bioRxiv Subject Collection: Neuroscience's Journal

Adducins regulate morphology and fate of neural progenitors during neocortical neurogenesis
The evolutionary expansion of the mammalian neocortex is mediated by an increase in the proliferative capacity of neural progenitor cells. However, the molecular machinery controlling the proliferation of apical and basal progenitors during neocortical development is still poorly understood. Here we show that the three actin-associated morpho-regulatory adducins (ADD1-3), underlie the abundance of basal progenitors in developing mouse and ferret neocortex in vivo and in human cortical organoids. Over expression of adducins in embryonic mouse neocortex increases the number of protrusions of basal progenitors, leading to an increase in their proliferative capacity and neuronal output. Conversely, knock-out of ADD1 in human cortical organoids, which also leads to down-regulation of other adducins, results in reduced proliferation of progenitors and aberrant neurogenesis. Hence, we show that adducins underlie the proliferation and fate of neural progenitors, which are key cellular features underlying the progression of mammalian neurogenesis.

Paired and solitary ionocytes in the zebrafish olfactory epithelium
The sense of smell is generated by electrical currents that are influenced by the concentration of ions in olfactory sensory neurons and mucus. In contrast to the extensive morphological and molecular characterization of sensory neurons, there has been little description of the cells that control ion concentrations in the zebrafish olfactory system. Here, we report the molecular and ultrastructural characterization of zebrafish olfactory ionocytes. Transcriptome analysis suggests that the zebrafish olfactory epithelium contains at least three different ionocyte types, which resemble Na+/K+-ATPase-rich (NaR), Na+/Cl- cotransporter (NCC), and H+-ATPase-rich (HR) cells, responsible for calcium, chloride, and pH regulation, respectively, in the zebrafish skin. NaR-like and HR-like ionocytes are usually adjacent to one another, whereas NCC-like cells are usually solitary. The distinct subtypes are differentially distributed: NaR-like/HR-like cell pairs are found broadly within the olfactory epithelium, whereas NCC-like cells reside within the peripheral non-sensory multiciliated cell zone. Comparison of gene expression and serial-section electron microscopy analysis indicates that the NaR-like cells wrap around the HR-like cells and are connected to them by shallow tight junctions. The development of olfactory ionocyte subtypes is also differentially regulated, as pharmacological Notch inhibition leads to a loss of NaR-like and HR-like cells, but does not affect NCC-like ionocyte number. These results provide a molecular and anatomical characterization of olfactory ionocytes in a stenohaline freshwater teleost. The paired ionocytes suggest that both transcellular and paracellular transport regulate ion concentrations in the olfactory epithelium, while the solitary ionocytes may enable independent regulation of multiciliated cells.

BLAKOR inputs to the BNST regulate social stress-escalated alcohol consumption
Background: Aversive social experiences can lead to escalated drug consumption and increase the risk of relapse to drug seeking. Individuals who consume alcohol to alleviate the effects of social stress are more likely to develop an alcohol use disorder (AUD). Repeated social defeat stress (SDS) enhances the rewarding and reinforcing effects of alcohol. However, the neural mechanisms that underlie social stress-escalated alcohol drinking are not well understood. Here we explored the role of the dynorphin/kappa opioid receptor (Dyn/KOR) system in regulating social stress-escalated alcohol consumption. Methods: Male and female mice were subjected to repeated SDS for 10 days following which they were left undisturbed in their home cages. They were then subject to intermittent access (IA) two-bottle choice alcohol consumption procedure. The effects of systemic and BNST-specific KOR antagonism using the selective KOR antagonist NorBNI on stress-escalated drinking were evaluated. Using chemogenetic approaches in Oprk1-Cre mice, we examined the role of KOR expressing cells in the basolateral amygdala (BLAKORs) and BLAKOR-BNST pathway in social stress-escalated alcohol consumption. Results: Repeated SDS increased alcohol consumption and preference in both males and females. Systemic KOR antagonism attenuated SDS- escalated alcohol consumption in both males and females. BNST -specific KOR antagonism also attenuated stress-escalated drinking in males. Finally, selective chemogenetic activation of BLAKORs and BKAKOR-BNST pathway attenuated social stress-escalated alcohol consumption in both sexes. Conclusion: Our results suggest a significant role for BLAKOR projections to the BNST in regulating social stress-escalated alcohol consumption. Our results provide further evidence that the Dyn/KOR system maybe a viable target for medications development to treat comorbid stress and AUD.

