Long-Term Impact of Early Life Stress on Serotonin Connectivity.

BACKGROUND: Chronic childhood stress is a prominent risk factor for developing affective disorders, yet mechanisms underlying this association remain unclear. Maintenance of optimal serotonin (5-HT) levels during early postnatal development is critical for the maturation of brain circuits. Understanding the long-lasting effects of early life stress (ELS) on serotonin-modulated brain connectivity is crucial to develop treatments for affective disorders arising from childhood stress. METHODS: Using a mouse model of chronic developmental stress, we determined the long-lasting consequences of ELS on 5-HT circuits and behavior in females and males. Using FosTRAP mice, we cross-correlated regional c-Fos density to determine brain-wide functional connectivity of the raphe nucleus. We next performed in vivo fiber photometry to establish ELS-induced deficits in 5-HT dynamics and optogenetics to stimulate 5-HT release to improve behavior. RESULTS: Adult female and male mice exposed to ELS showed heightened anxiety-like behavior. ELS further enhanced susceptibility to acute stress by disrupting the brain-wide functional connectivity of the raphe nucleus and the activity of 5-HT neuron population, in conjunction with increased orbitofrontal cortex (OFC) activity and disrupted 5-HT release in medial OFC. Optogenetic stimulation of 5-HT terminals in the medial OFC elicited an anxiolytic effect in ELS mice in a sex-dependent manner. CONCLUSIONS: These findings suggest a significant disruption in 5-HT-modulated brain connectivity in response to ELS, with implications for sex-dependent vulnerability. The anxiolytic effect of the raphe-medial OFC circuit stimulation has potential implications for developing targeted stimulation-based treatments for affective disorders that arise from early life adversities.

PAW, a cost-effective and open-source alternative to commercial rodent running wheels.

Voluntary wheel running is a common measure of general activity in many rodent models across neuroscience and physiology. However, current commercial wheel monitoring systems can be cost-prohibitive to many investigators, with many of these systems requiring investments of thousands of dollars. In recent years, several open-source alternatives have been developed, and while these tools are much more cost effective than commercial system, they often lack the flexibility to be applied to a wide variety of projects. Here, we have developed PAW, a 3D Printable Arduino-based Wheel logger. PAW is wireless, fully self-contained, easy to assemble, and all components necessary for its production can be obtained for only $75 CAD. Furthermore, with its compact internal electronics, the 3D printed casing can be easily modified to be used with a wide variety of running wheel designs for a wide variety of rodent species. Data recorded with the PAW system shows circadian patterns of activity which is expected from mice and is consistent with results found in the literature. Altogether, PAW is a flexible, low-cost system that can be beneficial to a broad range of researchers who study rodent models.

Protocol for the integration of fiber photometry and social behavior in rodent models.

Fiber photometry offers insight into cell-type-specific activity underlying social interactions. We provide a protocol for the integration of fiber photometry recordings into the analysis of social behavior in rodent models. This includes considerations during surgery, notes on synchronizing fiber photometry with behavioral recordings, advice on using multi-animal behavioral tracking software, and scripts for the analysis of fiber photometry recordings. For complete details on the use and execution of this protocol, please refer to Dawson et al. (2023).1.

Repeated episodes of postictal hypoxia are a mechanism for interictal cognitive impairments.

Comorbidities during the period between seizures present a significant challenge for individuals with epilepsy. Despite their clinical relevance, the pathophysiology of the interictal symptomatology is largely unknown. Postictal severe hypoxia (PIH) in those brain regions participating in the seizure has been indicated as a mechanism underlying several negative postictal manifestations. It is unknown how repeated episodes of PIH affect interictal symptoms in epilepsy. Using a rat model, we observed that repeated seizures consistently induced episodes of PIH that become increasingly severe with each seizure occurrence. Additionally, recurrent seizure activity led to decreased levels of oxygen in the hippocampus during the interictal period. However, these reductions were prevented when we repeatedly blocked PIH using either the COX-inhibitor acetaminophen or the L-type calcium channel antagonist nifedipine. Moreover, we found that interictal cognitive deficits caused by seizures were completely alleviated by repeated attenuation of PIH events. Lastly, mitochondrial dysfunction may contribute to the observed pathological outcomes during the interictal period. These findings provide evidence that seizure-induced hypoxia may play a crucial role in several aspects of epilepsy. Consequently, developing and implementing treatments that specifically target and prevent PIH could potentially offer significant benefits for individuals with refractory epilepsy.

