The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain and modulates diverse functions and brain states, including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single population. Using viral-genetic methods, we found that subcortical- and cortical-projecting serotonin neurons have distinct cell-body distributions within the DR and differentially co-express a vesicular glutamate transporter. Further, amygdala- and frontal-cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal-cortex-projecting neurons promote active coping in the face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.

Memories of fearful events can last a lifetime. The prelimbic (PL) subregion of prefrontal cortex plays a critical role in fear memory retrieval over time. Most studies have focused on acquisition, consolidation, and retrieval of recent memories, but much less is known about the neural mechanisms of remote memory. Using a new knock-in mouse for activity-dependent genetic labeling (TRAP2), we demonstrate that neuronal ensembles in PL are dynamic. PL neurons TRAPed during later memory retrievals are more likely to be reactivated and make larger behavioral contributions to remote memory retrieval compared to those TRAPed during learning or early memory retrieval. PL activity during learning is required to initiate this time-dependent reorganization in PL ensembles underlying memory retrieval. Finally, while neurons TRAPed during earlier and later retrievals have similar broad projections throughout the brain, PL neurons TRAPed later have a stronger functional recruitment of cortical targets.

Serotonin neurons of the dorsal and median raphe nuclei (DR, MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behavior. To gain a fundamental understanding of their molecular heterogeneity, we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR. These transcriptomic clusters differentially express a rich repertoire of neuropeptides, receptors, ion channels, and transcription factors. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus. Reconstruction of 50 individual DR serotonin neurons revealed diverse and segregated axonal projection patterns at the single-cell level. Together, these results provide a molecular foundation of the heterogenous serotonin neuronal phenotypes.

SUMMARY Background Developing neural networks display spontaneous and correlated rhythmic bursts of action potentials that are essential for circuit refinement. In the spinal cord, it is poorly understood how correlated activity is acquired and how its emergence relates to the formation of the spinal central pattern generator (CPG), the circuit that mediates rhythmic behaviors like walking and swimming. It is also unknown whether early, uncorrelated activity is necessary for the formation of the coordinated CPG. Results Time-lapse imaging in the intact zebrafish embryo with the genetically-encoded calcium indicator GCaMP3 revealed a rapid transition from slow, sporadic activity to fast, ipsilaterally correlated, and contralaterally anti-correlated activity, characteristic of the spinal CPG. Ipsilateral correlations were acquired through the coalescence of local microcircuits. Brief optical manipulation of activity with the light-driven pump Halorhodopsin revealed that the transition to correlated activity was associated with a strengthening of ipsilateral connections, likely mediated by gap junctions. Contralateral antagonism increased in strength at the same time. The transition to coordinated activity was disrupted by long-term optical inhibition of sporadic activity in motoneurons and VeLD interneurons, and resulted in more neurons exhibiting uncoordinated activity patterns at later time points. Conclusions These findings show that the CPG in the zebrafish spinal cord emerges directly from a sporadically active network as functional connectivity strengthens between local and then more distal neurons. These results also reveal that early, sporadic activity in a subset of ventral spinal neurons is required for the integration of maturing neurons into the coordinated CPG network.

Cerebellar evolution Cerebellar nuclei, substructures of the cerebellum, transfer information from the cerebellum to other parts of the brain. Using single-cell transcriptomics, Kebschull et al. have now identified a conserved pattern of cerebellar nuclei structure that has been repeated through evolution (see the Perspective by Hatten). Ranging from mice to chickens to humans, cerebellar nuclei are made up of region-specific excitatory neurons and region-invariant inhibitory neurons. In humans, a facet connecting the cerebellum to the frontal cortex is enhanced. Science , this issue p. eabd5059 ; see also p. 1411

Astrocytes, one of the most common cell types in the brain, are essential for processes ranging from neural development through potassium homeostasis to synaptic plasticity. Surprisingly, the developmental origins of astrocytes in the neocortex are still controversial. To investigate the patterns of astrocyte development in the neocortex we examined cortical development in a transgenic mouse in which a random, sparse subset of neural progenitors undergoes CRE/lox recombination, permanently labeling their progeny. We demonstrate that neural progenitors in neocortex generate discrete columnar structures that contain both projection neurons and protoplasmic astrocytes. Ninety five percent of developmental cortical columns labeled in our system contained both astrocytes and neurons. The astrocyte to neuron ratio of labeled cells in a developmental column was 1:7.4, similar to the overall ratio of 1:8.4 across the entire grey matter of the neocortex, indicating that column-associated astrocytes account for the majority of protoplasmic astrocytes in neocortex. Most of the labeled columns contained multiple clusters of several astrocytes. Dividing cells were found at the base of neuronal columns at the beginning of gliogenesis, and later within the cortical layers, suggesting a mechanism by which astrocytes could be distributed within a column. These data indicate that radial glia are the source of both neurons and astrocytes in the neocortex, and that these two cell types are generated in a spatially restricted manner during cortical development.

