A developmental brain-wide screen identifies retrosplenial cortex as a key player in the emergence of persistent memory.

Memories formed early in life are short-lived while those formed later persist. Recent work revealed that infant memories are stored in a latent state. But why they fail to be retrieved is poorly understood. Here we investigated brain-wide circuit mechanisms underlying infantile amnesia in mice. We performed a screen that combined activity-dependent neuronal tagging at different postnatal ages, tissue clearing and light sheet microscopy. We observed striking developmental transitions in the organization of fear memory networks and changes in the activity and functional connectivity of the retrosplenial cortex (RSP) that aligned with the emergence of persistent memory. 7 days after learning, chemogenetic reactivation of tagged RSP ensembles enhanced memory in adults but not in infants. But after 33 days, reactivating infant-tagged RSP ensembles recovered forgotten memories. These studies show that RSP ensembles store latent infant memories, reveal the time course of RSP functional maturation, and suggest that immature RSP functional networks contribute to infantile amnesia.

ATP-mediated increase in H+ efflux from retinal Müller cells of the axolotl.

Previous work has shown that activation of tiger salamander retinal radial glial cells by extracellular ATP induces a pronounced extracellular acidification, which has been proposed to be a potent modulator of neurotransmitter release. This study demonstrates that low micromolar concentrations of extracellular ATP similarly induce significant H+ effluxes from Müller cells isolated from the axolotl retina. Müller cells were enzymatically isolated from axolotl retina and H+ fluxes were measured from individual cells using self-referencing H+-selective microelectrodes. The increased H+ efflux from axolotl Müller cells induced by extracellular ATP required activation of metabotropic purinergic receptors and was dependent upon calcium released from internal stores. We further found that the ATP-evoked increase in H+ efflux from Müller cells of both tiger salamander and axolotl were sensitive to pharmacological agents known to interrupt calmodulin and protein kinase C (PKC) activity: chlorpromazine (CLP), trifluoperazine (TFP), and W-7 (all calmodulin inhibitors) and chelerythrine, a PKC inhibitor, all attenuated ATP-elicited increases in H+ efflux. ATP-initiated H+ fluxes of axolotl Müller cells were also significantly reduced by amiloride, suggesting a significant contribution by sodium-hydrogen exchangers (NHEs). In addition, α-cyano-4-hydroxycinnamate (4-cin), a monocarboxylate transport (MCT) inhibitor, also reduced the ATP-induced increase in H+ efflux in both axolotl and tiger salamander Müller cells, and when combined with amiloride, abolished ATP-evoked increase in H+ efflux. These data suggest that axolotl Müller cells are likely to be an excellent model system to understand the cell-signaling pathways regulating H+ release from glia and the role this may play in modulating neuronal signaling.NEW & NOTEWORTHY Glial cells are a key structural part of the tripartite synapse and have been suggested to regulate synaptic transmission, but the regulatory mechanisms remain unclear. We show that extracellular ATP, a potent glial cell activator, induces H+ efflux from axolotl retinal Müller (glial) cells through a calcium-dependent pathway that is likely to involve calmodulin, PKC, Na+/H+ exchange, and monocarboxylate transport, and suggest that such H+ release may play a key role in modulating neuronal transmission.

Developmentally distinct architectures in top-down circuits.

The medial prefrontal cortex (mPFC) plays a key role in learning, mood and decision making, including in how individuals respond to threats 1-6 . mPFC undergoes a uniquely protracted development, with changes in synapse density, cortical thickness, long-range connectivity, and neuronal encoding properties continuing into early adulthood 7-21 . Models suggest that before adulthood, the slow-developing mPFC cannot adequately regulate activity in faster-developing subcortical centers 22,23 . They propose that during development, the enhanced influence of subcortical systems underlies distinctive behavioural strategies of juveniles and adolescents and that increasing mPFC control over subcortical structures eventually allows adult behaviours to emerge. Yet it has remained unclear how a progressive strengthening of top-down control can lead to nonlinear changes in behaviour as individuals mature 24,25 . To address this discrepancy, here we monitored and manipulated activity in the developing brain as animals responded to threats, establishing direct causal links between frontolimbic circuit activity and the behavioural strategies of juvenile, adolescent and adult mice. Rather than a linear strengthening of mPFC synaptic connectivity progressively regulating behaviour, we uncovered multiple developmental switches in the behavioural roles of mPFC circuits targeting the basolateral amygdala (BLA) and nucleus accumbens (NAc). We show these changes are accompanied by axonal pruning coinciding with functional strengthening of synaptic connectivity in the mPFC-BLA and mPFC-NAc pathways, which mature at different rates. Our results reveal how developing mPFC circuits pass through distinct architectures that may make them optimally adapted to the demands of age-specific challenges.

Brain-Wide Projections and Differential Encoding of Prefrontal Neuronal Classes Underlying Learned and Innate Threat Avoidance.

