A whole-brain analysis of functional connectivity and immediate early gene expression reveals functional network shifts after operant learning.

Previous studies of operant learning have addressed neuronal activities and network changes in specific brain areas, such as the striatum, sensorimotor cortex, prefrontal/orbitofrontal cortices, and hippocampus. However, how changes in the whole-brain network are caused by cellular-level changes remains unclear. We, therefore, combined resting-state functional magnetic resonance imaging (rsfMRI) and whole-brain immunohistochemical analysis of early growth response 1 (EGR1), a marker of neural plasticity, to elucidate the temporal and spatial changes in functional networks and underlying cellular processes during operant learning. We used an 11.7-Tesla MRI scanner and whole-brain immunohistochemical analysis of EGR1 in mice during the early and late stages of operant learning. In the operant training, mice received a reward when they pressed left and right buttons alternately, and were punished with a bright light when they made a mistake. A group of mice (n = 22) underwent the first rsfMRI acquisition before behavioral sessions, the second acquisition after 3 training-session-days (early stage), and the third after 21 training-session-days (late stage). Another group of mice (n = 40) was subjected to histological analysis 15 min after the early or late stages of behavioral sessions. Functional connectivity increased between the limbic areas and thalamus or auditory cortex after the early stage of training, and between the motor cortex, sensory cortex, and striatum after the late stage of training. The density of EGR1-immunopositive cells in the motor and sensory cortices increased in both the early and late stages of training, whereas the density in the amygdala increased only in the early stage of training. The subcortical networks centered around the limbic areas that emerged in the early stage have been implicated in rewards, pleasures, and fears. The connectivities between the motor cortex, somatosensory cortex, and striatum that consolidated in the late stage have been implicated in motor learning. Our multimodal longitudinal study successfully revealed temporal shifts in brain regions involved in behavioral learning together with the underlying cellular-level plasticity between these regions. Our study represents a first step towards establishing a new experimental paradigm that combines rsfMRI and immunohistochemistry to link macroscopic and microscopic mechanisms involved in learning.

Defensive behaviors and c-fos expression in the midbrain.

Pavlovian fear conditioning serves as a valuable method for investigating species-specific defensive reactions (SSDRs) such as freezing and flight responses. The present study examines the role of white noise under different experimental conditions. Given that white noise has been shown to elicit both conditional (associative) and unconditional (nonassociative) defensive responses, we compared the response to noise following three separate training conditions: shock-only, white noise paired with shock, and context-only. Results showed that baseline freezing level significantly changed across groups: Both the shock-only group and the white noise paired with shock group froze more than the context-only group on the test day. White noise evoked differential freezing between groups on day 2: The shock-only group froze more than the context-only group although both groups were never exposed to white noise during training. Further, an activity burst triggered by white noise was similar for the shock-only and white noise paired with shock groups during testing, although shock-only group was never exposed to white noise stimuli during training. This aligned with c-fos data, indicating similar c-fos activity levels across different periaqueductal gray (PAG) regions for both shock-only and white noise paired with shock groups. However, the driving force behind c-fos activation-whether freezing, activity burst, or a combination of both-remains uncertain, warranting further analysis to explore specific correlations between SSDRs and c-fos activity within the PAG and related brain areas.

Sex-Specific Effects of Anxiety on Cognition and Activity-Dependent Neural Networks: Insights from (Female) Mice and (Wo)Men.

INTRODUCTION: Neuropsychiatric symptoms (NPS), such as depression and anxiety, are observed in 90% of Alzheimer's disease (AD) patients, two-thirds of whom are women. NPS usually manifest long before AD onset creating a therapeutic opportunity. Here, we examined the impact of anxiety on AD progression and the underlying brain-wide neuronal mechanisms. METHODS: To gain mechanistic insight into how anxiety impacts AD progression, we performed a cross-sectional analysis on mood, cognition, and neural activity utilizing the ArcCreERT2 x enhanced yellow fluorescent protein (eYFP) x APP/PS1 (AD) mice. The ADNI dataset was used to determine the impact of anxiety on AD progression in human subjects. RESULTS: Female APP/PS1 mice exhibited anxiety-like behavior and cognitive decline at an earlier age than control (Ctrl) mice and male mice. Brain-wide analysis of c-Fos+ revealed changes in regional correlations and overall network connectivity in APP/PS1 mice. Sex-specific eYFP+/c-Fos+ changes were observed; female APP/PS1 mice exhibited less eYFP+/c-Fos+ cells in dorsal CA3 (dCA3), while male APP/PS1 mice exhibited less eYFP+/c-Fos+ cells in the dorsal dentate gyrus (dDG). In the ADNI dataset, anxiety predicted transition to dementia. Female subjects positive for anxiety and amyloid transitioned more quickly to dementia than male subjects. CONCLUSIONS: While future studies are needed to understand whether anxiety is a predictor, a neuropsychiatric biomarker, or a comorbid symptom that occurs during disease onset, these results suggest that there are sex differences in AD network dysfunction, and that personalized medicine may benefit male and female AD patients rather than a one size fits all approach.

