bioRxiv Subject Collection: Neuroscience's Journal

Neurons in the medial prefrontal cortex that are not modulated by hippocampal sharp-wave ripples are involved in spatial tuning and signaling upcoming choice.
The hippocampus is known to encode spatial information and reactivate experienced trajectories during sharp-wave ripple events. These events are thought to be key time-points at which information about learned trajectories is transferred to the neocortex for long-term storage. It is unclear, however, how this information may be transferred and integrated in downstream cortical regions. In this study, we performed high-density probe recordings across the full depth of the medial prefrontal cortex and in the hippocampus simultaneously in rats while they were performing a task of spatial navigation. We find that neurons in the medial prefrontal cortex encode spatial information and reliably predict upcoming choice on a maze, and we find that a subset of neurons in the mPFC is modulated by hippocampal sharp-wave ripples. However, the neurons that are involved in predicting upcoming choice are not the neurons that are modulated by hippocampal sharp-wave ripples. This indicates that the integration of spatial information requires the collaboration of different specialized populations of neurons.

Multiple Functions of Cerebello-Thalamic Neurons in Learning and Offline Consolidation of a Motor Skill in mice.
Motor skill learning is a complex and gradual process that involves the cortex and basal ganglia, both crucial for the acquisition and long-term retention of skills. The cerebellum, which rapidly learns to adjust the movement, connects to the motor cortex and the striatum via the ventral and intralaminar thalamus respectively. Here, we evaluated the contribution of cerebellar neurons projecting to these thalamic nuclei in a skilled locomotion task in mice. Using a targeted chemogenetic inhibition that preserves the motor abilities, we found that cerebellar nuclei neurons projecting to the intralaminar thalamus contribute to learning and expression, while cerebellar nuclei neurons projecting to the ventral thalamus contribute to offline consolidation. Asymptotic performance, however, required each type of neurons. Thus, our results show that cerebellar neurons belonging to two parallel cerebello-thalamic pathways play distinct, but complementary, roles functioning on different timescales and both necessary for motor skill learning.

Intergenerational transmission of the structure of the auditory cortex and reading skills
High-level cognitive skill development relies on genetic and environmental factors, tied to brain structure and function. Inter-individual variability in language and music skills has been repeatedly associated with the structure of the auditory cortex: the shape, size and asymmetry of the transverse temporal gyrus (TTG) or gyri (TTGs). TTG is highly variable in shape and size, some individuals having one single gyrus (also referred to as Heschls gyrus, HG) while others presenting duplications (with a common stem or fully separated) or higher-order multiplications of TTG. Both genetic and environmental influences on childrens cognition, behavior, and brain can to some to degree be traced back to familial and parental factors. In the current study, using a unique MRI dataset of parents and children (135 individuals from 37 families), we ask whether the anatomy of the auditory cortex is related to reading skills, and whether there are intergenerational effects on TTG(s) anatomy. For this, we performed detailed, automatic segmentations of HG and of additional TTG(s), when present, extracting volume, surface area, thickness and shape of the gyri. We tested for relationships between these and reading skill, and assessed their degree of familial similarity and intergenerational transmission effects. We found that volume and area of all identified left TTG(s) combined was positively related to reading scores, both in children and adults. With respect to intergenerational similarities in the structure of the auditory cortex, we identified structural brain similarities for parent-child pairs of the 1st TTG (Heschls gyrus, HG) (in terms of volume, area and thickness for the right HG, and shape for the left HG) and of the lateralization of all TTG(s) surface area for father-child pairs. Both the HG and TTG-lateralization findings were significantly more likely for parent-child dyads than for unrelated adult-child pairs. Furthermore, we established characteristics of parents TTG that are related to better reading abilities in children: fathers small left HG, and a small ratio of HG to planum temporale. Our results suggest intergenerational transmission of specific structural features of the auditory cortex; these may arise from genetics and/or from shared environment.

