bioRxiv Subject Collection: Neuroscience's Journal

Experience-dependent sex differences in the role of dorsal striatal dopamine D1 receptor activity in methamphetamine self-administration revealed by a novel TREND model.
BACKGROUND: The role of dorsal striatal dopamine D1 receptor systems in the mechanism of methamphetamine self-administration (METH SA), and sex differences in this role, are unclear. We hypothesized that this role would be sex and METH experience-dependent. Because prior experience regulates subsequent effects of drugs, we developed a novel model to account for this interaction, termed the TREND model (Time-Related-Experience-Normalized-Dynamics) for drug SA analysis. We tested our hypothesis by comparing results from the new TREND model and the current model. METHODS: For model validation, we reanalyzed previous data (Job et al., 2020) with the aim of determining which model (current or TREND) was more effective as an analytical tool. We compared variables from each model with the effect of Clozapine-N-Oxide (CNO, chemogenetic ligand) on METH SA. We employed regression analysis, median split, ANOVA to see which could reveal sex and experience dependency of dorsal striatal dopamine D1 receptor system. RESULTS: The current model variables were unrelated to CNO effect, with no sex differences in these relationships. TREND model revealed new variables that were unrelated to current variables but related to CNO effect on METH in males and females, with sex differences in these relationships. TREND, but not the current model, detected sex differences when comparing males and females with prior high, but not low, behavioral response variables. CONCLUSIONS: TREND model is more sensitive than the current model for detecting experience-dependent sex differences in the role of the dorsal striatal dopamine D1 receptor systems in the mechanism of METH SA.

Mouse parasubthalamic Crh neurons drive alcohol drinking escalation and behavioral disinhibition
Corticotropin-releasing factor (CRF, encoded by Crh) signaling is thought to play a critical role in the development of excessive alcohol drinking and the emotional and physical pain associated with alcohol withdrawal. Here, we investigated the parasubthalamic nucleus (PSTN) as a potential source of CRF relevant to the control of alcohol consumption, affect, and nociception in mice. We identified PSTN Crh neurons as a neuronal subpopulation that exerts a potent and unique influence on behavior by promoting not only alcohol but also saccharin drinking, while PSTN neurons are otherwise known to suppress consummatory behaviors. Furthermore, PSTN Crh neurons are causally implicated in the escalation of alcohol and saccharin intake produced by chronic intermittent ethanol (CIE) vapor inhalation, a mouse model of alcohol use disorder. In contrast to our predictions, the ability of PSTN Crh neurons to increase alcohol drinking is not mediated by CRF1 signaling. Moreover, the pattern of behavioral disinhibition and reduced nociception driven by their activation does not support a role of negative reinforcement as a motivational basis for the concomitant increase in alcohol drinking. Finally, silencing Crh expression in the PSTN slowed down the escalation of alcohol intake in mice exposed to CIE and accelerated their recovery from withdrawal-induced mechanical hyperalgesia. Altogether, our results suggest that PSTN Crh neurons may represent an important node in the brain circuitry linking alcohol use disorder with sweet liking and novelty seeking.

Decoding the Epigenetic Landscape: Insights into 5mC and 5hmC Patterns in Mouse Cortical Cell Types
The DNA modifications, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), represent powerful epigenetic regulators of temporal and spatial gene expression. Yet, how the cooperation of these genome-wide, epigenetic marks determine unique transcriptional signatures across different brain cell populations is unclear. Here we applied Nanopore sequencing of native DNA to obtain a complete, genome-wide, single-base resolution atlas of 5mC and 5hmC modifications in neurons, astrocytes and microglia in the mouse cortex (99% genome coverage, 40 million CpG sites). In tandem with RNA sequencing, analysis of 5mC and 5hmC patterns across cell types reveals astrocytes drive uniquely high brain 5hmC levels and support two decades of research regarding methylation patterns, gene expression and alternative splicing, benchmarking this resource. As such, we provide the most comprehensive DNA methylation data in mouse brain as an interactive, online tool (NAM-Me, https://olsenlab.shinyapps.io/NAMME/) to serve as a resource dataset for those interested in the methylome landscape.

