Canada

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 &quot;core-shell&quot; 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 &quot;core-shell&quot; 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.

CAN-ACN 2021

Spatially patterned excitatory neuron subtypes and circuits within the claustrum

neuroscience; mfish; molecular biology

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

![](https://assets.underline.io/uploads/markdown_image/1/image/b9aea25343a87d5aac3b9367dc780974.jpg)



Please head to https://can-acn.org/meeting-2021/registration-2021/ to register. 

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

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.

poster

Though cannabis use during pregnancy has increased, our understanding of its effects on offspring is incomplete. Gestational THC injection studies found altered goal-directed behaviour and mesolimbic dopamine reward circuitry in offspring of various ages. To investigate whether oral cannabis exposure during pregnancy alters ventral tegmental area (VTA) dopamine neuron activity, mice were exposed to 5 mg/kg THC cannabis extract in peanut butter or peanut butter alone from GD0-PD10. Whole cell patch clamp electrophysiology was performed in VTA slices from adolescent (P42-P46) offspring. Maternal weight gain, food intake, litter size, frequency of miscarriages, or maternal behaviour in a pup retrieval test were not different between groups, suggesting the effects of cannabis are not secondary to poor maternal care or health. VTA dopamine neurons of male mice exposed to cannabis from GD0 to PD10 had a more depolarised resting membrane potential, increased spontaneous firing, decreased latency to fire, and increased after-hyperpolarisation potential height, but not width. In contrast, dopamine neurons from female mice had shorter after-hyperpolarisation potentials, without changes in resting membrane potential, spontaneous firing frequency or other firing properties. Consistent with previous injection studies, prenatal exposure to oral cannabis differently alters the firing properties of male and female VTA dopamine neurons. Future research will examine synaptic changes and the behavioural implications of cannabis-induced differences in VTA dopamine neuronal activity.

Prenatal cannabis exposure alters the firing properties of adolescent ventral tegmental area dopamine neurons

Astrocytes express different neurotransmitter receptors that serve to integrate these cells into neuronal networks. Many of these receptors, when sensing neuronal activity, induce elevations in intracellular astrocyte Ca2+, which leads to the release of gliotransmitters that modulate nearby neurons. Ionotropic N-methyl-D-aspartate (NMDA) receptors are found on astrocytes and are activated by glutamate and D-serine or glycine, and conduct Ca2+ into astrocytes. In brain slices, astrocyte NMDAR activation causes depolarization and Ca2+ elevations. However, its role in astrocytes Ca2+ transients and feedback modulation to neurons in vivo is not characterized. Therefore, we used a novel NMDAR knockdown (KD) construct to reduce NMDAR expression specifically in cortical astrocytes. Then, using dual calcium imaging of astrocytes and neurons each expressing a unique genetically encoded calcium indicator (Lck-GCaMP6f and RCaMP1.07 respectively) we determined the impact of astrocytes NMDARs on astrocytes Ca2+ transients and nearby neuronal activity. Two-photon microscopy of the barrel cortex of awake mice revealed that NMDAR KD reduced Ca2+ responses to whisker stimulation in both astrocytes and neurons. This highlights the importance of NMDAR in astrocyte Ca2+ signalling and astrocyte-neuron communication and suggests that astroglial NMDAR KD could cause deficits in sensory perception. This work contributes to a deeper knowledge of mechanisms underlying astrocyte Ca2+ signalling and provides novel directions to study the role of astrocytes in neuronal circuits.

Astroglial ionotropic NMDA receptor calcium signalling and modulation of cortical neuron activity

Spikes are usually initiated at the axon initial segment (AIS), the most excitable site of a neuron. Yet other regions of the neuron are also excitable. While there are many studies on somatic and dendritic excitability, axon excitability has yet to be thoroughly investigated in most neurons because the small size of the axon precludes intracellular recordings. Using a novel optogenetic approach, recent experiments from our lab have shown that axon spikes transiently in response to sustained depolarization. Although the optogenetic method has many advantages, it still has some limitations: first, light is not focused on one point, meaning stray light may hit other regions of the neuron; second, since voltage changes are recorded from the soma, the location of spike initiation must be inferred indirectly. These experimental limitations necessitated simulations to definitively interpret the experimental results. We built a multicompartment model of a CA1 pyramidal neuron with a detailed myelinated axon that reproduced our experimental data. The model confirmed the site of spike initiation based on the shape (kinkiness) of spikes recorded in the soma. Simulations also confirmed that even when targeting the axon for photostimulation, a small degree of stray light can hit the dendrites and evoke spikes in the AIS. The results ultimately confirm that the AIS spikes repetitively during sustained depolarization, consistent with analog to digital transduction of information, whereas the axon responds with transient spiking only during abrupt changes in depolarization. Simulations show that the stimulus the axon receives during spike propagation is a very intense and short depolarization, which is precisely the input to which it is tuned to respond.