Genetic deletion of NAPE-PLD induces context-dependent dysregulation of anxiety-like behaviors, stress responsiveness, and HPA-axis functionality in mice
The endocannabinoid (eCB) system regulates stress responsiveness and hypothalamic-pituitary-adrenal (HPA) axis activity. The enzyme N-acyl phosphatidylethanolamine phospholipase-D (NAPE-PLD) is primarily responsible for the synthesis of the endocannabinoid signaling molecule anandamide (AEA) and other structurally related lipid signaling molecules known as N-acylethanolamines (NAEs). However, little is known about how activity of this enzyme affects behavior. As AEA plays a regulatory role in stress adaptation, we hypothesized that reducing synthesis of AEA and other NAEs would dysregulate stress reactivity. To test this hypothesis, we evaluated wild type (WT) and NAPE-PLD knockout (KO) mice in behavioral assays that assess stress responsiveness and anxiety-like behavior. NAPE-PLD KO mice exhibited anxiety-like behaviors in the open field test and the light-dark box test after a period of single housing. NAPE-PLD KO mice exhibited a heightened freezing response to the testing environment that was further enhanced by exposure to 2,3,5-trimethyl-3-thiazoline (TMT) predator odor. NAPE-PLD KO mice exhibited an exaggerated freezing response at baseline but blunted response to TMT when compared to WT mice. NAPE-PLD KO mice also exhibited a context-dependent dysregulation of HPA axis in response to TMT in the paraventricular hypothalamic nucleus at a neuronal level, as measured by c-Fos immunohistochemstry. Male, but not female, NAPE-PLD knockout mice showed higher levels of circulating corticosterone relative to same-sex wildtype mice in response to TMT exposure, suggesting a sexually-dimorphic dysregulation of the HPA axis at the hormonal level. Together, these findings suggest the enzymatic activity of NAPE-PLD regulates emotional resilience and recovery from both acute and sustained stress.

Abrogation of presynaptic facilitation at hippocampal mossy fiber synapses impacts neural ensemble activity and spatial memory
Presynaptic short-term plasticity is thought to play a major role in the process of spike transfer within local circuits. Mossy fiber synapses between the axons of dentate gyrus (DG) granule cells and CA3 pyramidal cells (Mf-CA3 synapses) display a remarkable extent of presynaptic plasticity which endows these synaptic connections with detonator properties. The pattern of action potential firing, in the form of high frequency bursts in the DG, strongly controls the amplitude of synaptic responses and information transfer to CA3. Here we have investigated the role of presynaptic facilitation at Mf-CA3 synapses in the operation of CA3 circuits in vivo and in memory encoding. Syt7, a calcium sensor necessary for presynaptic facilitation, was selectively abrogated, in DG granule cells using Syt7 conditional KO mice (DG Syt7 KO mice). In hippocampal slices, we extend previous analysis to show that short-term presynaptic facilitation is selectively suppressed at Mf-CA3 synapses in the absence of Syt7, without any impact on basal synaptic properties and long-term potentiation. Short-term plasticity was found to be crucial for spike transfer between the DG and CA3 in conditions of naturalistic patterns of presynaptic firing. At the network level, in awake head-fixed mice, the abrogation of short-term plasticity largely reduced the co-activity of CA3 pyramidal cells. Finally, whereas DG Syt7 KO mice are not impaired in behavioral tasks based on pattern separation, they show deficits in spatial memory tasks which rely on the process of pattern completion. These results shed new light on the role of the detonator properties of DG-CA3 synapses, and give important insights into how this key synaptic feature translate at the population and behavioral levels.

The transcriptome of playfulness is sex-biased in the juvenile rat medial amygdala: a role for inhibitory neurons
Social play is a dynamic behavior known to be sexually differentiated; in most species, males play more than females, a sex difference driven in large part by the medial amygdala (MeA). Despite the well-conserved nature of this sex difference and the importance of social play for appropriate maturation of brain and behavior, the full mechanism establishing the sex bias in play is unknown. Here, we explore the transcriptome of playfulness in the juvenile rat MeA, assessing differences in gene expression between high- and low-playing animals of both sexes via bulk RNA-sequencing. Using weighted gene co-expression network analysis (WGCNA) to identify gene modules combined with analysis of differentially expressed genes (DEGs), we demonstrate that the transcriptomic profile in the juvenile rat MeA associated with playfulness is largely distinct in males compared to females. Of the 13 play-associated WGCNA networks identified, only two were associated with play in both sexes, and very few DEGs associated with playfulness were shared between males and females. Data from our parallel single-cell RNA-sequencing experiments using amygdala samples from newborn male and female rats suggests that inhibitory neurons drive this sex difference, as the majority of sex-biased DEGs in the neonatal amygdala are enriched within this population. Supporting this notion, we demonstrate that inhibitory neurons comprise the majority of play-active cells in the juvenile MeA, with males having a greater number of play-active cells than females, of which a larger proportion are GABAergic. Through integrative bioinformatic analyses, we further explore the expression, function, and cell-type specificity of key play-associated modules and the regulator hub genes predicted to drive them, providing valuable insight into the sex-biased mechanisms underlying this fundamental social behavior.