Hair Cells Loss Estimation from Audiograms
Age-related hearing loss is characterized by a progressive loss of threshold sensitivity, especially at high frequencies. There is increasing evidence that the loss of cilia in the inner and outer hair cells is the dominant cause of hearing loss. We present a framework for calculating the human auditory threshold based on a non-linear time-domain cochlear model that incorporates hair cell damage along the cochlear partition. We successfully predicted the audiogram measured prior to death by substituting the postmortem percentage of surviving hair cells, using data from Wu et al. (2020). We also present an algorithm for estimating the percentage of hair cells from a measured audiogram. Comparison with the data from Wu et al. revealed that the algorithm accurately predicted the surviving inner hair cells along the entire cochlear partition and the outer hair cells at the basal part of the cochlea.

Sexually dimorphic auditory representation in Aedes aegypti brains
Male attraction to female flight sounds is a vital, reproducible component of courtship in many species of mosquitoes; however, female acoustic behaviours have proven challenging to define. To investigate sexual dimorphisms in acoustic behaviours, previous reports have largely focused on differences in mosquito peripheral ear anatomy and function. Whilst molecular investigations have recently begun on the auditory periphery, sexual dimorphisms in central processing of acoustic information have not yet been explored. Here we used a combination of neurotracing, calcium imaging and molecular analyses to examine sexual dimorphisms in auditory processing in the yellow fever mosquito Aedes aegypti. We identified shared and dimorphic neurons connecting male and female ears to the primary auditory processing centre in the brain, and defined multiple distinct neuronal clusters based on responses to auditory stimulation. We finally used transcriptomic and proteomic analyses to investigate the molecular factors underlying these differences, with motile ciliary-related terms significantly enriched in males.

Dihydromyricetin alleviated the damage of hypoxia-induced mouse neurons by reducing ROS levels and inhibiting the expression of PAR and gammaH2AX
Objective: To investigate the effect of dihydromyricetin on hypoxia-induced neurons to understand the effect of dihydromyricetin on hypoxic-ischemic encephalopathy (HIE). Methods: Cortical neurons were isolated from C57BL/6j mice (24 hour-year old), cultured, and subjected to 4h hypoxia and 20h reoxygenation to mimic the neonatal hypoxic-ischemic encephalopathy. After dihydromyricetin (20 umol/L) treatment of hypoxia-induced neurons for 2h, CCK-8 assay was used to analyze the neuronal viability, Hoechst33342/PI double staining assay was used to analyze the neuronal death, Western blotting was used to analyze the expression of Poly ADP-ribose (PAR) polymer protein and gammaH2AX, comet assay was used to detect DNA damage, immunofluorescence staining was used to observe the nuclear translocation of apoptosis inducing factor, and 2',7'-dichlorodihydrofluorescein diacetate was used to detect the expression of reactive oxygen species (ROS). Results: Compared with the control groups, hypoxia-treated neurons exhibited significantly lower activity, higher neuronal death rate and the high expressions of PAR and {gamma}H2AX, hypoxia could also induce AIF nuclear translocation, increase tail DNA content and tail length, increase the expression of ROS in neurons; after dihydromyricetin treatment, neuronal activity were significantly increased, neuronal death rate, ROS levels, and the expressions of PAR and gammaH2AX were also decreased, AIF nuclear translocation was inhibited, the tail DNA content and tail length were also decreased. Conclusion: Dihydromyricetin could alleviate the damage of hypoxia-induced neurons through decreasing the levels of ROS and inhibiting the expressions of PAR and gammaH2AX, suggesting that dihydromyricetin may have the protective effect on HIE.

Xenon and nitrous oxide induced changes in resting EEG activity can be explained by systematic increases in the relaxation rates of stochastically driven alpha band oscillatory activity
Resting electroencephalographic activity is typically indistinguishable from a filtered linear random process across a diverse range of behavioural and pharmacological states, suggesting that the power spectral density of the resting electroencephalogram (EEG) can be modelled as the superposition of multiple, stochastically driven and independent, alpha band (8 - 13 Hz) relaxation oscillators. This simple model can account for variations in alpha band power and `1/f scaling' in eyes-open/eyes-closed conditions in terms of alterations in the distribution of the alpha band oscillatory relaxation rates. As changes in alpha band power and `1/f scaling' have been reported in anaesthesia we hypothesise that such changes may also be accounted for by alterations in alpha band relaxation oscillatory rate distributions. On this basis we choose to study the EEG activity of xenon and nitrous oxide, gaseous anaesthetic agents that have been reported to produce different EEG effects, notable given they are both regarded as principally acting via N-methyl-D-aspartate (NMDA) receptor antagonism. By recording high density EEG from participants receiving equilibrated step-level increases in inhaled concentrations of xenon (n = 24) and nitrous oxide (n = 20), alpha band relaxation rate (damping) distributions were estimated by solving an inhomogeneous integral equation describing the linear superposition of multiple alpha-band relaxation oscillators having a continuous distribution of dampings. For both agents, level-dependent reductions in alpha band power and spectral slope exponent (15-40 Hz) were observed, that were accountable by increases in mean alpha band damping. These shared increases suggest that, consistent with their identified molecular targets of action, xenon and nitrous oxide are mechanistically similar, a conclusion further supported by neuronal population modelling in which NMDA antagonism is associated with increases in damping and reductions in peak alpha frequency. Alpha band damping may provide an important link between experiment and theories of consciousness, such as the global neuronal network theory, where the likelihood of a globally excited state (`conscious percept'), is inversely related to mean damping.

