Canada

Bipolar disorder (BD) is characterized by alternating cases of depression and mania. Although deviations in neurodevelopment are suspected to contribute to the pathophysiology of BD, the precise molecular underpinnings remain unknown. In cells, calcium plays an influential role in development via signalling cascades. As such, calcium accumulates in the endoplasmic reticulum (ER) from where it can be rapidly released in response to a second messenger. Calcium levels are, therefore, tightly regulated through store-operated calcium entry (SOCE)--a mechanism that promotes calcium influx through ORAI channels when the ER is depleted. Recently, we found striking evidence of aberrant SOCE activity in BD patient-derived induced pluripotent stem cells (iPSCs) differentiated into neural progenitor cells (NPCs) in comparison to iPSC-NPCs derived from healthy control individuals. This led us to discover unsuspected cellular phenotypes. Here, we first summarize calcium imaging data revealing the sharp difference in SOCE between BD patient-derived and control iPSC-NPCs. Second, we show that lower SOCE activity in BD cells correlates with reduced STIM/Orai puncta formation. Third, we reveal decreased proliferation in BD-derived progenitors, which we then connect to a premature neuronal differentiation phenotype affecting migration. Finally, we support this dysregulated development with cerebral organoid modelling. Together, our findings suggest a contribution of SOCE to BD through dysregulation in the neurodevelopment of NPCs.

CAN-ACN 2021

Neurodevelopmental impact of aberrant calcium mobilization in bipolar disorder patient-derived iPSC-neural progenitor cells

neural progenitor cells

soce

bipolar disorder

neurodevelopment

calcium

Colleagues and Friends:

I am very happy to welcome you to the 14th annual Canadian Neuroscience Meeting, and to the first virtual edition of our meeting. I want to highlight the important work the CAN meeting chair, **Roger Thompson** and co-chair, **Alyson Fournier**, did to put together the great program we are offering this year. Moving to a virtual format required a complete re-design of the program, and both Roger and Alyson did a splendid job in providing us with an appealing and well adapted program for our first virtual event. I wish to thank them for this very important effort.

Highlights of this year’s scientific meeting include invited speakers **Michael Hausser, Franck Polleux, Gwyneth Card, Magdalena Götz**and Brain Prize winner **Huda Zoghbi**. Plenary and parallel symposia, proposed by our members complete our diverse [scientific program.](https://can-acn.org/meeting-2021/program-2021/)

The 2021 Advocacy session, chaired and organized by CAN advocacy committee chair **Karun Singh**, will feature a special lecture and question period with Member of Parliament and past Science Minister **Kirsty Duncan**. This will be followed by a short overview of CAN’s advocacy activities over the last year. We invite you to participate in this session for insight from Dr. Duncan on the workings of Parliament Hill, and to learn more about what CAN has been doing to promote neuroscience research in Canada.

We are also excited to host **Lisa Willis** in the Equity Diversity and Inclusion session, organized by EDI committee chair **Jibran Khokhar**.

A special highlight of our meeting will be the lectures by award-winning young neuroscientists. We are please to host the CAN 2020 Young Investigator Award winner **Mihaela Iordanova**, and CAN 2021 Young Investigator Award winners **Aaron Phillips** and **Tabrez Siddiqui**. Highlights will also include presentation by the top three winners of the CAN-CIHR-INMHA Brain Star Award winners, **Claire Gizowski, Nuria Daviu Abant** and **Lukas Tamayo-Orrego**.

Independently organized satellite symposia complement and complete this exciting program, check them out here: https://can-acn.org/meeting-2021/2021-satellite-symposia/

Some of the most engaging sessions are always the poster sessions featuring the work of our trainees, so make sure to log in to Gather Town during the poster sessions to chat with the presenters.
We hope you enjoy this program, and look forward to welcoming you, virtually.

Shernaz Bamji

President of the Canadian Association for Neuroscience

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Please head to https://can-acn.org/meeting-2021/registration-2021/ to register. 

