GABA signaling sustains fundamental brain functions, from nervous system development to the synchronization of population activity and synaptic plasticity. Despite these pivotal features, molecular determinants underscoring the rapid and cell-autonomous replenishment of the vesicular neurotransmitter GABA and its impact on synaptic plasticity remain elusive. Here, we show that genetic disruption of the glutamine transporter Slc38a1 in mice hampers GABA synthesis, modifies synaptic vesicle morphology in GABAergic presynapses and impairs critical period plasticity. We demonstrate that Slc38a1-mediated glutamine transport regulates vesicular GABA content, induces high-frequency membrane oscillations and shapes cortical processing and plasticity. Taken together, this work shows that Slc38a1 is not merely a transporter accumulating glutamine for metabolic purposes, but a key component regulating several neuronal functions.

Axon guidance requires interactions between extracellular signaling molecules and transmembrane receptors, but how appropriate context-dependent decisions are coordinated outside the cell remains unclear. Here we show that the transmembrane glycoprotein Dystroglycan interacts with a changing set of environmental cues that regulate the trajectories of extending axons throughout the mammalian brain and spinal cord. Dystroglycan operates primarily as an extracellular scaffold during axon guidance, as it functions non-cell autonomously and does not require signaling through its intracellular domain. We identify the transmembrane receptor Celsr3/Adgrc3 as a binding partner for Dystroglycan, and show that this interaction is critical for specific axon guidance events . These findings establish Dystroglycan as a multifunctional scaffold that coordinates extracellular matrix proteins, secreted cues, and transmembrane receptors to regulate axon guidance.

Widefield imaging of calcium dynamics is an emerging method for mapping regional neural activity but is currently limited to restrained animals. Here we describe cScope, a head-mounted widefield macroscope developed to image large-scale cortical dynamics in rats during natural behavior. cScope provides a 7.8 × 4 mm field of view and dual illumination paths for both fluorescence and hemodynamic correction and can be fabricated at low cost using readily attainable components. We also report the development of Thy-1 transgenic rat strains with widespread neuronal expression of the calcium indicator GCaMP6f. We combined these two technologies to image large-scale calcium dynamics in the dorsal neocortex during a visual evidence accumulation task. Quantitative analysis of task-related dynamics revealed multiple regions having neural signals that encode behavioral choice and sensory evidence. Our results provide a new transgenic resource for calcium imaging in rats and extend the domain of head-mounted microscopes to larger-scale cortical dynamics.

Calcium imaging is commonly used to measure the neural activity of large groups of neurons in mice. Genetically encoded calcium indicators (GECIs) can be delivered for this purpose using non-invasive genetic methods. Compared to viral gene transfer, transgenic targeting of GECIs provides stable long-term expression and obviates the need for invasive viral injections. Transgenic mice expressing the green GECI GCaMP6 are already widely used. Here we present the generation and characterization of transgenic mice expressing the sensitive red GECI jRGECO1a, driven by the Thy1 promoter. Four transgenic lines with different expression patterns showed sufficiently high expression for cellular in vivo imaging. We used two-photon microscopy to characterize visual responses of individual neurons in the visual cortex in vivo. The signal-to-noise ratio in transgenic mice was comparable to, or better than, mice transduced with adeno-associated virus. In addition, we show that Thy1-jRGECO1a transgenic mice are useful for transcranial population imaging and functional mapping using widefield fluorescence microscopy. We also demonstrate imaging of visual responses in retinal ganglion cells in vitro. Thy1-jRGECO1a transgenic mice are therefore a useful addition to the toolbox for imaging activity in intact neural networks.

Hunger and pain are two competing signals that individuals must resolve to ensure survival. However, the neural processes that prioritize conflicting survival needs are poorly understood. We discovered that hunger attenuates behavioral responses and affective properties of inflammatory pain without altering acute nociceptive responses. This effect is centrally controlled, as activity in hunger-sensitive agouti-related protein (AgRP)-expressing neurons abrogates inflammatory pain. Systematic analysis of AgRP projection subpopulations revealed that the neural processing of hunger and inflammatory pain converge in the hindbrain parabrachial nucleus (PBN). Strikingly, activity in AgRP → PBN neurons blocked the behavioral response to inflammatory pain as effectively as hunger or analgesics. The anti-nociceptive effect of hunger is mediated by neuropeptide Y (NPY) signaling in the PBN. By investigating the intersection between hunger and pain, we have identified a neural circuit that mediates competing survival needs and uncovered NPY Y1 receptor signaling in the PBN as a target for pain suppression.

Neural stem cells show age-dependent developmental potentials, as evidenced by their production of distinct neuron types at different developmental times. Drosophila neuroblasts produce long, stereotyped lineages of neurons. We searched for factors that could regulate neural temporal fate by RNA-sequencing lineage-specific neuroblasts at various developmental times. We found that two RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp), display opposing high-to-low and low-to-high temporal gradients with lineage-specific temporal dynamics. Imp and Syp promote early and late fates, respectively, in both a slowly progressing and a rapidly changing lineage. Imp and Syp control neuronal fates in the mushroom body lineages by regulating the temporal transcription factor Chinmo translation. Together, the opposing Imp/Syp gradients encode stem cell age, specifying multiple cell fates within a lineage.

Comprehensive measurement of neural activity remains challenging due to the large numbers of neurons in each brain area. We used volumetric two-photon imaging in mice expressing GCaMP6s and nuclear red fluorescent proteins to sample activity in 75% of superficial barrel cortex neurons across the relevant cortical columns, approximately 12,000 neurons per animal, during performance of a single whisker object localization task. Task-related activity peaked during object palpation. An encoding model related activity to behavioral variables. In the column corresponding to the spared whisker, 300 layer (L) 2/3 pyramidal neurons (17%) each encoded touch and whisker movements. Touch representation declined by half in surrounding columns; whisker movement representation was unchanged. Following the emergence of stereotyped task-related movement, sensory representations showed no measurable plasticity. Touch direction was topographically organized, with distinct organization for passive and active touch. Our work reveals sparse and spatially intermingled representations of multiple tactile features.

Cortical cells integrate synaptic input from multiple sources, but how these different inputs are distributed across individual neurons is largely unknown. Differences in input might account for diverse responses in neighboring neurons during behavior. We present a strategy for comparing the strengths of multiple types of input onto the same neuron. We developed methods for independent dual-channel photostimulation of synaptic inputs using ChR2 together with ReaChR, a red-shifted channelrhodopsin. We used dual-channel photostimulation to probe convergence of sensory information in the mouse primary motor cortex. Input from somatosensory cortex and thalamus converges in individual neurons. Similarly, inputs from distinct somatotopic regions of the somatosensory cortex are integrated at the level of single motor cortex neurons. We next developed a ReaChR transgenic mouse under the control of both Flp- and Cre-recombinases that is an effective tool for circuit mapping. Our approach to dual-channel photostimulation enables quantitative comparison of the strengths of multiple pathways across all length scales of the brain.

Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.