Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

Molecular processes regulating the gain of NMDA receptors modulate diverse physiological and pathological responses in the CNS. Group I metabotropic glutamate receptors (mGluRs), which neighbor NMDA receptors and which can be coactivated by synaptically released glutamate, couple to several different second messenger pathways, each of which could target NMDA receptors. In CA3 pyramidal cells we show that the activation of mGluR1 potentiates NMDA current via a G-protein-independent mechanism involving Src kinase activation. In contrast, mGluR5-mediated enhancement of NMDA current requires G-protein activation, triggering a signaling cascade including protein kinase C and Src. These results indicate that one neurotransmitter, glutamate, can activate two distinct and independent signaling systems to target the same effector. These two pathways are likely to contribute significantly to the highly differentiated control of NMDA receptor function.

Coordinated activity patterns in the developing brain may contribute to the wiring of neuronal circuits underlying future behavioural requirements. However, causal evidence for this hypothesis has been difficult to obtain owing to the absence of tools for selective manipulation of oscillations during early development. We established a protocol that combines optogenetics with electrophysiological recordings from neonatal mice in vivo to elucidate the substrate of early network oscillations in the prefrontal cortex. We show that light-induced activation of layer II/III pyramidal neurons that are transfected by in utero electroporation with a high-efficiency channelrhodopsin drives frequency-specific spiking and boosts network oscillations within beta–gamma frequency range. By contrast, activation of layer V/VI pyramidal neurons causes nonspecific network activation. Thus, entrainment of neonatal prefrontal networks in fast rhythms relies on the activation of layer II/III pyramidal neurons. This approach used here may be useful for further interrogation of developing circuits, and their behavioural readout.

In CA3 pyramidal neurons from organotypic slice cultures, activation of G_q‐coupled group I metabotropic glutamate receptors (mGluRs) induces a non‐selective cationic conductance that enhances excitability. We have found that this response shares several properties with conductances that are mediated by the transient receptor potential (TRP) family of ion channels, including inhibition by La³⁺, 2‐aminoethoxydiphenylborane (2APB), cis‐N‐(2‐phenylcyclopentyl)azacyclotridec‐1‐en‐2‐amine (MDL 12,330A) and a doubly rectifying current‐voltage relationship. Stimulation of mGluR1 and mGluR5 converged to activate the TRP‐like conductance in a synergistic manner, and activation of either subtype alone produced only a fraction of the normal response. Activation of the cationic current required elevated intracellular Ca²⁺. Chelating intracellular Ca²⁺ or blocking Ca²⁺ entry through voltage‐gated Ca²⁺ channels attenuated responses to the activation of mGluRs. Conversely, raising intracellular Ca²⁺ potentiated mGluR activation of the TRP‐like conductance. Under control conditions, blocking G protein activation using intracellular GDPβS with or without N‐(2, 6‐dimethylphenylcarbamoylmethyl) triethylammonium chloride (QX‐314) prevented mGluR‐mediated activation of the TRP‐like conductance. Following G protein blockade, however, the coupling between mGluRs 1 and/or 5 and the TRP‐like conductance was rescued by increasing intracellular Ca²⁺. This suggests that a G protein‐independent signalling pathway is also activated by group I mGluRs. Such a pathway may represent an alternative transduction mechanism to maintain metabotropic responses under conditions where G proteins are functionally uncoupled from their cognate receptors.