14th Canadian Neuroscience Meeting from August 23rd-25th

poster

Various biological functions in organisms, ranging from the level of gene expression to physiological and behavioral processes, undergo daily variations generated by endogenous circadian clocks. Disruptions to circadian clock gene expression in the striatum were associated with depressive- and anxiety-like conditions, as well as abnormalities in reward-related processes. Recent findings in our lab show that the conditional knockout of the core clock gene Period2 (Per2) in the striatum alters ethanol consumption in a sexual dimorphic manner, but has only minor effects on mood- and anxiety related behaviors. Originally, it has been suggested that the nucleus accumbens (NAc), located in the ventral part of the striatum, is strongly involved in the control of the above-mentioned neuropsychiatric conditions, which are known to establish comorbidities. To further disentangle the role of Per2 on the pathophysiology of mood and anxiety disorders and alcohol abuse, Per2-floxed male and female mice received bilateral intracerebral injections of adeno-associated viral vectors expressing Cre-recombinase and eGFP or control vectors expressing eGFP only and were tested in a battery of behavioral assays to evaluate depressive- and anxiety-like behaviors, as well as alcohol consumption. Preliminary results suggest Per2- knockouts specific to the NAc consume more ethanol than controls, but has no effects on anxiety- and depressive-like behaviors. Further studies including gene expression analyses are needed to elucidate the underlying mechanisms driving these altered ethanol drinking behaviors in striatal Per2 knockout mice.

The role of Per2 on mood and alcohol related behaviours in males and females

Introduction: This research investigates an anomalous pyramidal cell type, dubbed "deep cells" in the subiculum of the hippocampus. Deep cells are structurally unique in that they completely lack radial oblique dendrites. This work will use in-vivo calcium imaging to elucidate how this remarkable change in structure effects neuronal function in vivo in the subiculum and its possible role during behaviour. Methods: Our lab has constructed a transgenic cre line to access this deep cell type. I use this mouse line to image deep cells during spatial navigation and novel object tasks. During these experiments, I use wire-free 1-photon miniscopes to image the dorsal subiculum while mice undergo an open field task with two local objects, repeated over several timepoints. Results: Data from this project show that deep cells act on very slow timescales and have robust, sustained activity that appear to respond to encounters with novel, local objects . Deep cells show large increases in activity after interaction with a novel object on day one and this activity is substantially reduced by day four. Intriguingly, this cellular signature is still reduced 100 days after initial introduction. In comparison to control cells, deep cells show novel object-centered activity and no classic spatial phenotypes expected from pyramidal cells in the subiculum. Conclusion: This data leaves us with intriguing evidence of a seemingly spatially uninvolved, novelty driven and morphologically distinct cell type previously undescribed in the subiculum. Future work will be integral to identifying the role of this cell in memory processes/dysfunction and the role of this novel cell type in other brain regions.

An investigation into an anomalous pyramidal cell type: atypical morphology and sustained cellular activity to novel stimuli

The formation and remodeling of synaptic contacts requires the precise distribution and trafficking of proteins to specialized compartments. Recent work demonstrates that post-translational modifications, including protein S-acylation, play a key role in strengthening and weakening of synaptic connections, known to underlie the cellular basis of learning and memory. To determine how the dynamic palmitoylation of neuronal proteins contributes to synapse plasticity, we utilized an unbiased, proteomic approach to identify 121 hippocampal proteins that were differentially palmitoylated 1 hour after a hippocampal-dependent fear conditioning learning paradigm. Gene Ontology analysis revealed that majority of proteins that exhibited an increase in palmitoylation in the fear conditioned group compared to control were synaptic proteins involved in neurotransmission and plasticity, whereas those that exhibited decreased palmitoylation were primarily involved in metabolic functions. We further validated activity-induced differential palmitoylation of a subset of the 121 identified proteins in vitro and then investigated the role of dynamic palmitoylation on the function of a validated protein, lipid phosphate phosphatase-related protein 4 (LPPR4-also called plasticity-related gene 1, PRG-1). We identified cysteine residues in PRG-1 that are palmitoylated and found that palmitoylation at these sites is important for spine formation. Furthermore, we show that activity-mediated PRG-1 palmitoylation alters the phospholipid processing function of PRG-1 and is essential for activity-induced insertion of AMPA receptors into the post-synaptic membrane. Together, this study identifies networks of synaptic proteins whose dynamic palmitoylation may play a central role in learning and memory.