Parvalbumin as a sex-specific target in Alzheimer's disease research - A mini-review.

Alzheimer's disease (AD) is the most common form of dementia, and both the incidence of this disease and its associated cognitive decline disproportionally effect women. While the etiology of AD is unknown, recent work has demonstrated that the balance of excitatory and inhibitory activity across the brain may serve as a strong predictor of cognitive impairments in AD. Across the cortex, the most prominent source of inhibitory signalling is from a class of parvalbumin-expressing interneurons (PV+). In this mini-review, the impacts of sex- and age-related factors on the function of PV+ neurons are examined within the context of vulnerability to AD pathology. These primary factors of influence include changes in brain metabolism, circulating sex hormone levels, and inflammatory response. In addition to positing the increased vulnerability of PV+ neurons to dysfunction in AD, this mini-review highlights the critical importance of presenting sex stratified data in the study of AD.

Classifying high-dimensional phenotypes with ensemble learning.

Classification is a fundamental task in biology used to assign members to a class. While linear discriminant functions have long been effective, advances in phenotypic data collection are yielding increasingly high-dimensional datasets with more classes, unequal class covariances, and non-linear distributions. Numerous studies have deployed machine learning techniques to classify such distributions, but they are often restricted to a particular organism, a limited set of algorithms, and/or a specific classification task. In addition, the utility of ensemble learning or the strategic combination of models has not been fully explored.We performed a meta-analysis of 33 algorithms across 20 datasets containing over 20,000 high-dimensional shape phenotypes using an ensemble learning framework. Both binary (e.g., sex, environment) and multi-class (e.g., species, genotype, population) classification tasks were considered. The ensemble workflow contains functions for preprocessing, training individual learners and ensembles, and model evaluation. We evaluated algorithm performance within and among datasets. Furthermore, we quantified the extent to which various dataset and phenotypic properties impact performance.We found that discriminant analysis variants and neural networks were the most accurate base learners on average. However, their performance varied substantially between datasets. Ensemble models achieved the highest performance on average, both within and among datasets, increasing average accuracy by up to 3% over the top base learner. Higher class R2 values, mean class shape distances, and between- vs. within-class variances were positively associated with performance, whereas higher class covariance distances were negatively associated. Class balance and total sample size were not predictive.Learning-based classification is a complex task driven by many hyperparameters. We demonstrate that selecting and optimizing an algorithm based on the results of another study is a flawed strategy. Ensemble models instead offer a flexible approach that is data agnostic and exceptionally accurate. By assessing the impact of various dataset and phenotypic properties on classification performance, we also offer potential explanations for variation in performance. Researchers interested in maximizing performance stand to benefit from the simplicity and effectiveness of our approach made accessible via the R package pheble.

Hypocretin/orexin neurons encode social discrimination and exhibit a sex-dependent necessity for social interaction.

The hypothalamus plays a crucial role in the modulation of social behavior by encoding internal states. The hypothalamic hypocretin/orexin neurons, initially identified as regulators of sleep and appetite, are important for emotional and motivated behaviors. However, their role in social behavior remains unclear. Using fiber photometry and behavioral analysis, we show here that hypocretin neurons differentially encode social discrimination based on the nature of social encounters. The optogenetic inhibition of hypocretin neuron activity or blocking of hcrt-1 receptors reduces the amount of time mice are engaged in social interaction in males but not in females. Reduced hcrt-1 receptor signaling during social interaction is associated with altered activity in the insular cortex and ventral tegmental area in males. Our data implicating hypocretin neurons as sexually dimorphic regulators within social networks have significant implications for the treatment of neuropsychiatric diseases with social dysfunction, particularly considering varying prevalence among sexes.

