In the last decade, activity-dependent strategies for labelling multiple immediate early gene (IEG) ensembles in mice have generated unprecedented insight into the mechanisms of memory encoding, storage, and retrieval. However, few strategies exist for brain-wide mapping of multiple ensembles, including their overlapping population, and none incorporate capabilities for downstream network analysis. Here, we introduce a scalable workflow to analyze traditionally coronally-sectioned datasets produced by activity-dependent tagging systems. Intrinsic to this pipeline is simple multi-ensemble atlas registration and statistical testing in R (SMARTR), an R package which wraps mapping capabilities with functions for statistical analysis and network visualization. We demonstrate the versatility of SMARTR by mapping the ensembles underlying the acquisition and expression of learned helplessness (LH), a robust stress model. Applying network analysis, we find that exposure to inescapable shock (IS), compared to context training (CT), results in decreased centrality of regions engaged in spatial and contextual processing and higher influence of regions involved in somatosensory and affective processing. During LH expression, the substantia nigra emerges as a highly influential region which shows a functional reversal following IS, indicating a possible regulatory function of motor activity during helplessness. We also report that IS results in a robust decrease in reactivation activity across a number of cortical, hippocampal, and amygdalar regions, indicating suppression of ensemble reactivation may be a neurobiological signature of LH. These results highlight the emergent insights uniquely garnered by applying our analysis approach to multiple ensemble datasets and demonstrate the strength of our workflow as a hypothesis-generating toolkit.

Primary cultured odontoblasts rapidly lose their tissue-specific phenotype. To identify transcription factors (TF) that are important for the maintenance of the odontoblast phenotype, primary cultures of C57BL/6 J mouse dental mesenchymal cells (DMC) were isolated, and expression of TF and odontoblast marker genes in cells immediately after isolation and 2 days after culture were comprehensively evaluated and compared using RNA-sequencing (RNA-seq). The expression of odontoblast markers in mouse dental mesenchymal cells decreased rapidly after isolation. In addition, the expression of Hedgehog-related, Notch-related, and immediate- early gene (IEG)-related transcription factors significantly decreased. Forced expression of these genes in lentiviral vectors, together with fibroblast growth factor 4 (FGF4), fibroblast growth factor 9 (FGF9), and the Wnt pathway activator CHIR99021, significantly induced the expression of odontogenic marker genes. These results indicate, for the first time, that Notch signaling and early genes may be important for maintaining odontoblast cultures. Furthermore, simultaneous stimulation of FGF, Wnt, Hedgehog, Notch pathways, and IEG transcription factors cooperatively promoted the maintenance of the odontoblast phenotype. These results suggest that the Hedgehog and Notch signaling pathways may play an important role in maintaining odontoblast phenotypes, in addition to FGF and Wnt signaling.

Psilocybin, ketamine, and MDMA are psychoactive compounds that exert behavioral effects with distinguishable but also overlapping features. The growing interest in using these compounds as therapeutics necessitates preclinical assays that can accurately screen psychedelics and related analogs. We posit that a promising approach may be to measure drug action on markers of neural plasticity in native brain tissues. We therefore developed a pipeline for drug classification using light sheet fluorescence microscopy of immediate early gene expression at cellular resolution followed by machine learning. We tested male and female mice with a panel of drugs, including psilocybin, ketamine, 5-MeO-DMT, 6-fluoro-DET, MDMA, acute fluoxetine, chronic fluoxetine, and vehicle. In one-versus-rest classification, the exact drug was identified with 67% accuracy, significantly above the chance level of 12.5%. In one-versus-one classifications, psilocybin was discriminated from 5-MeO-DMT, ketamine, MDMA, or acute fluoxetine with >95% accuracy. We used Shapley additive explanation to pinpoint the brain regions driving the machine learning predictions. Our results support a novel approach for screening psychoactive drugs with psychedelic properties.

