Viruses have proved instrumental to elucidating neuronal connectivity relationships in a variety of organisms. Recent advances in genetic technologies have facilitated analysis of neurons directly connected to a defined starter population. These advances have also made viral transneuronal mapping available to the broader neuroscience community, where one-step rabies virus mapping has become routine. This method is commonly used to identify inputs onto defined cell populations, to demonstrate the quantitative proportion of inputs coming from specific brain regions, or to compare input patterns between two or more cell populations. Furthermore, the number of inputs labeled is often assumed to reflect the number of synaptic connections, and these viruses are commonly believed to label strong synapses more efficiently than weak synapses. While these maps are often interpreted to provide a quantitative estimate of the synaptic landscape onto starter cell populations, in fact very little is known about how transneuronal transmission takes place. We do not know how these viruses transmit between neurons, if they display biases in the cell types labeled, or even if transmission is synapse-specific. In this review, we discuss the experimental evidence against or in support of key concepts in viral tracing, focusing mostly on the use of one-step rabies input mapping and related methods. Does spread of these viruses occur specifically through synaptic connections, preferentially through synapses, or non-specifically? How efficient is viral transneuronal transmission, and is this efficiency equal in all cell types? And lastly, to what extent does viral labeling reflect functional connectivity?

Graduate physiology programs strive to provide students with in-depth expertise in a particular academic discipline, often facilitating this process in the form of a departmental seminar course. Within the Department of Physiology and Biophysics at the University of California Irvine (UCI), students are required to attend a seminar course, most often designed as a journal club, each quarter until they are ready to graduate. While this format may work well in departments where research topics are closely related, it has historically been less successful in UCI's Department of Physiology and Biophysics, where wide-ranging interests make for little overlap in foundational knowledge, limiting meaningful engagement with the material or with peers in the class. In this paper, we describe a complementary approach of developing a syllabus around student interests and covering topics that are critical for student success but often omitted from graduate curricula, such as interview skills, grant writing, and scientific communication. Results from our preclass survey motivated this approach to the class, and our retrospective survey demonstrated the substantial differences in student engagement, enthusiasm, and perceived benefits of this course relative to the journal club style course. We hope that the success of our course may serve as an exemplar for strategies to engage students more effectively and provide critical training in diverse skillsets that will help students after graduation.

Although midbrain dopamine (DA) circuits are central to motivated behaviors, our knowledge of how experience modifies these circuits to facilitate subsequent behavioral adaptations is limited. Here we demonstrate the selective role of a ventral tegmental area DA projection to the amygdala (VTA^DA→amygdala) for cocaine-induced anxiety but not cocaine reward or sensitization. Our rabies virus-mediated circuit mapping approach reveals a persistent elevation in spontaneous and task-related activity of inhibitory GABAergic cells from the bed nucleus of the stria terminalis (BNST) and downstream VTA^DA→amygdala cells that can be detected even after a single cocaine exposure. Activity in BNST^GABA→midbrain cells is related to cocaine-induced anxiety but not reward or sensitization, and silencing this projection prevents development of anxiety during protracted withdrawal after cocaine administration. Finally, we observe that VTA^DA→amygdala cells are strongly activated after a challenge exposure to cocaine and that activity in these cells is necessary and sufficient for reinstatement of cocaine place preference.

Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic's function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity.

Astrocytes respond to injury and disease in the central nervous system with reactive changes that influence the outcome of the disorder^1-4. These changes include differentially expressed genes (DEGs) whose contextual diversity and regulation are poorly understood. Here we combined biological and informatic analyses, including RNA sequencing, protein detection, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and conditional gene deletion, to predict transcriptional regulators that differentially control more than 12,000 DEGs that are potentially associated with astrocyte reactivity across diverse central nervous system disorders in mice and humans. DEGs associated with astrocyte reactivity exhibited pronounced heterogeneity across disorders. Transcriptional regulators also exhibited disorder-specific differences, but a core group of 61 transcriptional regulators was identified as common across multiple disorders in both species. We show experimentally that DEG diversity is determined by combinatorial, context-specific interactions between transcriptional regulators. Notably, the same reactivity transcriptional regulators can regulate markedly different DEG cohorts in different disorders; changes in the access of transcriptional regulators to DNA-binding motifs differ markedly across disorders; and DEG changes can crucially require multiple reactivity transcriptional regulators. We show that, by modulating reactivity, transcriptional regulators can substantially alter disorder outcome, implicating them as therapeutic targets. We provide searchable resources of disorder-related reactive astrocyte DEGs and their predicted transcriptional regulators. Our findings show that transcriptional changes associated with astrocyte reactivity are highly heterogeneous and are customized from vast numbers of potential DEGs through context-specific combinatorial transcriptional-regulator interactions.

Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury1-3, but efficient reversal of this regrowth failure remains elusive4. Here we show that three factors that are essential for axon growth during development but are attenuated or lacking in adults-(i) neuron intrinsic growth capacity2,5-9, (ii) growth-supportive substrate10,11 and (iii) chemoattraction12,13-are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI14,15; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor16,17 delivered via spatially and temporally controlled release from biomaterial depots18,19, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesion cores of non-neural tissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits.

Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury^1-3, but efficient reversal of this regrowth failure remains elusive⁴. Here we show that three factors that are essential for axon growth during development but are attenuated or lacking in adults-(i) neuron intrinsic growth capacity^2,5-9, (ii) growth-supportive substrate^10,11 and (iii) chemoattraction^12,13-are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI^14,15; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor^16,17 delivered via spatially and temporally controlled release from biomaterial depots^18,19, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesion cores of non-neural tissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits.