Neurons of the hypothalamic arcuate nucleus (ARC) integrate nutritional signals to modulate energy balance. Evidences suggest that the underlying mechanisms involve the release of fatty acids (FA) from intracellular lipid droplets (LD). This model is supported by data from our lab showing that neurons can form and accumulate triglycerides in LD. In addition, our data and single-cell RNAseq studies show that Adipose Triglyceride Lipase (ATGL) is expressed in ARC pro-opiomelanocortin (POMC) neurons that play a crucial role in energy balance. Thus, we propose that ATGL in ARC POMC neurons is involved in energy balance control by regulating LD lipolysis. To test this, we quantified LD and oxidation of FA in hypothalamic neurons (cell lines and primary cultures) treated with oleate, forskolin (ATGL activator in adipose tissues), or Atglistatin (ATGL inhibitor). Also, we generated POMC specific ATGL KO mice (Cre-Lox) which were subjected to metabolic phenotyping. Our results show that hypothalamic neurons accumulate LD in response to Atglistatin/oleate, forskolin decreases LDs via ATGL and Atglistatin decreases the oxidation rate of LD-derived FA. Our lipidomics data show that palmitate and oleate are the major FA in neuronal LD. In our mouse model, ATGL KO in POMC neurons does not affect body weight or food intake in chow-fed males. These animals will be subjected to metabolic challenges (high fat diet) to assess the role of ATGL in adaptive metabolic responses. Our data show for the first time that hypothalamic neurons can accumulate LD and that ATGL activates LD lipolysis.

Feeling hungry? The role of fat stores in hypothalamic neurons on energy balance

Social defeat is a preclinical model to study the effects of chronic psychosocial stress in rodents, as it recapitulates many stress-induced pathologies observed in humans, including metabolic changes. This model, however, is based off male territorial aggression that is not applicable to female rodents. To investigate how females respond to psychosocial stress, we tested two adaptations to the social defeat paradigm to study the effect of chronic stress on metabolism in females. In the first paradigm, fighting females, a female and castrated male CD-1 are cohoused for several days, priming the CD-1 female to display territorial behaviors towards an intruding C57 female mouse. In the second paradigm, non-discriminatory social defeat, we introduced a C57 male and female simultaneously to a CD-1 male mouse. The intruding male provokes territorial aggression towards both the male and female mice. We tested both paradigms for 21-days and gave mice access to chow and a high fat diet, ad lib. Females in the non-discriminatory model displayed changes to hormone levels and metabolism commonly associated with chronic stress. These mice increased their consumption of the standard chow diet, high in carbohydrates, when stressed and had elevated ghrelin and corticosterone levels. Females from the fighting female's paradigm, however, did not display the same markers of chronic stress. Our results highlight discrepancies in the magnitude of stress elicited by each paradigm, with the second paradigm inducing metabolic changes in females commonly observed in males following social defeat.

Chronic stress & metabolism in females: evaluating two novel models of female social defeat stress

Circadian rhythms, 24-hour oscillations within an organism generated by biological clocks, keep temporal order of molecular, physiological, and behavioral processes with respect to daily changes in the environment, thus enabling systemic homeostasis and proper function of the biological system. A multi-oscillatory network of circadian pacemakers, comprising a central clock in the suprachiasmatic nucleus of the hypothalamus, and peripheral clocks located in other brain regions and tissues throughout the body, sustains tissue-specific rhythms, such as in the striatum. The striatum, a region of the basal ganglia that receives dopaminergic input from the midbrain, controls motor functions and behavioral processes related to mood and reward. Proper function of the striatum is presumably maintained by a mutual interaction of components of the circadian clock and the dopamine signalling pathway. Using a conditional knockout of the core clock gene Bmal1 within medium spiny neurons (MSNs) of the striatum, we assessed the molecular, physiological, and behavioural consequences of striatal clock malfunction. Male and female knockout mice showed deficits in motor coordination and control, but were less affected in mood-related tests. Daily rhythms of gene expression, primarily targeting the circadian clockwork and cell signalling pathways, were also studied, in addition to measurements of mitochondrial respiration in MSNs. Preliminary results indicate changes in molecular and physiological processes in MSNs of knockout animals, that may contribute to the observed behavioural phenotypes.