Although electroconvulsive therapy (ECT) is one of the most effective treatments for severe mood and psychotic disorders, the mechanisms underlying its therapeutic effects remain unknown. Electroconvulsive stimulation (ECS), the animal model for ECT, can be used to investigate the potential therapeutic mechanisms of ECT in rodents. ECS produces numerous effects in the brain, such as increasing levels of growth factors, inducing dendritic sprouting, and stimulating neurogenesis. It also induces high-level expression of immediate early genes (IEGs) that have been implicated in the pathogenesis of schizophrenia, such as early growth response 3 (Egr3) and activity-regulated cytoskeleton-associated protein (Arc), a validated downstream target of Egr3 [1-3]. However, the effect of isoflurane anesthesia preceding ECS on IEG response in mice has not been well characterized. This article provides immunofluorescent data of the activity responsive IEG ARC in the dorsal and ventral dentate gyrus of wildtype (WT) mice following ECS with or without anesthesia, as well as following sham ECS. The data in this article relate to a published article that employed serial ECS in mice to investigate the requirement of Egr3 in the neurobiological effects of this model of ECT [4]. The ability to study the effects of serial ECS has been limited in mice due to high rates of mortality during seizure. Administration of isoflurane anesthesia prior to ECS significantly reduces rodent mortality, irrespective of the number of times ECS is applied [5]. Since general anesthesia is administered to patients prior to ECT, use of isoflurane prior to ECS also more closely models the clinical use of ECT [6].

Obsessive-compulsive disorder (OCD) is a debilitating psychiatric disorder characterized by intrusive obsessive thoughts and compulsive behaviors. Multiple studies have shown the association of polymorphisms in the SLC1A1 gene with OCD. The most common of these OCD-associated polymorphisms increases the expression of the encoded protein, excitatory amino acid transporter 3 (EAAT3), a neuronal glutamate transporter. Previous work has shown that increased EAAT3 expression results in OCD-relevant behavioral phenotypes in rodent models. In this study, we created a novel mouse model with targeted, reversible overexpression of Slc1a1 in forebrain neurons. The mice do not have a baseline difference in repetitive behavior but show increased hyperlocomotion following a low dose of amphetamine (3 mg/kg) and increased stereotypy following a high dose of amphetamine (8 mg/kg). We next characterized the effect of amphetamine on striatal cFos response and found that amphetamine increased cFos throughout the striatum in both control and Slc1a1-overexpressing (OE) mice, but Slc1a1-OE mice had increased cFos expression in the ventral striatum relative to controls. We used an unbiased machine classifier to robustly characterize the behavioral response to different doses of amphetamine and found a unique response to amphetamine in Slc1a1-OE mice, relative to controls. Lastly, we found that the differences in striatal cFos expression in Slc1a1-OE mice were driven by cFos expression specifically in D1 neurons, as Slc1a1-OE mice had increased cFos in D1 ventral medial striatal neurons, implicating this region in the exaggerated behavioral response to amphetamine in Slc1a1-OE mice.

Sexual bonds are central to the social lives of many species, including humans, and monogamous prairie voles have become the predominant model for investigating such attachments. We developed an automated whole-brain mapping pipeline to identify brain circuits underlying pair-bonding behavior. We identified bonding-related c-Fos induction in 68 brain regions clustered in seven major brain-wide neuronal circuits. These circuits include known regulators of bonding, such as the bed nucleus of the stria terminalis, paraventricular hypothalamus, ventral pallidum, and prefrontal cortex. They also include brain regions previously unknown to shape bonding, such as ventromedial hypothalamus, medial preoptic area, and the medial amygdala, but that play essential roles in bonding-relevant processes, such as sexual behavior, social reward, and territorial aggression. Contrary to some hypotheses, we found that circuits active during mating and bonding were largely sexually monomorphic. Moreover, c-Fos induction across regions was strikingly consistent between members of a pair, with activity best predicted by rates of ejaculation. A novel cluster of regions centered in the amygdala remained coordinated after bonds had formed, suggesting novel substrates for bond maintenance. Our tools and results provide an unprecedented resource for elucidating the networks that translate sexual experience into an enduring bond.

The rodent hippocampus is a spatially organized neuronal network that supports the formation of spatial and episodic memories. We conducted bulk RNA sequencing and spatial transcriptomics experiments to measure gene expression changes in the dorsal hippocampus following the recall of active place avoidance (APA) memory. Through bulk RNA sequencing, we examined the gene expression changes following memory recall across the functionally distinct subregions of the dorsal hippocampus. We found that recall induced differentially expressed genes (DEGs) in the CA1 and CA3 hippocampal subregions were enriched with genes involved in synaptic transmission and synaptic plasticity, while DEGs in the dentate gyrus (DG) were enriched with genes involved in energy balance and ribosomal function. Through spatial transcriptomics, we examined gene expression changes following memory recall across an array of spots encompassing putative memory-associated neuronal ensembles marked by the expression of the IEGs Arc , Egr1 , and c-Jun . Within samples from both trained and untrained mice, the subpopulations of spatial transcriptomic spots marked by these IEGs were transcriptomically and spatially distinct from one another. DEGs detected between Arc + and Arc -spots exclusively in the trained mouse were enriched in several memory-related gene ontology terms, including \"regulation of synaptic plasticity\" and \"memory.\" Our results suggest that APA memory recall is supported by regionalized transcriptomic profiles separating the CA1 and CA3 from the DG, transcriptionally and spatially distinct IEG expressing spatial transcriptomic spots, and biological processes related to synaptic plasticity as a defining the difference between Arc + and Arc -spatial transcriptomic spots.