Perineuronal nets on CA2 pyramidal cells and parvalbumin-expressing cells differentially regulate hippocampal dependent memory
Perineuronal nets (PNNs) are a specialized extracellular matrix that surround certain populations of neurons, including (inhibitory) parvalbumin (PV) expressing-interneurons throughout the brain and (excitatory) CA2 pyramidal neurons in hippocampus. PNNs are thought to regulate synaptic plasticity by stabilizing synapses and as such, could regulate learning and memory. Most often, PNN functions are queried using enzymatic degradation with chondroitinase, but that approach does not differentiate PNNs on CA2 neurons from those on adjacent PV cells. To disentangle the specific roles of PNNs on CA2 pyramidal cells and PV neurons in behavior, we generated conditional knockout mouse strains with the primary protein component of PNNs, aggrecan (Acan), deleted from either CA2 pyramidal cells (Amigo2 Acan KO) or from PV cells (PV Acan KO). Male and female animals of each strain were tested for social, fear, and spatial memory, as well as for reversal learning. We found that Amigo2 Acan KO animals, but not PV Acan KO animals, had impaired social memory and reversal learning. PV Acan KOs, but not Amigo2 Acan KOs had impaired contextual fear memory. These findings demonstrate independent roles for PNNs on each cell type in regulating hippocampal-dependent memory. We further investigated a potential mechanism of impaired social memory in the Amigo2 Acan KO animals and found reduced input to CA2 from the supramammillary nucleus (SuM), which signals social novelty. Additionally, Amigo2 Acan KOs lacked a social novelty-related local field potential response, suggesting that CA2 PNNs may coordinate functional SuM connections and associated physiological responses to social novelty.

Automated cell detection for immediate early gene-expressing neurons using inhomogeneous background subtraction in fluorescent images
Although many methods for automated fluorescent-labeled cell detection have been proposed, not all of them assume a highly inhomogeneous background arising from complex biological structures. Here, we propose an automated cell detection algorithm that accounts for and subtracts the inhomogeneous background by avoiding high-intensity pixels in the blur filtering calculation. Cells were detected by intensity thresholding in the background-subtracted image, and the algorithm's performance was tested on NeuN- and c-Fos-stained images in the mouse prefrontal cortex and hippocampal dentate gyrus. In addition, applications in c-Fos positive cell counting and the quantification for the expression level in double-labeled cells were demonstrated. Our method of automated detection after background assumption (ADABA) offers the advantage of high-throughput and unbiased analysis in regions with complex biological structures that produce inhomogeneous background.

Ccp1 depletion disrupts network integration of hippocampal parvalbumin interneurons
Post-translational modifications (PTMs) of microtubules (MTs) endow them with specific properties that are essential for key cellular functions, such as axonal transport. Polyglutamylation, a PTM that accumulates in long-lived MTs, has been linked to neurodegeneration in the cerebellum when in excess. While hyperglutamylation of MTs leads to neurodegeneration and disrupts the function of specific neuronal subtypes like Purkinje cells, cortical neurons, and hippocampal excitatory neurons, little is known about its impact on inhibitory interneurons and their functional integration into local networks. In this study, we generated a conditional knockout mouse model to deplete cytosolic carboxypeptidase 1 (Ccp1) in GABAergic neurons, a key MT deglutamylase expressed by hippocampal interneurons. Our findings reveal that the loss of Ccp1 has a profound effect on hippocampal parvalbumin (PV)-expressing interneurons, impairing their axonal transport and reducing their perisomatic inhibition of pyramidal cells (PCs) in the CA2 region of the hippocampus.