Neural correlates of phenomenological attitude toward perceptual experience
Phenomenology is one of the most promising approaches to study conscious experience. It holds that a rigorous study of conscious experience requires a transition in the subject from the "natural attitude" (NA) to the "phenomenological attitude" (PA). NA describes our ordinary stance, in which our attention is directed at external objects and events. PA is a distinctive, reflective stance in which our attention is directed at our conscious experience itself. Despite its theoretical importance in philosophy and science of consciousness, the neural mechanisms underlying PA remain unknown. To clarify this point, we developed a novel behavioral task in which participants alternate between NA and PA in relation to their stimulus-evoked subjective experiences. Participants are presented with two sentences and requested to identify the one that best captures their experience. These sentences are designed to induce either NA or PA. We found that participants had lower error rates but slower reaction times in the PA condition compared to the NA condition, suggesting a difference beyond task difficulty. Using fMRI, we also found that multivoxel activation patterns in the premotor cortex, posterior parietal cortex, supplementary motor area, and cerebellum successfully classified the task conditions. Furthermore, the activation strength in these regions was lower in the PA condition, indicating that PA depends on neural processes that suppress action-related information. These findings provide the first evidence for the neural signature of PA, contributing to a better understanding of phenomenological method and its underlying neural mechanisms.

Transcriptome signatures of the medial prefrontal cortex underlying GABAergic control of resilience to chronic stress exposure
Analyses of postmortem human brains and preclinical studies of rodents have identified somatostatin (SST)-positive interneurons as key elements that regulate the vulnerability to stress-related psychiatric disorders. Conversely, genetically induced disinhibition of SST neurons or brain region-specific chemogenetic activation of SST neurons in mice results in stress resilience. Here, we used RNA sequencing of mice with disinhibited SST neurons to characterize the transcriptome changes underlying GABAergic control of stress resilience. We found that stress resilience of male but not female mice with disinhibited SST neurons is characterized by resilience to chronic stress-induced transcriptome changes in the medial prefrontal cortex. Interestingly, the transcriptome of non-stressed stress-resilient male mice resembled the transcriptome of chronic stress-exposed stress-vulnerable mice. However, the behavior and the serum corticosterone levels of non-stressed stress-resilient mice showed no signs of physiological stress. Most strikingly, chronic stress exposure of stress-resilient mice was associated with an almost complete reversal of their chronic stress-like transcriptome signature, along with pathway changes indicating stress-induced enhancement of mRNA translation. Behaviorally, the mice with disinhibited SST neurons were not only resilient to chronic stress-induced anhedonia -- they also showed an inversed anxiolytic-like response to chronic stress exposure that mirrored the chronic stress-induced reversal of the chronic stress-like transcriptome signature. We conclude that GABAergic dendritic inhibition by SST neurons exerts bidirectional control over behavioral vulnerability and resilience to chronic stress exposure that is mirrored in bidirectional changes in expression of putative stress resilience genes, through a sex-specific brain substrate.