Comprehensive analysis of transcription factors involved in odontoblast differentiation mechanism.

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.

A SMARTR workflow for multi-ensemble atlas mapping and brain-wide network analysis.

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.

Development of ocular dominance columns across rodents and other species: revisiting the concept of critical period plasticity.

The existence of cortical columns, regarded as computational units underlying both lower and higher-order information processing, has long been associated with highly evolved brains, and previous studies suggested their absence in rodents. However, recent discoveries have unveiled the presence of ocular dominance columns (ODCs) in the primary visual cortex (V1) of Long-Evans rats. These domains exhibit continuity from layer 2 through layer 6, confirming their identity as genuine ODCs. Notably, ODCs are also observed in Brown Norway rats, a strain closely related to wild rats, suggesting the physiological relevance of ODCs in natural survival contexts, although they are lacking in albino rats. This discovery has enabled researchers to explore the development and plasticity of cortical columns using a multidisciplinary approach, leveraging studies involving hundreds of individuals-an endeavor challenging in carnivore and primate species. Notably, developmental trajectories differ depending on the aspect under examination: while the distribution of geniculo-cortical afferent terminals indicates matured ODCs even before eye-opening, consistent with prevailing theories in carnivore/primate studies, examination of cortical neuron spiking activities reveals immature ODCs until postnatal day 35, suggesting delayed maturation of functional synapses which is dependent on visual experience. This developmental gap might be recognized as 'critical period' for ocular dominance plasticity in previous studies. In this article, I summarize cross-species differences in ODCs and geniculo-cortical network, followed by a discussion on the development, plasticity, and evolutionary significance of rat ODCs. I discuss classical and recent studies on critical period plasticity in the venue where critical period plasticity might be a component of experience-dependent development. Consequently, this series of studies prompts a paradigm shift in our understanding of species conservation of cortical columns and the nature of plasticity during the classical critical period.

Classification of psychedelic drugs based on brain-wide imaging of cellular c-Fos expression.

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.

Nr4a1 regulates cell-specific transcriptional programs in inhibitory GABAergic interneurons.

The patterns of synaptic connectivity and physiological properties of diverse neuron types are shaped by distinct gene sets. Our study demonstrates that, in the mouse forebrain, the transcriptional profiles of inhibitory GABAergic interneurons are regulated by Nr4a1, an orphan nuclear receptor whose expression is transiently induced by sensory experiences and is required for normal learning. Nr4a1 exerts contrasting effects on the local axonal wiring of parvalbumin- and somatostatin-positive interneurons, which innervate different subcellular domains of their postsynaptic partners. The loss of Nr4a1 activity in these interneurons results in bidirectional, cell-type-specific transcriptional switches across multiple gene families, including those involved in surface adhesion and repulsion. Our findings reveal that combinatorial synaptic organizing codes are surprisingly flexible and highlight a mechanism by which inducible transcription factors can influence neural circuit structure and function.

Sensory Stimulation-dependent Npas4 Expression in the Olfactory Bulb during Early Postnatal Development.

The development of the olfactory system is influenced by sensory inputs, and it maintains neuronal generation and plasticity throughout the lifespan. The olfactory bulb contains a higher proportion of interneurons than other brain regions, particularly during the early postnatal period of neurogenesis. Although the relationship between sensory stimulation and olfactory bulb development during the postnatal period has been well studied, the molecular mechanisms have yet to be identified. In this study, we used western blotting and immunohistochemistry to analyze the expression of the transcription factor Npas4, a neuron-specific immediate-early gene that acts as a developmental regulator in many brain regions. We found that Npas4 is highly expressed in olfactory bulb interneurons during the early postnatal stages and gradually decreases toward the late postnatal stages. Npas4 expression was observed in all olfactory bulb layers, including the rostral migratory stream, where newborn neurons are generated and migrate to the olfactory bulb. Under sensory deprivation, the olfactory bulb size and the number of olfactory bulb interneurons were reduced. Furthermore, Npas4 expression and the expression of putative Npas4 downstream molecules were decreased. Collectively, these findings indicate that Npas4 expression induced by sensory input plays a role in the formation of neural circuits with excitatory mitral/tufted cells by regulating the survival of olfactory bulb interneurons during the early stages of postnatal development.

Characterization of the Ictalurid herpesvirus 1 immediate-early gene ORF24 and its potential role in transcriptional regulation in yeast.