To understand how the brain produces behavior, we must elucidate the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) is critical for complex functions including decision-making and mood. mPFC projection neurons collateralize extensively, but the relationships between mPFC neuronal activity and brain-wide connectivity are poorly understood. We performed whole-brain connectivity mapping and fiber photometry to better understand the mPFC circuits that control threat avoidance in male and female mice. Using tissue clearing and light sheet fluorescence microscopy (LSFM), we mapped the brain-wide axon collaterals of populations of mPFC neurons that project to nucleus accumbens (NAc), ventral tegmental area (VTA), or contralateral mPFC (cmPFC). We present DeepTraCE (deep learning-based tracing with combined enhancement), for quantifying bulk-labeled axonal projections in images of cleared tissue, and DeepCOUNT (deep-learning based counting of objects via 3D U-net pixel tagging), for quantifying cell bodies. Anatomical maps produced with DeepTraCE aligned with known axonal projection patterns and revealed class-specific topographic projections within regions. Using TRAP2 mice and DeepCOUNT, we analyzed whole-brain functional connectivity underlying threat avoidance. PL was the most highly connected node with functional connections to subsets of PL-cPL, PL-NAc, and PL-VTA target sites. Using fiber photometry, we found that during threat avoidance, cmPFC and NAc-projectors encoded conditioned stimuli, but only when action was required to avoid threats. mPFC-VTA neurons encoded learned but not innate avoidance behaviors. Together our results present new and optimized approaches for quantitative whole-brain analysis and indicate that anatomically defined classes of mPFC neurons have specialized roles in threat avoidance.SIGNIFICANCE STATEMENT Understanding how the brain produces complex behaviors requires detailed knowledge of the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) plays a key role in learning, mood, and decision-making, including evaluating and responding to threats. mPFC dysfunction is strongly linked to fear, anxiety and mood disorders. Although mPFC circuits are clear therapeutic targets, gaps in our understanding of how they produce cognitive and emotional behaviors prevent us from designing effective interventions. To address this, we developed a high-throughput analysis pipeline for quantifying bulk-labeled fluorescent axons [DeepTraCE (deep learning-based tracing with combined enhancement)] or cell bodies [DeepCOUNT (deep-learning based counting of objects via 3D U-net pixel tagging)] in intact cleared brains. Using DeepTraCE, DeepCOUNT, and fiber photometry, we performed detailed anatomic and functional mapping of mPFC neuronal classes, identifying specialized roles in threat avoidance.

ATP-mediated increase in H+ flux from retinal Müller cells: a role for Na+/H+ exchange.

Small alterations in extracellular H+ can profoundly alter neurotransmitter release by neurons. We examined mechanisms by which extracellular ATP induces an extracellular H+ flux from Müller glial cells, which surround synaptic connections throughout the vertebrate retina. Müller glia were isolated from tiger salamander retinae and H+ fluxes examined using self-referencing H+-selective microelectrodes. Experiments were performed in 1 mM HEPES with no bicarbonate present. Replacement of extracellular sodium by choline decreased H+ efflux induced by 10 µM ATP by 75%. ATP-induced H+ efflux was also reduced by Na+/H+ exchange inhibitors. Amiloride reduced H+ efflux initiated by 10 µM ATP by 60%, while 10 µM cariporide decreased H+ flux by 37%, and 25 µM zoniporide reduced H+ flux by 32%. ATP-induced H+ fluxes were not significantly altered by the K+/H+ pump blockers SCH28080 or TAK438, and replacement of all extracellular chloride with gluconate was without effect on H+ fluxes. Recordings of ATP-induced H+ efflux from cells that were simultaneously whole cell voltage clamped revealed no effect of membrane potential from -70 mV to 0 mV. Restoration of extracellular potassium after cells were bathed in 0 mM potassium produced a transient alteration in ATP-dependent H+ efflux. The transient response to extracellular potassium occurred only when extracellular sodium was present and was abolished by 1 mM ouabain, suggesting that alterations in sodium gradients were mediated by Na+/K+-ATPase activity. Our data indicate that the majority of H+ efflux elicited by extracellular ATP from isolated Müller cells is mediated by Na+/H+ exchange.NEW & NOTEWORTHY Glial cells are known to regulate neuronal activity, but the exact mechanism(s) whereby these "support" cells modulate synaptic transmission remains unclear. Small changes in extracellular levels of acidity are known to be particularly powerful regulators of neurotransmitter release. Here, we show that extracellular ATP, known to be a potent activator of glial cells, induces H+ efflux from retinal Müller (glial) cells and that the bulk of the H+ efflux is mediated by Na+/H+ exchange.

EZcalcium: Open-Source Toolbox for Analysis of Calcium Imaging Data.

Fluorescence calcium imaging using a range of microscopy approaches, such as two-photon excitation or head-mounted "miniscopes," is one of the preferred methods to record neuronal activity and glial signals in various experimental settings, including acute brain slices, brain organoids, and behaving animals. Because changes in the fluorescence intensity of genetically encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on inferring such spiking from changes in pixel intensity values across time within different regions of interest. However, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines. For decades, the only way to perform these analyses was for individual laboratories to write their custom code. These routines were typically not well annotated and lacked intuitive graphical user interfaces (GUIs), which made it difficult for scientists in other laboratories to adopt them. Although the panorama is changing with recent tools like CaImAn, Suite2P, and others, there is still a barrier for many laboratories to adopt these packages, especially for potential users without sophisticated programming skills. As two-photon microscopes are becoming increasingly affordable, the bottleneck is no longer the hardware, but the software used to analyze the calcium data optimally and consistently across different groups. We addressed this unmet need by incorporating recent software solutions, namely NoRMCorre and CaImAn, for motion correction, segmentation, signal extraction, and deconvolution of calcium imaging data into an open-source, easy to use, GUI-based, intuitive and automated data analysis software package, which we named EZcalcium.