Adaptive arousal regulation: Pharmacologically shifting the peak of the Yerkes-Dodson curve by catecholaminergic enhancement of arousal
Performance typically peaks at moderate arousal levels, consistent with the Yerkes-Dodson law, as confirmed by recent human and mouse pupillometry studies. Arousal states are influenced by neuromodulators like catecholamines (noradrenaline; NA and dopamine; DA) and acetylcholine (ACh). To explore their causal roles in this law, we pharmacologically enhanced arousal while measuring human decision-making and spontaneous arousal fluctuations via pupil size. The catecholaminergic agent atomoxetine (ATX) increased overall arousal and shifted the entire arousal-performance curve, suggesting a relative arousal mechanism where performance adapts to arousal fluctuations within arousal states. In contrast, the cholinergic agent donepezil (DNP) did not affect arousal or the curve. We modeled these findings in a neurobiologically plausible computational framework, showing how catecholaminergic modulation alters a disinhibitory neural circuit that encodes sensory evidence for decision-making. This work suggests that performance adapts flexibly to arousal fluctuations, ensuring optimal performance in each and every arousal state.

Adaptive protein synthesis in genetic models of copper deficiency and childhood neurodegeneration
Rare inherited diseases caused by mutations in the copper transporters SLC31A1 (CTR1) or ATP7A induce copper deficiency in the brain and throughout the body, causing seizures and neurodegeneration in infancy. The mechanistic underpinnings of such neuropathology remains unclear. Here, we characterized the molecular mechanisms by which neuronal cells respond to copper depletion in multiple genetic model systems. Targeted deletion of CTR1 in neuroblastoma clonal cell lines produced copper deficiency that was associated with compromised copper-dependent Golgi and mitochondrial enzymes and a metabolic shift favoring glycolysis over oxidative phosphorylation. Proteomic and transcriptomic analysis revealed simultaneous upregulation of mTORC1 and S6K signaling, along with reduced PERK signaling in CTR1 KO cells. Patterns of gene and protein expression and pharmacogenomics show increased activation of the mTORC1-S6K pathway as a pro-survival mechanism, ultimately resulting in increased protein synthesis as measured by puromycin labeling. These effects of copper depletion were corroborated by spatial transcriptomic profiling of the cerebellum of Atp7aflx/Y :: Vil1Cre/+ mice, in which copper-deficient Purkinje cells exhibited upregulated protein synthesis machinery and expression of mTORC1-S6K pathway genes. We tested whether increased activity of mTOR in copper-deficient neurons was adaptive or deleterious by genetic epistasis experiments in Drosophila. Copper deficiency dendritic phenotypes in class IV neurons are partially rescued by increased S6k expression or 4E-BP1 (Thor) RNAi, while epidermis phenotypes are exacerbated by Akt, S6k, or raptor RNAi. Overall, we demonstrate that increased mTORC1-S6K pathway activation and protein synthesis is an adaptive mechanism by which neuronal cells respond to copper depletion.