The impact of loneliness and social isolation on the development of cognitive decline and Alzheimer's Disease.

Alzheimer's Disease (AD) is the leading cause of dementia, observed at a higher incidence in women compared with men. Treatments aimed at improving pathology in AD remain ineffective to stop disease progression. This makes the detection of the early intervention strategies to reduce future disease risk extremely important. Isolation and loneliness have been identified among the major risk factors for AD. The increasing prevalence of both loneliness and AD emphasizes the urgent need to understand this association to inform treatment. Here we present a comprehensive review of both clinical and preclinical studies that investigated loneliness and social isolation as risk factors for AD. We discuss that understanding the mechanisms of how loneliness exacerbates cognitive impairment and AD with a focus on sex differences will shed the light for the underlying mechanisms regarding loneliness as a risk factor for AD and to develop effective prevention or treatment strategies.

Low intensity repetitive transcranial magnetic stimulation modulates brain-wide functional connectivity to promote anti-correlated c-Fos expression.

Repetitive transcranial magnetic stimulation (rTMS) induces action potentials to induce plastic changes in the brain with increasing evidence for the therapeutic importance of brain-wide functional network effects of rTMS; however, the influence of sub-action potential threshold (low-intensity; LI-) rTMS on neuronal activity is largely unknown. We investigated whether LI-rTMS modulates neuronal activity and functional connectivity and also specifically assessed modulation of parvalbumin interneuron activity. We conducted a brain-wide analysis of c-Fos, a marker for neuronal activity, in mice that received LI-rTMS to visual cortex. Mice received single or multiple sessions of excitatory 10 Hz LI-rTMS with custom rodent coils or were sham controls. We assessed changes to c-Fos positive cell densities and c-Fos/parvalbumin co-expression. Peak c-Fos expression corresponded with activity during rTMS. We also assessed functional connectivity changes using brain-wide c-Fos-based network analysis. LI-rTMS modulated c-Fos expression in cortical and subcortical regions. c-Fos density changes were most prevalent with acute stimulation, however chronic stimulation decreased parvalbumin interneuron activity, most prominently in the amygdala and striatum. LI-rTMS also increased anti-correlated functional connectivity, with the most prominent effects also in the amygdala and striatum following chronic stimulation. LI-rTMS induces changes in c-Fos expression that suggest modulation of neuronal activity and functional connectivity throughout the brain. Our results suggest that LI-rTMS promotes anticorrelated functional connectivity, possibly due to decreased parvalbumin interneuron activation induced by chronic stimulation. These changes may underpin therapeutic rTMS effects, therefore modulation of subcortical activity supports rTMS for treatment of disorders involving subcortical dysregulation.

Brain-wide neuronal activation and functional connectivity are modulated by prior exposure to repetitive learning episodes.

Memory storage and retrieval are shaped by past experiences. Prior learning and memory episodes have numerous impacts on brain structure from micro to macroscale. Previous experience with specific forms of learning increases the efficiency of future learning. It is less clear whether such practice effects on one type of memory might also have transferable effects to other forms of memory. Different forms of learning and memory rely on different brain-wide networks but there are many points of overlap in these networks. Enhanced structural or functional connectivity caused by one type of learning may be transferable to another type of learning due to overlap in underlying memory networks. Here, we investigated the impact of prior chronic spatial training on the task-specific functional connectivity related to subsequent contextual fear memory recall in mice. Our results show that mice exposed to prior spatial training exhibited decreased brain-wide activation compared to control mice during the retrieval of a context fear memory. With respect to functional connectivity, we observed changes in several network measures, notably an increase in global efficiency. Interestingly, we also observed an increase in network resilience based on simulated targeted node deletion. Overall, this study suggests that chronic learning has transferable effects on the functional connectivity networks of other types of learning and memory. The generalized enhancements in network efficiency and resilience suggest that learning itself may protect brain networks against deterioration.