Hormonal contraceptive intake during adolescence and cortical brain measures in the ABCD Study
Adolescence is a critical period in human development, marked by rapid changes in brain structure and function, influenced, in part, by sex steroids. During adolescence, females may initiate the use of hormonal contraception, which suppresses ovarian production of estradiol and progesterone. Here, we examined the impact of hormonal contraceptive use on adolescent brain structures using magnetic resonance imaging data from 1,234 individuals from the ABCD Study (average age = 14 years). We investigated differences in cortical thickness, surface area, and volume in hormonal contraceptive-users compared to non-users. Statistically significant differences were observed in several regions, and differences in cortical thickness of the paracentral gyrus survived familywise error correction. Estradiol, testosterone and DHEA levels negatively correlated with cortical thickness, surface area, and volume across both hormonal contraceptive-users and non-users only at an uncorrected threshold. These findings highlight the need to further study longitudinal effects of hormonal contraceptives and endogenous hormone changes on brain development in the ABCD Study and related datasets as these data become available.

Cortical contributions to attentional orienting and response cancellation in action stopping
Action cancellation involves the termination of planned or initiated movement. Contemporary models of action cancellation, such as the Pause-then-Cancel model, propose that this occurs via a two-stage process, initiated in the cortex by the pre-supplementary motor area (preSMA) and inferior frontal gyrus (IFG). Previous experimental work using electromyography (EMG) has identified that the cancellation of actions can involve the partial activation of the responding muscles, which does not result in an overt behavioural response. In this study, we used functional near-infrared spectroscropy (fNIRS) to investigate the neural correlates of these partial responses in a modified stopping task (a response- and stimulus-selective stop-signal task). We controlled for the attentional effects that have long confounded action cancellation research by comparing responses to stop stimuli with those to ignore stimuli. We identified stopping-related activity in the preSMA but not the IFG, consistent with predictions of the Pause-then-Cancel model. Additionally, we observed increased preSMA activity in trials where no partial responses occurred, potentially due to the cumulative effect of different inhibitory processes in those trials. This study highlights the utility of combining fNIRS and EMG in examining the cortical correlates and dynamic processes involved in action cancellation.

Convolutional neural network models describe the encoding subspace of local circuits in auditory cortex
Auditory cortex encodes information about nonlinear combinations of spectro-temporal sound features. Convolutional neural networks (CNNs) provide an architecture for generalizable encoding models that can predict time-varying neural activity evoked by natural sounds with substantially greater accuracy than established models. However, the complexity of CNNs makes it difficult to discern the computational properties that support their improved performance. To address this limitation, we developed a method to visualize the tuning subspace captured by a CNN. Single-unit data was recorded using high channel-count microelectrode arrays from primary auditory cortex (A1) of awake, passively listening ferrets during presentation of a large natural sound set. A CNN was fit to the data, replicating approaches from previous work. To measure the tuning subspace, the dynamic spectrotemporal receptive field (dSTRF) was measured as the locally linear filter approximating the input-output relationship of the CNN at each stimulus timepoint. Principal component analysis was then used to reduce this very large set of filters to a smaller subspace, typically requiring 2-10 filters to account for 90% of dSTRF variance. The stimulus was projected into the subspace for each neuron, and a new model was fit using only the projected values. The subspace model was able to predict time-varying spike rate nearly as accurately as the full CNN. Sensory responses could be plotted in the subspace, providing a compact model visualization. This analysis revealed a diversity of nonlinear responses, consistent with contrast gain control and emergent invariance to spectrotemporal modulation phase. Within local populations, neurons formed a sparse representation by tiling the tuning subspace. Narrow spiking, putative inhibitory neurons showed distinct patterns of tuning that may reflect their position in the cortical circuit. These results demonstrate a conceptual link between CNN and subspace models and establish a framework for interpretation of deep learning-based models.

Axonal spheroids are regulated by Schwann cells after peripheral nerve injury
Axonal spheroids are hallmark features of neurodegeneration, forming along degenerating axons and contributing to disease progression. Despite their ubiquity across degenerative etiologies, the dynamics of spheroid disappearance, as well as their interactions with glial cells, remain poorly understood. Here, using an in vivo zebrafish model of peripheral nerve injury, we identified several patterns of spheroid disappearance that are regulated by Schwann cells. These results describe spheroid dynamics across their lifetimes, establish a role for the extra-axonal environment in altering spheroid outcomes, and identify a cellular mechanism whereby spheroid fates are altered.

