Multiplex imaging of neural activity and signaling dynamics

Deciphering the intricate and interactive relationship between the information encoded in the genome and the ongoing synaptic activity is critical for understanding the molecular and cellular signaling underlying long-term memory formation and maintenance. To systematically dissect this question, we have investigated the molecular basis of the signaling from synapses to the nucleus and from the nucleus to the synapses, which crucially determines the persistence of synaptic plasticity. Recent evidence suggests that a synaptic activity-dependent protein kinase cascade CaMKK-CaMKIV critically controls the amplitude and time course of phosphorylation of a nuclear transcription factor CREB. We found that during fear memory formation, another route mediated by calcineurin and CRTC1, a CREB co-activator, may also independently contribute to transcriptional activation downstream of synaptic activity, thus indicating that a plethora of Ca2+-driven signal transduction mechanisms regulate transcriptionally-controlled aspects of long-term memory. What then lies downstream of CREB? The immediate early gene Arc’s rapid induction following strong physiological stimuli is dictated by a potent synaptic activity-responsive element (SARE) present in its enhancer/promoter region, which strikingly harbors a unique cluster of binding sites for CREB, MEF2 and SRF/TCF. This modularity could be artificially enhanced, to create a synthetic promoter E-SARE, which now allows to map, label, record and manipulate active neuronal ensembles in various areas of the brain in vivo. Within an active neuronal circuit, Arc’s biological function is further determined by an “inverse” synaptic tagging mechanism, through which one of CREB’s target gene, Arc, acts as a brake that helps weaken the non-potentiated synapses during the maintenance phase of synaptic plasticity, thereby regulating long-term memory. To decipher dynamic brain information processing in active ensembles, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 msec of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo. Using an RGB combination, recording from up to three distinct ensembles was possible in freely moving mice. In vivo paired recording of preand postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. These findings collectively shed light on new multiplex imaging technologies that will help address in the near future key molecular and cellular steps governing the formation and maintenance of long-term memory in vivo.