Toxoplasma infection in male mice alters dopamine-sensitive behaviors and host gene expression patterns associated with neuropsychiatric disease.

During chronic infection, the single celled parasite, Toxoplasma gondii, can migrate to the brain where it has been associated with altered dopamine function and the capacity to modulate host behavior, increasing risk of neurocognitive disorders. Here we explore alterations in dopamine-related behavior in a new mouse model based on stimulant (cocaine)-induced hyperactivity. In combination with cocaine, infection resulted in heightened sensorimotor deficits and impairment in prepulse inhibition response, which are commonly disrupted in neuropsychiatric conditions. To identify molecular pathways in the brain affected by chronic T. gondii infection, we investigated patterns of gene expression. As expected, infection was associated with an enrichment of genes associated with general immune response pathways, that otherwise limits statistical power to identify more informative pathways. To overcome this limitation and focus on pathways of neurological relevance, we developed a novel context enrichment approach that relies on a customized ontology. Applying this approach, we identified genes that exhibited unexpected patterns of expression arising from the combination of cocaine exposure and infection. These include sets of genes which exhibited dampened response to cocaine in infected mice, suggesting a possible mechanism for some observed behaviors and a neuroprotective effect that may be advantageous to parasite persistence. This model offers a powerful new approach to dissect the molecular pathways by which T. gondii infection contributes to neurocognitive disorders.

New neurons in old brains: implications of age in the analysis of neurogenesis in post-mortem tissue.

Adult neurogenesis, the proliferation and integration of newly generated neurons, has been observed in the adult mammalian hippocampus of many species. Numerous studies have also found adult neurogenesis in the human hippocampus, but several recent high-profile studies have suggested that this process is considerably reduced in humans, occurring in children but not in adults. In comparison, rodent studies also show age-related decline but a greater degree of proliferation of new neurons in adult animals. These differences may represent biological species differences or could alternatively be explained by methodological differences in tissue handling and fixation. Here, we examine whether differences in the post-mortem interval between death and tissue fixation might impact subsequent detection of adult neurogenesis due to increased tissue degradation. Because there are fewer new neurons present in older subjects to begin with we hypothesized that, subject age might interact significantly with post-mortem interval in the detection of adult neurogenesis. We analyzed neurogenesis in the hippocampus of rats that were either perfusion-fixed or the brains extracted and immersion-fixed at various post-mortem intervals. We observed an interaction between animal age and the time delay between death and tissue fixation. While similar levels of neurogenesis were observed in young rats regardless of fixation, older rats had significantly fewer labeled neurons when fixation was not immediate. Furthermore, the morphological detail of the labeled neurons was significantly reduced in the delayed fixation conditions at all ages. This study highlights critical concerns that must be considered when using post-mortem tissue to quantify adult neurogenesis.

Neurogenesis mediated plasticity is associated with reduced neuronal activity in CA1 during context fear memory retrieval.

Postnatal hippocampal neurogenesis has been demonstrated to affect learning and memory in numerous ways. Several studies have now demonstrated that increased neurogenesis can induce forgetting of memories acquired prior to the manipulation of neurogenesis and, as a result of this forgetting can also facilitate new learning. However, the mechanisms mediating neurogenesis-induced forgetting are not well understood. Here, we used a subregion-based analysis of the immediate early gene c-Fos as well as in vivo fiber photometry to determine changes in activity corresponding with neurogenesis induced forgetting. We found that increasing neurogenesis led to reduced CA1 activity during context memory retrieval. We also demonstrate here that perineuronal net expression in areas CA1 is bidirectionally altered by the levels or activity of postnatally generated neurons in the dentate gyrus. These results suggest that neurogenesis may induce forgetting by disrupting perineuronal nets in CA1 which may otherwise protect memories from degradation.