RID-Rihaczek Phase Synchrony Method Applied to Resting-State EEG: Simultaneous prediction of visuospatial tracking, verbal communication, executive function, neuro-cognitive health, and intelligence
Brain activity at a resting state and functional connectivity represent individuals' neural configurations that can be used to predict various performance and trait measures. Yet, most of the previous empirical demonstrations of this phenomena are limited to using resting-state neuroimaging to predict a single task performance/trait measure, and a few studies that predicted multiple task performance/traits measures employed conceptually similar tasks to produce converging evidence for their topic of investigation. In the current paper, we applied RID-Rihazcek phase synchrony, a nonlinear time-frequency-based method for neural computation, to resting-state EEG data and simultaneously predicted a wide variety of measures: standard shooting task performance and novel verbal communication-based team shooting task performance in a simulator, visuo-spatial tracking task performance (Neurotracker), executive function (verbal fluency task), fluid and crystalized Intelligence (Multidimensional Aptitude Battery II: MAB-II), and neurocognitive functioning (Automated Neuropsychological Assessment Metrics-4: ANAM-4) with the average R2 = .60. Our findings show great promise for RID-Rihazcek phase synchrony and resting-state EEG in general to be used in aptitude assessment.

Pathological tau alters head direction signaling and induces spatial disorientation
Disorientation is an early symptom of dementia, suggesting impairments in neural circuits responsible for head direction signaling. The anterodorsal thalamic nucleus (ADn) exhibits early and selective vulnerability to pathological misfolded forms of tau (ptau), a major hallmark of Alzheimer's disease and ageing. The ADn contains a high density of head direction (HD) cells; their disruption may contribute to spatial disorientation. To test this, we virally expressed human tau in the ADn of adult mice. HD-ptau mice were defined by ptau+ cells in the ADn and ptau+ axon terminals in postsynaptic target regions. Despite being able to learn spatial memory tasks, HD-ptau mice exhibited increased looping behavior during spatial learning and made a greater number of head turns during memory recall, consistent with disorientation. Using in vivo extracellular recordings, we identified ptau-expressing ADn cells and found that ADn cells from HD-ptau mice had reduced directionality and altered burst firing. These data suggest that ptau alters HD signaling, leading to impairments in spatial orientation.

Mental Tasks Induce Common Modulations of Oscillations in Cortex and Spinal Cord
We investigated whether the same modulations in spinal motor neurons parallel power modulations of cortical oscillations induced by mental tasks. We recruited 15 participants and recorded high-density electromyography signals (HD-EMG) from the tibialis anterior muscle, as well as electroencephalography (EEG) signals. The cumulative spike train (CST) was computed from the activity of spinal motor neurons decoded from HD-EMG signals. The participants performed sustained dorsiflexion concurrent with foot motor imagery, hand motor imagery, mental arithmetic, or no specific mental task. We found significant power correlations between CST and EEG across trials irrespective of the mental task and across mental tasks at the intra-muscular coherence peak ({tau}_trial = 0.08 {+/-} 0.10, {tau}_task = 0.33 {+/-} 0.19, respectively; mean {+/-} std. dev.). CST power in beta and low-gamma bands could provide a novel control signal for neural interface applications, as power changes in these bands are not translated into actual force changes. To evaluate the potential of CST bands as a control signal, we classified the mental tasks from CST bandpower with a linear classifier and obtained classification accuracies slightly but significantly above chance level (30% {+/-} 5%; chance level = 25%). These results show for the first time that mental tasks can modulate the power of cortical and spinal oscillations concurrently. This supports the notion that movement-unrelated oscillations can leak down from the cortex to the spinal level. We further show that mental tasks can be classified from CST, although further research is necessary to boost the classification performance to an adequate level for neural interface applications.

Sudden sensory events trigger modality-independent responses across layers in the mouse neocortex
Sudden sensory events trigger widespread electrocortical responses and noticeable changes in an animal's behavior. It remains unclear, though, how this "surprise" signal is generated via afferent sensory input and represented by neuronal ensembles. Using in vivo electrophysiology, here we recorded the activity of different cortical areas across layers in awake head-fixed mice, while presenting sensory stimuli of different modalities and saliencies. When brief, salient stimuli were delivered at long intervals, we found prominent responses across cortical areas regardless of the stimulus modalities, resembling those observed in human electroencephalography. These responses were larger and emerged earlier in the infragranular layer than in the granular layer of the primary sensory cortex. At short inter-stimulus intervals, in contrast, only modality-specific responses were observed in the corresponding primary sensory cortex. These results indicate that sensory information travels through multiple ascending pathways, in particular, supra-modal surprise signals via non-classical sensory pathways to the cortex.