Herpesviruses adhere to a precise temporal expression model in which immediate-early (IE) genes play a crucial role in regulating the viral life cycle. However, there is a lack of functional research on the IE genes in Ictalurid herpesvirus 1 (IcHV-1). In this study, we identified the IcHV-1 ORF24 as an IE gene via a metabolic inhibition assay, and subcellular analysis indicated its predominant localisation in the nucleus. To investigate its function, we performed yeast reporter assays using an ORF24 fusion protein containing the Gal4-BD domain and found that BD-ORF24 was able to activate HIS3/lacZ reporter genes without the Gal4-AD domain. Our findings provide concrete evidence that ORF24 is indeed an IE gene that likely functions as a transcriptional regulator during IcHV-1 infection. This work contributes to our understanding of the molecular mechanisms underlying fish herpesvirus IE gene expression.

Data on electroconvulsive seizure in mice, effects of anesthesia on immediate early gene expression.

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].

Forebrain EAAT3 Overexpression Increases Susceptibility to Amphetamine-Induced Repetitive Behaviors.

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.

Effects of the Phosphodiesterase 10A Inhibitor MR1916 on Alcohol Self-Administration and Striatal Gene Expression in Post-Chronic Intermittent Ethanol-Exposed Rats.

Alcohol use disorder (AUD) requires new neurobiological targets. Problematic drinking involves underactive indirect pathway medium spiny neurons (iMSNs) that subserve adaptive behavioral selection vs. overactive direct pathway MSNs (dMSNs) that promote drinking, with a shift from ventromedial to dorsolateral striatal (VMS, DLS) control of EtOH-related behavior. We hypothesized that inhibiting phosphodiesterase 10A (PDE10A), enriched in striatal MSNs, would reduce EtOH self-administration in rats with a history of chronic intermittent ethanol exposure. To test this, Wistar rats (n = 10/sex) with a history of chronic intermittent EtOH (CIE) vapor exposure received MR1916 (i.p., 0, 0.05, 0.1, 0.2, and 0.4 µmol/kg), a PDE10A inhibitor, before operant EtOH self-administration sessions. We determined whether MR1916 altered the expression of MSN markers (Pde10a, Drd1, Drd2, Penk, and Tac1) and immediate-early genes (IEG) (Fos, Fosb, ΔFosb, and Egr1) in EtOH-naïve (n = 5-6/grp) and post-CIE (n = 6-8/grp) rats. MR1916 reduced the EtOH self-administration of high-drinking, post-CIE males, but increased it at a low, but not higher, doses, in females and low-drinking males. MR1916 increased Egr1, Fos, and FosB in the DLS, modulated by sex and alcohol history. MR1916 elicited dMSN vs. iMSN markers differently in ethanol-naïve vs. post-CIE rats. High-drinking, post-CIE males showed higher DLS Drd1 and VMS IEG expression. Our results implicate a role and potential striatal bases of PDE10A inhibitors to influence post-dependent drinking.

Sexual coordination in a whole-brain map of prairie vole pair bonding.

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 guanine nucleotide exchange factor RapGEF2 is required for ERK-dependent immediate-early gene (Egr1) activation during fear memory formation.

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.

Spatially Resolved Transcriptomic Signatures of Hippocampal Subregions and Arc-Expressing Ensembles in Active Place Avoidance Memory.

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.

Specific RNA structures in the 5' untranslated region of the human cytomegalovirus major immediate early transcript are critical for efficient virus replication.

Human cytomegalovirus (HCMV) requires the robust expression of two immediate early proteins, IE1 and IE2, immediately upon infection to suppress the antiviral response and promote viral gene expression. While transcriptional control of IE1 and IE2 has been extensively studied, the role of post-transcriptional regulation of IE1 and IE2 expression is relatively unexplored. We previously found that the shared major immediate early 5' untranslated region (MIE 5' UTR) of the mature IE1 and IE2 transcripts plays a critical role in facilitating the translation of the IE1 and IE2 mRNAs. As RNA secondary structure in 5' UTRs can regulate mRNA translation efficiency, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to identify RNA structures in the shared MIE 5' UTR. We found that the MIE 5' UTR contains three stable stem loop structures. Using a series of recombinant viruses to investigate the role of each stem loop in IE1 and IE2 protein synthesis, we found that the stem loop closest to the 5' end of the MIE 5' UTR (SL1) is both necessary and sufficient for efficient IE1 and IE2 mRNA translation and HCMV replication. The positive effect of SL1 on mRNA translation and virus replication was dependent on its location within the 5' UTR. Surprisingly, a synthetic stem loop with the same free energy as SL1 in its native location also supported wild type levels of IE1 and IE2 mRNA translation and virus replication, suggesting that the presence of RNA structure at a specific location in the 5' UTR, rather than the primary sequence of the RNA, is critical for efficient IE1 and IE2 protein synthesis. These data reveal a novel post-transcriptional regulatory mechanism controlling IE1 and IE2 expression and reinforce the critical role of RNA structure in regulating HCMV protein synthesis and replication.IMPORTANCEThese results reveal a new aspect of immediate early gene regulation controlled by non-coding RNA structures in viral mRNAs. Previous studies have largely focused on understanding viral gene expression at the level of transcriptional control. Our results show that a complete understanding of the control of viral gene expression must include an understanding of viral mRNA translation, which is driven in part by RNA structure(s) in the 5' UTR of viral mRNAs. Our results illustrate the importance of these additional layers of regulation by defining specific 5' UTR RNA structures regulating immediate early gene expression in the context of infection and identify important features of RNA structure that govern viral mRNA translation efficiency. These results may therefore broadly impact current thinking on how viral gene expression is regulated for human cytomegalovirus and other DNA viruses.