Activity-dependent changes in the hippocampal palmitoylome

Introduction: The retrosplenial cortex (RSC) participates in many higher-order cognitive functions and is host to many neurons with complex firing properties. However the organizational principles of this region, which underlies a multiplicity of functionality, are not well defined. The aim of this project is to employ a bottom-up approach in order to resolve the cellular composition of the RSC. Our hope is to generate a multi-level, comprehensive view of the mechanisms underlying RSC functionality from molecules, to cells, and circuits. Methods: We utilized a combination of single cell RNA sequencing and multiplexed in situ hybridization in order to reveal the identity and organization of transcriptomically unique subpopulations of neurons within the region. Using retrograde AAV tracing, we are mapping the long range projections of cells in the region. Results: We uncovered 9 unique subpopulations of excitatory neurons within the RSC. Contrary to canonical thought on gene expression and cell identity within the cortex, we found that the RSC exhibits finer-grained changes than expected from a cortical region.

The cellular landscape of the retrosplenial cortex

uthorDespite being implicated in various functions, the structural organization of the claustrum remains largely unknown. It is crucial that we first understand the intrinsic neural organization of the claustrum to better elucidate its functional complexity. Thus, we sought to investigate the transcriptomic breakdown of the claustrum through single cell RNA-sequencing (scRNA-seq). In our analysis, we uncovered a previously unknown excitatory neuronal subtype, suggesting the claustrum is composed of 2 transcriptomically distinct excitatory neurons. To investigate the spatial organization of these subtypes, we used multiplexed single-molecule fluorescence in situ hybridization (smFISH), targeting RNA from 12 different marker genes in individual brain sections. We found that the gene expression patterns of the claustral neurons correlated strongly with the scRNA-seq predictions, organizing into a "core-shell" spatial configuration that was consistent across the anterior-posterior axis. To determine if these transcriptomic signatures corresponded to specific projection neuron populations within the claustrum, multicolour retrograde tracing in conjunction with smFISH was performed. Here, we found the core and shell subtypes correlated with distinct projection targets from the retrosplenial cortex and lateral entorhinal cortex, respectively. Thus, the claustrum exhibits a "core-shell" spatial organization with distinct molecular and circuit properties, which may drive its functional complexity. This spatial heterogeneity can be used in the future to examine subtype-specific function.

Spatially patterned excitatory neuron subtypes and circuits within the claustrum

At the onset of Alzheimer's disease (AD), accumulation of oligomeric amyloid-β (Aβ) correlates with local hyperexcitability, possibly due to dyshomeostasis of extracellular glutamate. However, the interplay between Aβ oligomers and extracellular glutamate accumulation remains unresolved. We use an in silico approach to characterize the putative underpinnings of Aβ-dependent modulations of extracellular glutamate. We first consider how Aβ modifies the expression of astrocytic glutamate transporters (GLT1) and how it impacts extracellular glutamate time course. Accordingly, we develop a mathematical model for glutamate diffusion and uptake at synaptic terminals and their surroundings. Our model predicts that above a threshold Aβ concentration, astrocytic GLT1 transporters cannot prevent the accumulation of extracellular glutamate. This, in turn, promotes a positive feedback on synaptic glutamate release that favors excitotoxicity and neuronal hyperactivity. Next, we complement our model including calcium-dependent Aβ production and glutamate release. The interaction of multiple positive feedback loops of signaling can account for different tissue states, overcoming the traditional dichotomy of healthy and excitotoxic conditions. Specifically, changes in the basal firing activity can promote intermediate conditions useful to either predict AD's degeneration or offer new therapeutic perspectives. Our results support the notion of AD as a multistage pathology where transitions can follow multiple pathways, such as Aβ accumulation, excitotoxicity, or calcium dysregulation.

Aβ-dependent alteration of astrocytic GLT1 expression can trigger the multistage progression of Alzheimer's disease

Dendritic spines are responsible for relaying excitatory input and regulating signalling between neurons and their morphology can give insight into synaptic function. Excitotoxicity brought on by brain injury results in synaptic dysfunction, as intracellular Ca2+ increases. This increase in intracellular Ca2+ over-activates various cellular kinases and proteases responsible for microtubule stabilization. Previous work in our lab has shown that after an impact, there is an increase in tensile strain within the depths of the porcine cingulate sulcus. Moreover, there was a marked decrease of the microtubule stabilizing protein MAP2 specifically within the apex of the sulcus. In our current work, we explored whether changes in spine morphology were associated with our impact model. The Golgi-Cox staining method was used to visualize dendritic spine morphology. Following impact, coronal slabs were placed into Golgi-Cox solution for 2 weeks. Cortical pyramidal neurons within the sulcus apex and arm were reconstructed. Preliminary data revealed a shift towards mushroom-type spines, compared to thin and stubby-type spines within the sulci of the brain after impact. These findings suggest a shift towards an excitatory synapse shortly after impact. Along with our previous findings of changes in MAP2 properties, the shift towards an excitatory synapse could implicate activation of kinases and proteases responsible for regulating MAP2 dynamics and altering spine morphology.