Molecular and behavioural consequences of a striatal bmal1 knockout in mice

Gonadal and adrenal steroids play a fundamental homeostatic role in the brain, maintaining the hippocampal-neocortical circuitry, which is important for learning, memory and mood regulation. Previous work in our laboratory has demonstrated changes in hippocampal dendritic morphology in males, but not females, following gonadectomy. Acute glucocorticoid exposure in male rats mirrored this effect, suggesting that surgical stress results in pruning of the hippocampal CA3 apical dendrites. The consequences of short-term stress in females have not been as well characterized. We investigated the effects of acute glucocorticoid exposure in cycling female Sprague-Dawley rats by treatment with the synthetic glucocorticoid Dexamethasone (DEX) in the drinking water for 16h, at a dose designed to mimic the effects of acute stress. The animals were sacrificed during proestrus, when circulating estradiol (E) levels are at a maximum. DEX significantly decreased apical dendritic spine density in CA1 and CA3 at 1 and 5 days following treatment. The dendritic branching patterns and length were significantly increased in CA3 dendrites at 1 day. These effects were accompanied by increased phosphorylation of the MAPK signaling proteins, ERK and JNK, as well as increased levels of the ERK-specific phosphatase, DUSP6. These results indicate differences between the sexes in the effects of brief glucocorticoid exposure on hippocampal neuronal structure, possibly as a result of E-induced increases in the activity of the MAP kinase pathways involved in dendrite growth, in females but not in males.

Effects of short-term glucocorticoid exposure on hippocampal neuronal structure and signaling pathways in female rats

Adult hippocampal neurogenesis (AHN) is the process that generates neurons from neural progenitor cells (NPCs) in the adult hippocampus. AHN is important for proper learning, memory, cognition, and mood regulation but is severely decreased in memory and mood disorders. Understanding how AHN is regulated may lead to treatments for these conditions. AHN entails multiple cell stages involving NPC proliferation and differentiation, followed by neuron maturation. The transition of these cell stages is regulated by expression of specific transcription factors. We found that the transcription factor capicua (CIC) is expressed in NPCs but is downregulated during NPC differentiation. Contrastingly, CIC is upregulated during neuron maturation. Such expression changes suggest CIC might play a role in AHN. Here we find that deleting CIC in the hippocampus, including the adult hippocampal neuronal lineage, results in reduced NPCs due to increased differentiation into immature neurons. These immature neurons have impaired dendrite complexity and abnormal migration, with delayed progression to fully matured granule neurons. Together, our results show that CIC plays a critical role in NPC differentiation and neuron maturation in AHN. Our study identifies a previously unknown role for CIC in AHN. Given that decreased AHN is associated with mood and memory disorders, future investigations into the genes and pathways that CIC regulates may reveal crucial molecular inroads and therapeutic entry points into these conditions.

Capicua is required for the regulation of adult hippocampal neurogenesis

The adult brain has a remarkable capacity to produce new cells that migrate and integrate into pre-existing neuronal circuits throughout the lifespan of animals. Adult neural stem cells (NSCs) in subventricular zone (SVZ) are a largely quiescent population and their activation and proliferation is modulated by a number of physical and chemical cues from different sources. However, the mechanisms that integrate those signals and regulate quiescent/proliferative (Q/A) states transition are poorly understood. In this study, using ex-vivo calcium imaging, we revealed the universal Ca2+ signature that characterize activated (aNSCs) and quiescent (qNSCs) NSCs states. We showed that qNSCs are characterized by higher frequency of Ca2+ fluctuations and lower intracellular Ca2+ level, compared to aNSCs. This different Ca2+ dynamics were regulated by intracellular IP3-sensitive Ca2+ stores. Pharmacological inhibition of IP3-sensitive stores decreased the frequency of Ca2+ fluctuations and increased intracellular Ca2+ level of qNSCs. Similarly, CRISPR-Cas9 mediated deletion of Itpr2, specifically in SVZ NSCs resulted in the same characteristic differences in the Ca2+ dynamics. Importantly, these changes were accompanied with increase in proliferation indicating a causal relationship between internal store-dependent Ca2+ dynamics and NSCs Q/A transition. We also showed that anti-proliferative pathways are keeping NSCs quiescent by modulating calcium dynamics. For instance, pharmacological inhibition of the melatonin signaling pathway activates NSCs by reducing the frequency of Ca2+ fluctuations and increasing intracellular Ca2+ levels. Altogether, our data suggest that Ca2+ signaling in NSCs may integrate and decode different signals to regulate NSCs Q/A transition.