Transient spike initiation supports digital information transfer in axons: simulations in a biophysically detailed model

Brain mural cells are important for the regulation of cerebral blood flow (CBF) and they participate in endogenous pathways like vasomotion and neurovascular coupling that direct CBF to highly activated areas of the brain. Pericytes are spatially isolated mural cells embedded on brain blood vessels and have possible contractile capacity; however, recent studies have identified different types of pericytes that differ in morphology and protein expression, creating a debate on their functional roles in CBF control. We evaluated the calcium signaling and hemodynamic influence of distinct pericyte populations: ensheathing pericytes and capillary pericytes, using transgenic mice with expression of genetically encoded calcium indicators (GCaMP6s and RCaMP1.07) in different pericyte types. Intracellular calcium signals were visualized via two-photon microscopy in brain slices and in vivo. By targeting different ion channel pathways pharmacologically, we found that ensheathing and capillary pericytes have different basal calcium pathways and different roles in hemodynamics at rest and during sensory stimulation to evoke neurovascular coupling. Our data provides novel insight into brain pericyte calcium signaling mechanisms and opens new avenues to clarify the role of pericytes in the regulation of CB. This may also contribute to the understanding of cerebrovascular and neurodegenerative diseases, like stroke, cerebral hypoperfusion or Alzheimer's disease, in which their pathogenesis remains to be fully elucidated.

The contribution of calcium ion channels to brain pericyte calcium signaling in vivo

Contexts associated with prior reinforcement can renew extinguished conditioned responding. Both the infralimbic medial prefrontal cortex (IL) and paraventricular nucleus of the thalamus (PVT) are implicated in this renewal effect. Here we examined if connectivity between these regions, namely an IL-to-PVT neural circuit, was involved in renewal. We trained male and female Long-Evans rats to associate a conditioned stimulus (CS; 10 s white noise) with the delivery of a 10% sucrose unconditioned stimulus (US; 0.2 mL/CS) to a fluid port in a distinct context (Context A; 14 trials/session; 12 sessions). We then extinguished responding by presenting the CS without the US in a different context (Context B; 14 trials/session; minimum 2 sessions). At test, we used optogenetics to unilaterally stimulate IL-to-PVT neurons during CS presentations (473 nm; 5 ms pulses at 20 Hz for 10.2 s) in Context A under extinction conditions. Optically stimulating the IL-to-PVT in rats expressing Channelrhodopsin-2 with enhanced yellow fluorescent protein (ChR2-eYFP) significantly attenuated renewal of CS-elicited port entries compared to rats expressing eYFP alone, and this effect was equivalent in males and females. Further, rats expressing ChR2-eYFP (1) nose-poked significantly more for optical self-stimulation of the IL-to-PVT, and (2) expressed greater Fos density (a marker of neural activity) in the IL and PVT. These results demonstrate a compelling, sex-independent role of the IL-to-PVT neural circuit in mediating renewal of appetitive Pavlovian conditioned responding following extinction.

Activation of a cortico-thalamic neural circuit attenuates renewal in male and female rats

Metabolic regulation is thought to be an important feature within stem cell niches. Recent work suggests that ependymal cells (ECs), that line the ventricular system of the brain, could be critical players in regulating the ventricular-subventricular zone (V-SVZ). ECs are multi-ciliated glial cells that are responsible for regulating the neural stem cell niche and propelling the cerebrospinal fluid. Transcriptional profiling of the adult SVZ niche showed that ECs are highly enriched in glucose transporter 1 (GLUT1), leading us to hypothesize that ECs may regulate NSC behavior by modulating metabolism within the V-SVZ. To test this, I performed a conditional deletion of GLUT1 in adult ECs in vivo using aSMACreERT2:ROSATdTomato:GLUT1flox/flox mice to delete GLUT1 in aSMA+ ECs. At 1-month post-GLUT1 deletion, an increase in overall proliferation (marked with Ki67) was observed within the V-SVZ niche. A sex dimorphic effect was observed on neurogenesis; with females displaying a reduction in the number of DCX+ neuroblasts, while males exhibited no change. Interestingly, this reduction was more pronounced in the anterior V-SVZ compared to posterior V-SVZ, suggesting sensitivity to GLUT1KO might be spatially dependent. There was also a marked increase in GFAP staining and an accumulation of lipid droplets within the V-SVZ post-GLUT1 deletion suggesting that disruption in glucose metabolism may perturb local lipid metabolism. Altogether, these results indicate that EC metabolism regulates the NSC niche and may play a broader role in maintaining brain homeostasis.

Impact of ependymal cell metabolic perturbation on brain function

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.

Spatially patterned excitatory neuron subtypes and circuits within the claustrum

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Prenatal cannabis exposure alters the firing properties of adolescent ventral tegmental area dopamine neurons

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