FASTMAP: Open-Source Flexible Atlas Segmentation Tool for Multi-Area Processing of Biological Images.

To better understand complex systems, such as the brain, studying the interactions between multiple brain regions is imperative. Such experiments often require delineation of multiple brain regions on microscopic images based on preexisting brain atlases. Experiments examining the relationships of multiple regions across the brain have traditionally relied on manual plotting of regions. This process is very intensive and becomes untenable with a large number of regions of interest (ROIs). To reduce the amount of time required to process multi-region datasets, several tools for atlas registration have been developed; however, these tools are often inflexible to tissue type, only supportive of a limited number of atlases and orientation, require considerable computational expertise, or are only compatible with certain types of microscopy. To address the need for a simple yet extensible atlas registration tool, we have developed FASTMAP, a Flexible Atlas Segmentation Tool for Multi-Area Processing. We demonstrate its ability to register images efficiently and flexibly to custom mouse brain atlas plates, to detect differences in the regional numbers of labels of interest, and to conduct densitometry analyses. This open-source and user-friendly tool will facilitate the atlas registration of diverse tissue types, unconventional atlas organizations, and a variety of tissue preparations.

Network Neuroscience Untethered: Brain-Wide Immediate Early Gene Expression for the Analysis of Functional Connectivity in Freely Behaving Animals.

Studying how spatially discrete neuroanatomical regions across the brain interact is critical to advancing our understanding of the brain. Traditional neuroimaging techniques have led to many important discoveries about the nature of these interactions, termed functional connectivity. However, in animal models these traditional neuroimaging techniques have generally been limited to anesthetized or head-fixed setups or examination of small subsets of neuroanatomical regions. Using the brain-wide expression density of immediate early genes (IEG), we can assess brain-wide functional connectivity underlying a wide variety of behavioural tasks in freely behaving animal models. Here, we provide an overview of the necessary steps required to perform IEG-based analyses of functional connectivity. We also outline important considerations when designing such experiments and demonstrate the implications of these considerations using an IEG-based network dataset generated for the purpose of this review.

Voluntary Exercise Increases Neurogenesis and Mediates Forgetting of Complex Paired Associates Memories.

The hippocampus is a critical structure involved in many forms of learning and memory. It is also one of the only regions in the mammalian brain that continues to generate new neurons throughout adulthood. This process of adult neurogenesis may increase the plasticity of the hippocampus which could be beneficial for learning but has also been demonstrated to decrease the stability of previously acquired memories. Here we test whether exposure to voluntary running (which increases the production of new neurons) following the formation of a gradually acquired paired associates task will result in forgetting of this type of memory. We trained mice in a touchscreen-based object/location task and then increased neurogenesis using voluntary running. Our results indicate that running increased neurogenesis and resulted in poor recall of the previously established memory. When subsequently exposed to a reversal task we also show that running reduced the number of correction trials required to acquire the new task contingencies. This suggests that prior forgetting reduces perseveration on the now outdated memory. Together our results add to a growing body of literature which indicates the important role of adult neurogenesis in destabilizing previously acquired memories to allow for flexible encoding of new memories.

Adult neurogenesis mediates forgetting of multiple types of memory in the rat.