Cerebellar-Prefrontal Connectivity Predicts Negative Symptom Severity Across the Psychosis Spectrum
Background: Negative symptom severity predicts functional outcome and quality life in people with psychosis. However, negative symptoms are poorly responsive to antipsychotic medication and existing literature has not converged on their neurobiological basis. Previous work in small schizophrenia samples has observed that lower cerebellar-prefrontal connectivity is associated with higher negative symptom severity and demonstrated in a separate neuromodulation experiment that increasing cerebellar-prefrontal connectivity reduced negative symptom severity. We sought to expand this finding to test associations between cerebellar-prefrontal connectivity with negative symptom severity and cognitive performance in a large, transdiagnostic sample of individuals with psychotic disorders. Methods: In this study, 260 individuals with psychotic disorders underwent resting-state MRI and clinical characterization. Negative symptom severity was measured using the Positive and Negative Symptoms Scale, and cognitive performance was assessed with the Screen for Cognitive Impairment in Psychiatry. Using a previously identified cerebellar region as a seed, we performed seed to whole brain analyses and regressed connectivity against negative symptom severity, using age and sex as covariates. Results: Consistent with prior work, we identified relationships between higher cerebellar-prefrontal connectivity and lower negative symptom severity (r=-0.17, p=.007). Higher cerebellar-prefrontal connectivity was also associated with better delayed verbal learning (r=.13, p=.034). Conclusions: Our results provide further evidence supporting the relationship between cerebellar-prefrontal connectivity and negative symptom severity and cognitive performance. Larger, randomized, sham-controlled neuromodulation studies should test if increasing cerebellar-prefrontal connectivity leads to reductions in negative symptoms in psychosis.

Targeted H3K9 acetylation at lncSox1 promoter by cell type-specific epigenome editing promotes intermediate progenitor proliferation in developing mouse cortex
The distribution and level of epigenetic (chromatin) marks have implications for differential regulatory effects at specific gene loci. Herein, we applied a protocol which combines in vivo electroporation and a CRISPR-dead (d)Cas9 system to probe and edit a specific chromatin mark in the epigenome of intermediate progenitor cells (IPCs) in developing mouse cortex. We found that the promoter of lncSox1, a long non-coding gene, is a key genomic locus for H3K9 acetylation (H3K9ac) during IPC amplification. CRISPR-dCas9-mediated addition of H3K9ac at lncSox1 promoter resulted in lncSox1 upregulation, with attendant increase in IPC pool and augmented neurogenesis. Thus, we have identified dynamic regulation of lncSox1 as a major downstream target of H3 acetylation, and as part of an epigenetic mechanism involved in IPC proliferation during neocortex expansion. This finding is a proof-of-concept that our epigenome editing-based method can be used for manipulating specific epigenetic effectors to determine their (neuro)biological significance.

MOTIVATIONThe abundance of basal progenitors is critical for cortical neurogenesis during brain development. There is growing interest in identifying how specific epigenetic factors regulate the genesis and expansion of basal progenitor cell sub-populations, including intermediate progenitor cells. We established a protocol that allowed us to identify the involvement of a long non-coding RNA (lncSox1) in regulating the proliferation of intermediate progenitor cells under the influence of H3K9 acetylation (H3K9ac). By enhancing H3K9ac at the promoter region of lncSox1 using a CRISPR-dCas9-mediated gene-editing tool, we were able to determine that lncSox1 upregulation is a downstream effect of H3K9ac acetylation and is necessary for intermediate progenitor pool amplification during cortical development.