Studies of amnesic patients and animal models support a systems consolidation model, which posits that explicit memories formed in hippocampus are transferred to cortex over time 1-6 . Prelimbic cortex (PL), a subregion of the medial prefrontal cortex, is required for the expression of learned fear memories from hours after learning until weeks later 7-12 . While some studies suggested that prefrontal cortical neurons active during learning are required for memory retrieval 13-15 , others provided evidence for ongoing cortical circuit reorganization during memory consolidation 10,16,17 . It has been difficult to causally relate the activity of cortical neurons during learning or recent memory retrieval to their function in remote memory, in part due to a lack of tools 18 . Here we show that a new version of 'targeted recombination in active populations', TRAP2, has enhanced efficiency over the past version, providing brain-wide access to neurons activated by a particular experience. Using TRAP2, we accessed PL neurons activated during fear conditioning or 1-, 7-, or 14-day memory retrieval, and assessed their contributions to 28-day remote memory. We found that PL neurons TRAPed at later retrieval times were more likely to be reactivated during remote memory retrieval, and more effectively promoted remote memory retrieval. Furthermore, reducing PL activity during learning blunted the ability of TRAPed PL neurons to promote remote memory retrieval. Finally, a series of whole-brain analyses identified a set of cortical regions that were densely innervated by memory-TRAPed PL neurons and preferentially activated by PL neurons TRAPed during 14-day retrieval, and whose activity co-varied with PL and correlated with memory specificity. These findings support a model in which PL ensembles underlying remote memory undergo dynamic changes during the first two weeks after learning, which manifest as increased functional recruitment of cortical targets.Targeted recombination in active populations (TRAP) allows permanent genetic access to neurons activated by a specific experience 19 . The TRAP system uses an immediate early gene locus to drive the expression of tamoxifen-inducible CreER, along with a transgenic or virally-delivered Cre-dependent effector. When a neuron is active in the presence of tamoxifen, CreER can enter the nucleus to catalyze recombination, resulting in permanent expression of the effector (Fig. 1a). Because the original FosTRAP (TRAP1) disrupts endogenous Fos 19 and does not efficiently access many brain regions, we developed a new mouse line, TRAP2 20 , that preserves endogenous Fos, including the highly conserved first intron 21 and the 3' untranslated region critical for mRNA destabilization 22 (Fig. 1b; Extended Data Fig. 1). Further, we replaced the original Cre with a codon-optimized iCre for improved expression 23 .To characterize TRAP2, we first determined the time course of TRAPing and sensitivity of TRAP2 using the tdTomato Cre reporter Ai14 24 . We dark adapted TRAP1;Ai14 and TRAP2;Ai14 dou...

The projection targets of a neuronal population are a key feature of its anatomical characteristics. Historically, tissue sectioning, confocal microscopy, and manual scoring of specific regions of interest have been used to generate coarse summaries of mesoscale projectomes. We present here TrailMap, a three-dimensional (3D) convolutional network for extracting axonal projections from intact cleared mouse brains imaged by light-sheet microscopy. TrailMap allows region-based quantification of total axon content in large and complex 3D structures after registration to a standard reference atlas. The identification of axonal structures as thin as one voxel benefits from data augmentation but also requires a loss function that tolerates errors in annotation. A network trained with volumes of serotonergic axons in all major brain regions can be generalized to map and quantify axons from thalamocortical, deep cerebellar, and cortical projection neurons, validating transfer learning as a tool to adapt the model to novel categories of axonal morphology. Speed of training, ease of use, and accuracy improve over existing tools without a need for specialized computing hardware. Given the recent emphasis on genetically and functionally defining cell types in neural circuit analysis, TrailMap will facilitate automated extraction and quantification of axons from these specific cell types at the scale of the entire mouse brain, an essential component of deciphering their connectivity.