The formation and retention of hippocampus-dependent memories is impacted by neurogenesis, a process that involves the production of new neurons in the dentate gyrus of the hippocampus. Recent studies demonstrate that increasing neurogenesis after memory formation induces forgetting of previously acquired memories. Neurogenesis-induced forgetting was originally demonstrated in mice, but a recent report suggests that the same effect may be absent in rats. Although a general species difference is possible, other potential explanations for these incongruent findings are that memories which are more strongly reinforced become resilient to forgetting or that perhaps only certain types of memories are affected. Here, we investigated whether neurogenesis-induced forgetting occurs in rats using several hippocampus-dependent tasks including contextual fear conditioning (CFC), the Morris Water Task (MWT), and touchscreen paired associates learning (PAL). Neurogenesis was increased following training using voluntary exercise for 4 weeks before recall of the previous memory was assessed. We show that voluntary running causes forgetting of context fear memories in a neurogenesis-dependent manner, and that neurogenesis-induced forgetting is present in rats across behavioral tasks despite differences in complexity or reliance on spatial, context, or object memories. In addition, we asked whether stronger memories are less susceptible to forgetting by varying the strength of training. Even with a very strong training protocol in the CFC task, we still observed enhanced forgetting related to increased neurogenesis. These results suggest that forgetting due to neurogenesis is a conserved mechanism that aids in the clearance of memories.

Disrupted Neurogenesis in Germ-Free Mice: Effects of Age and Sex.

The gut microbiome has profound effects on development and function of the nervous system. Recent evidence indicates that disruption of the gut microbiome leads to altered hippocampal neurogenesis. Here, we examined whether the effects of gut microbiome disruption on neurogenesis are age-dependent, given that both neurogenesis and the microbiome show age-related changes. Additionally, we examined memory induced functional connectivity of hippocampal networks. Control and germ-free mice at three different ages (4, 8, and 12 weeks) were trained in contextual fear-conditioning, then subsequently tested the following day. Hippocampal neurogenesis, quantified via BrdU and doublecortin, exhibited age-dependent changes relative to controls, with the established age-dependent decrease in neurogenesis being delayed in germ-free mice. Moreover, we found sex-dependent effects of germ-free status on neurogenesis, with 4 week old male germ-free mice having decreased neurogenesis and 8 week old female germ-free mice having increased neurogenesis. To assess systems-level consequences of disrupted neurogenesis, we assessed functional connectivity of hippocampal networks by inducing c-Fos expression with contextual memory retrieval and applying a previously described network analysis. Our results indicate impaired connectivity of the dentate gyrus in germ-free mice in a pattern highly correlated with adult neurogenesis. In control but not germ-free mice, functional connectivity became more refined with age, indicating that age dependent network refinement is disrupted in germ-free mice. Overall, the results show that disruption of the gut microbiome affects hippocampal neurogenesis in an age- and sex-dependent manner and that these changes are also related to changes in the dentate gyrus functional network.

Assessing Individual Neuronal Activity Across the Intact Brain: Using Hybridization Chain Reaction (HCR) to Detect Arc mRNA Localized to the Nucleus in Volumes of Cleared Brain Tissue.

Arc (activity-regulated cytoskeleton-associated protein) is an immediate early gene that may be used to label recently active neurons. Arc is transcribed following neuronal activity, and its mRNA is then rapidly transported to dendrites. This feature allows nuclear-localized Arc mRNA to define ensembles of recently active neurons in systems or circuit neuroscience. However, typical in situ hybridization techniques severely constrain the thickness of the tissue specimen (typically 20-µm brain slices). Here, we describe a protocol for visualizing intranuclear Arc mRNA in large (4 × 4 × 3 mm) volumes of intact mouse brain tissue. We combined a tissue clearing protocol (iDISCO+) with an advanced in situ hybridization technique (hybridization chain reaction [HCR]) to detect nuclear-localized Arc mRNA in whole, intact brain regions without the need for brain sectioning or reconstruction. We successfully applied this protocol to image ensembles of neurons of the basolateral amygdala in mice that are active following the recall of a conditioned fear memory. © 2018 by John Wiley & Sons, Inc.

Re-engineering the Hippocampus.

As adult-generated neurons integrate into hippocampal circuits, they compete with mature neurons for inputs from the entorhinal cortex. By reducing spines on mature granule cells, McAvoy et al. (2016) find that new neurons integrate more efficiently, and this facilitates learning.