Highlights- Identification of H3 acetylation-dependent expression of ncRNAs in developing cortex.
- Establishment of cell Cre/LoxP and CRISPR-dCas9-dependent H3K9ac epigenome editing.
- CRISPR-dCas9-mediated addition of H3K9ac at lncSox1 promoter resulted in lncSox1 upregulation.
- H3K9 acetylation at lncSox1 promoter enhances proliferation of TBR2-expressing IPCs.
- Targeted epigenome editing revealed lncSox1 as a key regulator of cortical development.

Challenges in inferring breathing rhythms from olfactory bulb local field potentials
Odors convey useful navigational and episodic information, yet much of the chemical world remains inaccessible without active sampling through sniffing. To effectively interpret olfactory cues, the brain must unify odor-driven activity with respiratory cycles, making accurate respiratory measurements critical in understanding olfactory bulb (OB) dynamics. Previous studies have shown that behavioral signals are often present in primary sensory areas, and OB local field potentials (LFPs) have long been known to couple with respiration. Here we investigated whether OB LFPs can reliably recover the precise timing and frequency of respiration. Our results indicate that OB LFPs across multiple frequency bands align with respiratory cycles. Using time and frequency domain methods, we show that 2-12 Hz LFP oscillations effectively track respiratory frequency. However, a monotonic relationship between LFP-respiratory delay and sniffing frequency, which varies across animals, renders the recovery of precise respiratory events challenging. This work demonstrates the complex and individualized relationship between rodent respiration and OB LFPs, contributing to our understanding of how respiratory signals are represented in the OB.

Targeting Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1) Neurons: Validating the Specificity of a Novel Transgenic Crhr1-FlpO Mouse
Introduction: Corticotropin-releasing hormone (CRH) signaling through its cognate receptors, CRHR1 and CRHR2, contributes to diverse stress-related functions in the mammalian brain. Whereas CRHR2 is predominantly expressed in choroid plexus and blood vessels, CRHR1 is abundantly expressed in neurons in discrete brain regions, including the neocortex, hippocampus and nucleus accumbens. Activation of CRHR1 influences motivated behaviors, emotional states, and learning and memory. However, it is unknown whether alterations in CRHR1 signaling contribute to aberrant motivated behaviors observed, for example, in stressful contexts. These questions require tools to manipulate CRHR1 selectively. Here we describe and validate a novel Crhr1-FlpO mouse. Methods: Using bacterial artificial chromosome (BAC) transgenesis, we engineered a transgenic mouse that expresses FlpO recombinase in CRHR1-expressing cells. We used two independent methods to assess the specificity of FlpO to CRHR1-expressing cells. First, we injected Crhr1-FlpO mice with Flp-dependent viruses expressing fluorescent reporter molecules. Additionally, we crossed the Crhr1-FlpO mouse with a transgenic Flp-dependent reporter mouse. CRHR1 and reporter molecules were identified using immunocytochemistry and visualized via confocal microscopy in several brain regions in which CRHR1 expression and function is established. Results: Expression of Flp-dependent viral constructs was highly specific to CRHR1-expressing cells in all regions examined (over 90% co-localization). In accord, robust and specific expression of the Flp-dependent transgenic reporter was observed in a reporter mouse, recapitulating endogenous CRHR1 expression. Conclusions: The Crhr1-FlpO mouse enables selective genetic access to CRHR1-expressing cells within the mouse brain. When combined with Cre-lox or site-specific recombinases, the mouse facilitates intersectional manipulations of CRHR1-expressing neurons.

Projections between the globus pallidus externa and cortex span motor and non-motor regions
The globus pallidus externa (GPe) is a heterogenous nucleus of the basal ganglia, with intricate connections to other basal ganglia nuclei, as well as direct connections to the cortex. The anatomic, molecular and electrophysiologic properties of cortex-projecting pallidocortical neurons are not well characterized. Here we show that pallidocortical neurons project to diverse motor and non-motor cortical regions, are organized topographically in the GPe, and segregate into two distinct electrophysiological and molecular phenotypes. In addition, we find that the GPe receives direct synaptic input back from deep layers of diverse motor and non-motor cortical regions, some of which form reciprocal connections onto pallidocortical neurons. These results demonstrate the existence of a fast, closed-loop circuit between the GPe and the cortex which is ideally positioned to integrate information about behavioral goals, internal states, and environmental cues to rapidly modulate behavior.