Developmental dynamics of the prefrontal cortical SST and PV interneuron networks: Insights from the monkey highlight human-specific features
The primate prefrontal cortex (PFC) is a quintessential hub of cognitive functions. Amidst its intricate neural architecture, the interplay of distinct neuronal subtypes, notably parvalbumin (PV) and somatostatin (SST) interneurons (INs), emerge as a cornerstone in sculpting cortical circuitry and governing cognitive processes. While considerable strides have been made in elucidating the developmental trajectory of these neurons in rodent models, our understanding of their postmigration developmental dynamics in primates still needs to be studied. Disruptions to this developmental trajectory can compromise IN function, impairing signal gating and circuit modulation within cortical networks. This study examined the expression patterns of PV and SST, ion transporter KCC2, and ion channel subtypes Kv3.1b, and Nav1.1 - associated with morphophysiological stages of development in the postnatal marmoset monkey in different frontal cortical regions (granular areas 8aD, 8aV, 9, 46; agranular areas 11, 47L). Our results demonstrate that the maturation of PV+ INs extends into adolescence, characterized by discrete epochs associated with specific expression dynamics of ion channel subtypes. Interestingly, we observed a postnatal decrease in SST interneurons, contrasting with studies in rodents. This endeavor broadens our comprehension of primate cortical development and furnishes invaluable insights into the etiology and pathophysiology of neurodevelopmental disorders characterized by perturbations in PV and SST IN function.

Neuronal Hyperactivity in Neurons Derived from Individuals with Grey Matter Heterotopia
Periventricular heterotopia (PH), a common form of grey matter heterotopia associated with developmental delay and drug-resistant seizures, poses a challenge in understanding its neurophysiological basis. Human cerebral organoids (hCOs) derived from patients with causative mutations in FAT4 or DCHS1 mimic PH features. However, neuronal activity in these 3D models has not yet been investigated. Here, silicon probe recordings revealed exaggerated spontaneous spike activity in FAT4 and DCHS1 hCOs, suggesting functional changes in neuronal networks. Transcriptome and proteome analyses identified changes in gene ontology terms associated with neuronal morphology and synaptic function. Furthermore, patch-clamp recordings revealed a decreased spike threshold specifically in DCHS1 neurons, likely due to increased somatic voltage-gated sodium channels. Morphological reconstructions and immunostainings revealed greater morphological complexity of PH neurons and synaptic alterations contributing to hyperactivity, with morphological rescue observed in DCHS1 neurons by wild-type DCHS1 expression. Overall, we provide new comprehensive insights into the cellular changes underlying symptoms of grey matter heterotopia.

Functional ultrasound imaging and neuronal activity: how accurate is the spatiotemporal match?
Over the last decade, functional ultrasound (fUS) has risen as a critical tool in functional neuroimaging, leveraging hemodynamic changes to infer neural activity indirectly. Recent studies have established a strong correlation between neural spike rates (SR) and functional ultrasound signals. However, understanding their spatial distribution and variability across different brain areas is required to thoroughly interpret fUS signals. In this regard, we conducted simultaneous fUS imaging and Neuropixels recordings during stimulus-evoked activity in awake mice within three regions the visual pathway. Our findings indicate that the temporal dynamics of fUS and SR signals are linearly correlated, though the correlation coefficients vary among visual regions. Conversely, the spatial correlation between the two signals remains consistent across all regions with a spread of approximately 300 micrometers. Finally, we introduce a model that integrates the spatial and temporal components of the fUS signal, allowing for a more accurate interpretation of fUS images.

Striatal endocannabinoid-long-term potentiation mediates one-shot learning
The ability to learn and adapt its behavior from salient events encountered only once is crucial for survival. Few action potentials may be elicited during such one-shot learning, challenging usual learning frameworks. While the striatum is classically associated with incremental learning, some activity changes have been detected during the initial learning phase, but following several and/or long trials. Hence, the striatal engram for one-shot learning per se remains to be elucidated. Here, we show in a novel one-shot behavioral test in mice, the sticky tape avoidance test, that in vivo striatal long-term potentiation (LTP) emerges after a single, spontaneous and brief contact with an uncomfortable stimulus, and is associated with its subsequent avoidance that can last more than a month. We found that the non-classical endocannabinoid-mediated LTP, shown to occur in vitro after a small number of action potentials, is engaged during one-shot learning, even for brief exposures to the stimulus, and that paired cortico-striatal network activity shows behaviorally-correlated changes in activity consistent with endocannabinoid-mediated LTP induction during contact. Furthermore, we found impairments of one-shot learning in conditional knock-out mice affecting endocannabinoid-mediated LTP. Our results thus reveal new functional relevance of endocannabinoids in activity-dependent boundaries delineating synaptic plasticity expression in one-shot learning. This opens new avenues for understanding the effects of endo- and phyto-cannabinoids, including endocannabinoid-based medicinal drugs, on learning and memory.