Differentiated somatic gene expression is triggered in the dorsal hippocampus and the anterior retrosplenial cortex by hippocampal synaptic plasticity prompted by spatial content learning.

Hippocampal afferent inputs, terminating on proximal and distal subfields of the cornus ammonis (CA), enable the functional discrimination of 'what' (item identity) and 'where' (spatial location) elements of a spatial representation. This kind of information is supported by structures such as the retrosplenial cortex (RSC). Spatial content learning promotes the expression of hippocampal synaptic plasticity, particularly long-term depression (LTD). In the CA1 region, this is specifically facilitated by the learning of item-place features of a spatial environment. Gene-tagging, by means of time-locked fluorescence in situ hybridization (FISH) to detect nuclear expression of immediate early genes, can reveal neuronal populations that engage in experience-dependent information encoding. In the current study, using FISH, we examined if learning-facilitated LTD results in subfield-specific information encoding in the hippocampus and RSC. Rats engaged in novel exploration of small items during stimulation of Schaffer collateral-CA1 synapses. This resulted in LTD (> 24 h). FISH, to detect nuclear expression of Homer1a, revealed that the distal-CA1 and proximal-CA3 subcompartments were particularly activated by this event. By contrast, all elements of the proximodistal cornus ammonis-axis showed equal nuclear Homer1a expression following LTD induction solely by means of afferent stimulation. The RSC exhibited stronger nuclear Homer1a expression in response to learning-facilitated LTD, and to novel item-place experience, compared to LTD induced by sole afferent stimulation in CA1. These results show that both the cornus ammonis and RSC engage in differentiated information encoding of item-place learning that is salient enough, in its own right, to drive the expression of hippocampal LTD. These results also reveal a novel role of the RSC in item-place learning.

Approaches and considerations of studying neuronal ensembles: a brief review.

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.

Brain regions controlling courtship behavior in the bluehead wrasse.

Bluehead wrasses (Thalassoma bifasciatum) follow a socially controlled mechanism of sex determination. A socially dominant initial-phase (IP) female is able to transform into a new terminal-phase (TP) male if the resident TP male is no longer present. TP males display an elaborate array of courtship behaviors, including both color changes and motor behaviors. Little is known concerning the neural circuits that control male-typical courtship behaviors. This study used glutamate iontophoresis to identify regions that may be involved in courtship. Stimulation of the following brain regions elicited diverse types of color change responses, many of which appear similar to courtship color changes: the ventral telencephalon (supracommissural nucleus of the ventral telencephalon [Vs], lateral nucleus of the ventral telencephalon [Vl], ventral nucleus of the ventral telencephalon [Vv], and dorsal nucleus of the ventral telencephalon [Vd]), parts of the preoptic area (NPOmg and NPOpc), entopeduncular nucleus, habenular nucleus, and pretectal nuclei (PSi and PSm). Stimulation of two regions in the posterior thalamus (central posterior thalamic [CP] and dorsal posterior thalamic [DP]) caused movements of the pectoral fins that are similar to courtship fluttering and vibrations. Furthermore, these responses were elicited in female IP fish, indicating that circuits for sexual behaviors typical of TP males exist in females. Immunohistochemistry results revealed regions that are more active in fish that are not courting: interpeduncular nucleus, red nucleus, and ventrolateral thalamus (VL). Taken together, we propose that the telencephalic-habenular-interpeduncular pathway plays an important role in controlling and regulating courtship behaviors in TP males; in this model, in response to telencephalic input, the habenular nucleus inhibits the interpeduncular nucleus, thereby dis-inhibiting forebrain regions and promoting the expression of courtship behaviors.