The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for neuronal ERK activation are undetermined. RapGEF2 is a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether it is required for cAMP-dependent ERK activation leading to other downstream neuronal signaling events occurring during associative learning, and if RapGEF2-dependent signaling impairments affect learned behavior. Camk2α-cre+/-::RapGEF2fl/fl mice with depletion of RapGEF2 in hippocampus and amygdala exhibit impairments in context- and cue-dependent fear conditioning linked to corresponding impairment in Egr1 induction in these two brain regions. Camk2α-cre+/-::RapGEF2fl/fl mice show decreased RapGEF2 expression in CA1 and dentate gyrus associated with abolition of pERK and Egr1, but not of c-Fos induction, following fear conditioning, impaired freezing to context after fear conditioning, and impaired cAMP-dependent long-term potentiation at perforant pathway and Schaffer collateral synapses in hippocampal slices ex vivo. RapGEF2 expression is largely eliminated in basolateral amygdala, also involved in fear memory, in Camk2α-cre+/-::RapGEF2fl/fl mice. Neither Egr1 nor c-fos induction in BLA after fear conditioning, nor cue-dependent fear learning, are affected by ablation of RapGEF2 in BLA. However, Egr1 induction (but not that of c-fos) in BLA is reduced after restraint stress-augmented fear conditioning, as is freezing to cue after restraint stress-augmented fear conditioning, in Camk2α-cre+/-::RapGEF2fl/fl mice. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. Here we show a functional link between one of them, RapGEF2, and cognitive processes involved in associative learning in amygdala and hippocampus.

First theorized by Hebb, neuronal ensembles have provided a framework for understanding how the mammalian brain operates, especially regarding learning and memory. Neuronal ensembles are discrete, sparsely distributed groups of neurons that become activated in response to a specific stimulus and are thought to provide an internal representation of the world. Beyond the study of region-wide or projection-wide activation, the study of ensembles offers increased specificity and resolution to identify and target specific memories or associations. Neuroscientists interested in the neurobiology of learning, memory, and motivated behavior have used electrophysiological-, calcium-, and protein-based proxies of neuronal activity in preclinical models to better understand the neurobiology of learned and motivated behaviors. Although these three approaches may be used to pursue the same general goal of studying neuronal ensembles, technical differences lead to inconsistencies in the output and interpretation of data. This mini-review highlights some of the methodologies used in electrophysiological-, calcium-, and protein-based studies of neuronal ensembles and discusses their strengths and weaknesses.

When living in urban habitats, \'urban adapter\' species often show greater aggression toward conspecifics, yet we do not understand the mechanisms underlying this behavioral shift. The neuroendocrine system regulates socio-sexual behaviors including aggression and thus could mediate behavioral responses to urbanization. Indeed, urban male song sparrows (Melospiza melodia), which are more territorially aggressive, also have greater abundance of the neuropeptide arginine vasotocin (AVT) in nodes of the brain social behavior network. Higher abundance of AVT could reflect long-term synthesis that underlies baseline territoriality or short-term changes that regulate aggression in response to social challenge. To begin to resolve the timeframe over which the AVT system contributes to habitat differences in aggression we used immediate early gene co-expression as a measure of the activation of AVT neurons. We compared Fos induction in AVT-immunoreactive neurons of the bed nucleus of the stria terminalis (BSTm) and paraventricular nucleus of the hypothalamus (PVN) between urban and rural male song sparrows in response to a short (< 5 min.) or long (> 30 min.) song playback to simulate territorial intrusion by another male. We found that urban males had a higher proportion of Fos-positive AVT neurons in both brain regions compared to rural males, regardless of the duration of song playback. Our results suggest that AVT neurons remain activated in urban males, independently of the duration of social challenge. These findings that Fos induction in AVT neurons differs between rural and urban male song sparrows further implicate this system in regulating behavioral responses to urbanization.