Significance: Phgdh is a crucial enzyme in the astrocytic synthesis of l -serine from glucose. Our study uncovers the role of glial-neuronal cross-talk in regulating NMDAR synaptic activation through the astrocytic export of Phgdh-derived l -serine to generate the coagonists d -serine and glycine. We clarify the relative roles of d -serine and glycine by showing that like d -serine, Phgdh-derived glycine mediates NMDAR synaptic activation in the mature hippocampus. We show that glycine is a significant regulator of d -serine synthesis and release, revealing a cross-talk between glycine and d -serine metabolism that fine tunes NMDAR activation. Our data reveal a multifaceted mechanism regulating NMDAR activity, which ultimately depends on a Phgdh-dependent serine shuttle.

NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity.

Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. “MAG” PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, l-MAG0₄₆₀, to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, d-MAG0₄₆₀. 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca²⁺-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca²⁺ imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.

The photoactivatable calcium sensors reported in this paper allow simultaneous highlighting of cellular morphology and recording of calcium activity, which is demonstrated in neuronal cultures, in Drosophila and in zebrafish. Circuit mapping requires knowledge of both structural and functional connectivity between cells. Although optical tools have been made to assess either the morphology and projections of neurons or their activity and functional connections, few probes integrate this information. We have generated a family of photoactivatable genetically encoded Ca2+ indicators that combines attributes of high-contrast photolabeling with high-sensitivity Ca2+ detection in a single-color protein sensor. We demonstrated in cultured neurons and in fruit fly and zebrafish larvae how single cells could be selected out of dense populations for visualization of morphology and high signal-to-noise measurements of activity, synaptic transmission and connectivity. Our design strategy is transferrable to other sensors based on circularly permutated GFP (cpGFP).