Assessing dendritic integrity and spine morphology following impact in the porcine cortex

Repeated mild traumatic brain injury (rmTBI) disrupts hippocampal function and can lead to long-lasting episodic memory impairments. We hypothesized that these deficits could arise as a result of microvascular damage and microglia activation leading to impaired synaptic plasticity. Using our Awake Closed Head Injury (ACHI) Model for repeated (8x over 4 days) head injury, we found that animals showed impaired neurological function after the first hit, that was sustained throughout all 8 impacts. Histological analysis showed that animals experienced diffuse microbleeds in the brain, including the hippocampus, and that this was accompanied by fibrinogen release and microglia activation. Furthermore, animals that experience rmTBI showed impaired bilateral synaptic plasticity, with the magnitude of both long-term depression (LTD) and long-term potentiation (LTP) of synaptic efficacy being impaired in the hippocampus. This work indicate that rmTBI can produce diffuse microbleeds, microglia activation, and impaired synaptic plasticity, giving insights into the mechanisms that can underlie the memory impairments that can accompany rmTBI. This work was supported by grants from the Canadian Institutes for Health Research (CIHR) to BRC.

Repeated traumatic brain injury causes diffuse microvascular damage and impairs bidirectional synaptic plasticity

Schizophrenia (SZ) is a serious mental disorder characterized by hallucinations, delusions, and impaired cognitive behavior. Substantial heterogeneity in the clinical phenotypes complicates its diagnosis and treatment. The pathogenesis results from a complex interaction between genetic vulnerability and environmental factors, altering neuronal development particularly. Indeed, several genes and proteins associated with neurite growth, neuronal migration, and synaptic development are altered in SZ. However, the processes of neurodifferentiation and functional maturation of neuronal networks are still largely unexplored. Therefore, the exploration with multimodal optical imaging tools of the in vitro of cortical neural cell generation using iPSCs from SZ patients and controls will help measure the maturation and organization of these cultured neuronal networks. Human iPSCs lines were differentiated into mixed cortical neural cells and characterized by flow cytometry, immunostaining, western blot, and patch-clamp techniques. To explore functional and morphological properties, we use quantitative phase digital holographic microscopy (QP-DHM) combined with fluorescence imaging. QP-DHM is a high-resolution, non-invasive technique allowing to assesse cell structure and dynamics with axial sensitivity at the nanoscale. These tools will serve to explore the excitatory and inhibitory (E/I) balance of these cultured neural networks. Such a multimodal imaging approach coupled with human iPSCs disease models holds great promises to identify cellular risk biomarkers for major psychiatric disorders, including schizophrenia.

Exploring neurodifferentiation and maturation process of iPSCs derived from schizophrenia patients with multimodal optical microscopy

The ventral tegmental area (VTA) contains a heterogenous population of GABA neurons, making up about 30% of cells in the VTA. These GABA neurons regulate the activity of VTA dopamine neurons, affecting the release of dopamine in the nucleus accumbens (NAc). The release of dopamine in the NAc plays an important role in energizing goal directed behaviour towards food. GABA neurons, projecting from the lateral hypothalamus (LH), have been shown to preferentially synapse with VTA GABA neurons. Activation of LH GABA disinhibits the VTA GABA circuit to stimulate the release of dopamine in the NAc, resulting in consummatory behaviour. Acute fasting (AF) increases the incentive motivation for food. Despite the LH GABA to VTA GABA circuit being a powerful driver for food consumption, how it is affected by AF is unknown. Using patch clamp electrophysiology in midbrain slices expressing channelrhodopin in LH GABAergic terminals in the VTA, we demonstrate that following AF, LH GABA neurons have reduced inhibition of VTA GABA neuronal firing. Through train stimulations and strontium-induced asynchronous release, we show that this weakening of the LH GABA input is due to a scaling down of inhibitory postsynaptic current (IPSC) responses. Following AF, release probability of GABA from LH GABA neurons is decreased in female by not male mice. Taken together, these results show that the LH GABA to VTA GABA circuit is affected by acute energy deprivation and that males and females respond differently.

Neurodevelopmental impact of aberrant calcium mobilization in bipolar disorder patient-derived iPSC-neural progenitor cells

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