Quiescent and proliferative states of adult neural stem cells are characterized by different Ca2+ dynamics and steady-state intracellular levels

Haploinsufficiency of Syngap1 gene encoding the Synaptic Ras- GTPase Activating protein is associated with intellectual disability, autism spectrum disorder and epilepsy. Haploinsufficiency of Syngap1 leads to alterations in synaptic plasticity, behavioral abnormalities and cognitive deficits in mouse models. In particular, several studies have shown that Syngap1 regulates the time course of the maturation of dendritic spines and glutamatergic synapses in excitatory neurons; In contrast, the role of Syngap1 in inhibitory, GABAergic neurons is relatively uncharted. GABAergic neurons are a diverse class of neurons with different morphology, connectivity and physiological properties. They play an important role in neural circuit development and plasticity. Parvalbumin (PV)-expressing interneurons, one of the major classes of cortical GABAergic interneurons, form synapses onto the soma and proximal dendrites of pyramidal cells and are involved in the synchronization of the firing rate of pyramidal cell populations. Our aim is to study the role of Syngap1 in PV cell development In vivo, in establishing balanced synaptic connectivity and proper network in contributing to the behavioral endophenotype and cognition in mouse model by techniques like immunolabelling the synapses and behavioral studies. Preliminary data suggest that alteration in synaptic connectivity of PV cells in different cortical regions do contribute to observed overall behavioral and cognitive deficits. A better understanding of the role of Syngap1 in GABAergic cell development may shed light on the involvement of GABAergic circuit alterations in the cognitive deficits caused by Syngap1 haploinsufficiency in humans.

Understanding the role of SYNGAP1 in GABAergic circuit development and function

Astrocytes are involved in diverse and highly specialized functions in the brain. They display morphological and molecular heterogeneity that is reflective of the microenvironments in which they reside. Astrocytes also become reactive in many brain disorders and diseases, and contribute to disease progression. To better understand how astrocytes develop and specialize within brain microenvironments, we transplanted mouse astrocytes into the mouse cortex in the first postnatal week, and assessed their integration in the host brain at various time points. We found that transplanted astrocytes developed complex morphologies typical of cortical protoplasmic astrocytes and displayed tiling arrangements with astrocytes from the host brain. Also, at 110-130 days post-transplantation, transplanted astrocytes expressed the same levels of GLAST/EAAT1, GLT1/EAAT2, and GFAP as astrocytes in the host brain. Similarly, spatial territories covered by transplanted astrocytes displayed the same density of pre-synaptic terminals as territories covered by host brain astrocytes. Finally, transplanted astrocytes developed endfeet with high AQP4 expression that were tightly associated with capillaries, similar to what we observed with astrocytes in the host brain. Our results indicate that transplanted astrocytes successfully integrate and survive long-term within the mouse brain when transplanted in the immature cortex. These findings open the possibility of using transplanted astrocytes to monitor changes in brain activity/function, and potentially as therapeutic tool to tackle brain diseases.

Transplanted astrocytes develop, mature, and survive long-term in the mouse cortex

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.

Underline digital video library

Feeling hungry? The role of fat stores in hypothalamic neurons on energy balance

Chronic stress & metabolism in females: evaluating two novel models of female social defeat stress

Molecular and behavioural consequences of a striatal bmal1 knockout in mice

Effects of short-term glucocorticoid exposure on hippocampal neuronal structure and signaling pathways in female rats

Capicua is required for the regulation of adult hippocampal neurogenesis

Quiescent and proliferative states of adult neural stem cells are characterized by different Ca2+ dynamics and steady-state intracellular levels

Understanding the role of SYNGAP1 in GABAergic circuit development and function

Transplanted astrocytes develop, mature, and survive long-term in the mouse cortex

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

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

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

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

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