Cerebellar contribution to cognitive deficits and prefrontal cortex dysfunction in Spinocerebellar Ataxia Type 1 (SCA1)
The cerebellum role in cognition and its functional bi-directional connectivity with prefrontal cortex (PFC) is well recognized. However, how chronic cerebellar dysfunction affects PFC function and cognition remains less understood. Spinocerebellar ataxia type 1 (SCA1), is an inherited, fatal neurodegenerative disease caused by an abnormal expansion of glutamine (Q) encoding CAG repeats in the gene Ataxin-1 (ATXN1) and characterized by severe loss of Purkinje cells (PCs) in the cerebellum. Patients with SCA1 suffer from movement and balance deficits, cognitive decline and premature lethality. Cognitive deficits significantly impact patients quality of life, yet how exactly cerebellar degeneration contributes to cognitive deficits and PFC dysfunction in SCA1 is unknown. We have previously demonstrated that expression of mutant ATXN1 only in cerebellar Purkinje cells (PCs) is sufficient to cause cognitive deficits in a transgenic ATXN1[82Q] mouse line. To understand how cerebellar dysfunction impacts the PFC, we examined neuronal activity, synaptic density, and gene expression changes in the PFC of ATXN1[82Q] mice. Remarkably, we found decreased neuronal activity, reduced synaptic density, and altered expression of immediate early genes and pathways involved in glucose metabolism, inflammation and amphetamine in the PFC of ATXN1[82Q] mice. Furthermore, we characterized cellular and molecular PFC dysfunction in a novel conditional knock-in SCA1 line, f-ATXN1146Q mice, expressing floxed human expanded ATXN1 throughout the brain. Intriguingly, we found an increased number of neurons, increased synaptic density and large gene expression alterations in the PFC of f-ATXN1146Q mice. Finally, to precisely determine the role of cerebellar dysfunction in cognitive deficits and PFC dysfunction in SCA1, we crossed f-ATXN1146Q mice with Pcp2-Cre mice expressing Cre recombinase in PCs to delete expanded ATXN1 only in PCs. Surprisingly, we have found that deleting expanded ATXN1 in PCs exacerbated cognitive deficits and PFC dysfunction in these mice. Our findings demonstrate that circumscribed cerebellar dysfunction is sufficient to impact PFC activity and synaptic connectivity impairing cognition. However, when multiple brain regions are impacted in disease, cerebellar dysfunction may ameliorate PFC pathology and cognitive performance.

Golgi cells regulate timing and variability of information transfer in a cerebellar-behavioural loop
Golgi cells are inhibitory interneurons residing in the input layer of the cerebellar cortex. These neurons sit in a key position to govern the transformation of incoming information from extracerebellar regions and influence downstream cerebellar processing. Here, we examine the contribution of Golgi cells to network dynamics in Crus 1 of mouse lateral cerebellar cortex during free whisking. We recorded neuronal population activity using NeuroPixels probes before and after chemogenetic downregulation of Golgi cell activity. Under resting conditions, cerebellar population activity reliably encoded whisker movements. Reductions in Golgi cell activity produced mild increases in neural activity which did not significantly impair these sensorimotor representations. However, reduced Golgi cell inhibition did increase the temporal alignment of local population network activity at the initiation of movement. These network alterations had variable impacts on behaviour, producing both increases and decreases in whisking velocity. Our results suggest that Golgi cell inhibition primarily governs the temporal patterning of population activity, which in turn is required to support downstream cerebellar dynamics and behavioural coordination.

Hippocampal-prefrontal communication subspaces align with behavioral and network patterns in a spatial memory task
Rhythmic network states have been theorized to facilitate communication between brain regions, but how these oscillations influence communication subspaces, i.e, the low-dimensional neural activity patterns that mediate inter-regional communication, and in turn how subspaces impact behavior remains unclear. Using a spatial memory task in rats, we simultaneously recorded ensembles from hippocampal CA1 and the prefrontal cortex to address this question. We found that task behaviors best aligned with low-dimensional, shared subspaces between these regions, rather than local activity in either region. Critically, both network oscillations and speed modulated the structure and performance of this communication subspace. Contrary to expectations, theta coherence did not better predict CA1-PFC shared activity, while theta power played a more significant role. To understand the communication space, we visualized shared CA1-PFC communication geometry using manifold techniques and found ring-like structures. We hypothesize that these shared activity manifolds are utilized to mediate the task behavior. These findings suggest that memory-guided behaviors are driven by shared CA1-PFC interactions that are dynamically modulated by oscillatory states, offering a novel perspective on the interplay between rhythms and behaviorally relevant neural communication.