Association between maternal depressive symptoms and hair cortisol concentration during pregnancy with corpus callosum integrity in newborns
Maternal prenatal depressive symptoms are linked to neurodevelopmental impairments in offspring. Maternal cortisol levels are hypothesized to moderate this association, but its relationship with depressive symptoms is inconsistent. This study examined how maternal prenatal depressive symptoms and cortisol levels predict infant brain development, focusing on neonatal corpus callosum (CC) integrity. Using data from the FinnBrain Birth Cohort Study, we analyzed 37 mother-infant dyads. MRI data were collected from 2 to 5 weeks old infants, and DTI imaging estimated fractional anisotropy (FA) in CC regions (Genu, Body, and Splenium). Maternal cortisol levels were assessed through hair cortisol concentration (HCC) from a 5cm hair segment, reflecting cortisol over the last five months of pregnancy. A factor score of maternal depressive symptoms was computed from EPDS questionnaire data collected at gestational weeks 14, 24, and 34. We employed multivariate regression models with a Bayesian approach for statistical testing, controlling for maternal and infant attributes. Results indicated that maternal prenatal depressive symptoms and HCC interact negatively in predicting infants' FA across all CC regions. Infants exposed to high prenatal depressive symptoms and low HCC (1 SD below the mean) showed higher FA in all CC regions. These findings highlight the complex dynamics between maternal prenatal cortisol levels and depressive symptoms, revealing a nuanced impact of those factors on the structural integrity of infants' CC.

Dynamic imaging of myelin pathology in physiologically preserved human brain tissue using third harmonic generation microscopy
Myelin pathology is known to play a central role in disorders such as multiple sclerosis (MS) among others. Despite this, the pathological mechanisms underlying these conditions are often difficult to unravel. Conventional techniques like immunohistochemistry or dye-based approaches, do not provide a temporal characterization of the pathophysiological aberrations responsible for myelin changes in human specimens. Here, to circumvent this curb, we present a label-free, live-cell imaging approach of myelin using recent advancements in nonlinear harmonic generation microscopy applied to physiologically viable human brain tissue from post-mortem donors. Gray and white matter brain tissue from epilepsy surgery and post-mortem donors was excised. To sustain viability of the specimens for several hours, they were subjected to either acute or organotypic slice culture protocols in artificial cerebral spinal fluid. Imaging was performed using a femtosecond pulsed 1060 nm laser to generate second harmonic generation (SHG) and third harmonic generation (THG) signals directly from myelin and axon-like structures without the need to add any labels. Experiments on acute human brain slices and post-mortem human slice cultures reveal that myelin, along with lipid bodies, are the prime sources of THG signal. We show that tissue viability is maintained over extended periods during THG microscopy, and that prolonged THG imaging is able to detect experimentally induced subtle alterations in myelin morphology. Finally, we provide practical evidence that live-cell imaging of myelin with THG microscopy is a sensitive tool to investigate subtle changes in white matter of neurological donors. Overall, our findings support that nonlinear live-cell imaging is a suitable setup for researching myelin morphology in neurological conditions like MS.

Distinct input-specific mechanisms enable presynaptic homeostatic plasticity
Synapses are endowed with the flexibility to change through experience, but must be sufficiently stable to last a lifetime. This tension is illustrated at the Drosophila neuromuscular junction (NMJ), where two motor inputs that differ in structural and functional properties co-innervate most muscles to coordinate locomotion. To stabilize NMJ activity, motor neurons augment neurotransmitter release following diminished postsynaptic glutamate receptor functionality, termed presynaptic homeostatic potentiation (PHP). How these distinct inputs contribute to PHP plasticity remains enigmatic. We have used a botulinum neurotoxin to selectively silence each input and resolve their roles in PHP, demonstrating that PHP is input-specific: Chronic (genetic) PHP selectively targets the tonic MN-Ib, where active zone remodeling enhances Ca2+ influx to promote increased glutamate release. In contrast, acute (pharmacological) PHP selectively increases vesicle pools to potentiate phasic MN-Is. Thus, distinct homeostatic modulations in active zone nanoarchitecture, vesicle pools, and Ca2+ influx collaborate to enable input-specific PHP expression.

