to measure the number and morphology of presynaptic and postsynaptic compartments from super resolution light microscopy images of dense cortical tissue. Using these tools, combined with electrophysiological miniature excitatory postsynaptic current measurements, and synaptic imaging at the electron microscopy level, we assessed the functional and morphological results of prolonged deprivation. Excitatory synapses were strengthened both presynaptically and postsynaptically. Surprisingly, we also observed a decrement in the density of excitatory synapses, both as measured from colocalized staining of pre- and postsynaptic proteins in tissue, and from the number of dendritic spines. Overall, our results suggest that cortical networks deprived of activity progressively move towards a smaller population of stronger synapses.Significance statement Blocking activity in neocortical slice cultures produced coordinated pre and postsynaptic changes at excitatory synapses. Functional and structural assays suggest that deprivation results in fewer excitatory synapses, but each is strengthened both pre- and postsynaptically. This may contribute to the emergence of epileptiform activity.

Progressive Circuit Hyperexcitability in Mouse Neocortical Slice Cultures with Increasing Duration of Activity Silencing.

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 3 d after slices were released from 5 d of deprivation, they were less able to recover after 10 d 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 activation of Hebbian plasticity. 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 excitatory synapses. Putative excitatory synapses colocalizing PSD-95 and Bassoon declined by 39 and 56% following 5 and 10 d 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.

A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons.

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons - the most molecularly distinct subtype - possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

Impaired NMDA receptor transmission alters striatal synapses and DISC1 protein in an age-dependent manner.

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 age-dependent 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 synapse-specific 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.

Activation of CaMKII in single dendritic spines during long-term potentiation.

Calcium/calmodulin-dependent kinase II (CaMKII) plays a central part 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 two-photon fluorescence lifetime imaging microscopy, in combination with two-photon glutamate uncaging. Induction of LTP and associated spine enlargement in single spines triggered transient ( approximately 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 by nanodomain Ca(2+) 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.

Self-phase modulation signatures of neuronal activity.

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.