Role of ERK2 dimerization in synaptic plasticity and memory
Extensive research has focused on extracellular-signal regulated kinase 1/2 (ERK) phosphorylation in different memory and plasticity models. However, the precise mechanism by which ERK activity leads to memory stabilization and restabilization remains largely elusive, and little is known about the role of ERK 1/2 dimerization in those processes. ERK dimerization is critical for the binding and activation of extranuclear targets, some of which have been strongly associated with these processes. Here we report for the first time that ERK2 dimerization occurs in the context of the rodent nervous system and plays a critical role in plasticity and memory processes. ERK2 dimerization was blocked by DEL-22379 (DEL), a recently developed specific ERK dimerization inhibitor in mice hippocampus in vivo. Moreover, DEL impaired high frequency stimulation-induced long-term potentiation in acute hippocampal slices. However, inhibitory avoidance (IA) memory reactivation induced a significant decrease of ERK2 dimerization in hippocampi from weak IA-trained mice. Noteworthily, intrahippocampal infusion of the inhibitor after memory reactivation had a surprising bidirectional effect: while it blocked reconsolidation of a strong IA memory, the opposite effect was observed on reconsolidation of a weak IA memory, resulting in its enhancement. Although more research is needed, these initial findings suggest a relevant role of ERK dimerization in plasticity and memory.

Synapse-to-synapse plasticity variability balanced to generate input-wide constancy of transmitter release
Basal synaptic strength can vary greatly between synapses formed by an individual neuron because of diverse probabilities of action potential (AP) evoked transmitter release (Pr). Optical quantal analysis on large numbers of identified Drosophila larval glutamatergic synapses shows that short-term plasticity (STP) also varies greatly between synapses made by an individual type I motor neuron (MN) onto a single body wall muscle. Synapses with high and low Pr and different forms and level of STP have a random spatial distribution in the MN nerve terminal, and ones with very different properties can be located within 200 nm of one other. While synapses start off with widely diverse basal Pr at low MN AP firing frequency and change Pr differentially when MN firing frequency increases, the overall distribution of Pr remains remarkably constant due to a balance between the numbers of synapses that facilitate and depress as well as their degree of change and basal synaptic weights. This constancy in transmitter release can ensure robustness across changing behavioral conditions.

Spontaneously regenerative corticospinal neurons in mice
The spinal cord receives inputs from the cortex via corticospinal neurons (CSNs). While predominantly a contralateral projection, a less-investigated minority of its axons terminate in the ipsilateral spinal cord. We analyzed the spatial and molecular properties of these ipsilateral axons and their post-synaptic targets in mice and found they project primarily to the ventral horn, including directly to motor neurons. Barcode-based reconstruction of the ipsilateral axons revealed a class of primarily bilaterally-projecting CSNs with a distinct cortical distribution. The molecular properties of these ipsilaterally-projecting CSNs (IP-CSNs) are strikingly similar to the previously described molecular signature of embryonic-like regenerating CSNs. Finally, we show that IP-CSNs are spontaneously regenerative after spinal cord injury. The discovery of a class of spontaneously regenerative CSNs may prove valuable to the study of spinal cord injury. Additionally, this work suggests that the retention of juvenile-like characteristics may be a widespread phenomenon in adult nervous systems.

Synaptic inhibition in the accessory olfactory bulb regulates pheromone location learning and memory
Pheromone signaling is pivotal in driving social and reproductive behaviors of rodents. Learning and memorizing the pheromone locations involve olfactory subsystems. To study the neural basis of this behavior, we trained female heterozygous knockouts of GluA2 (AMPAR subunit) and NR1 (NMDAR subunit), targeting GAD65 interneuron population, in a pheromone place preference learning assay. We observed memory loss of pheromone locations on early and late recall periods, pointing towards the possible role of ionotropic glutamate receptors (iGluRs), and thereby the synaptic inhibition in pheromone location learning. Correlated changes were observed in the expression levels of activity-regulated cytoskeletal (Arc) protein, which is critical for memory consolidation, in the associated brain areas. Further, to probe the involvement of main olfactory bulb (MOB) and accessory olfactory bulb (AOB) in pheromone location learning, we knocked out NR1 and GluA2 from MOB and/or AOB neuronal circuits by stereotaxic injection of Cre-dependent AAV5 viral particles. Perturbing the inhibitory circuits of MOB and AOB & AOB-alone resulted in the loss of pheromone location memory. These results confirm the role of iGluRs and the synaptic inhibition exerted by the interneuron network of AOB in regulating learning and memory of pheromone locations.