Single-cell Calcium Imaging Research Articles

Parabrachial Calca neurons mediate second-order conditioning

Learning to associate cues, both directly and indirectly, with biologically significant events is essential for survival. Second-order conditioning (SOC) involves forming an association between a previously reinforced conditioned stimulus (CS1) and a new conditioned stimulus (CS2) without the presence of an unconditioned stimulus (US). The neural substrates mediating SOC, however, remain unclear. Parabrachial Calca neurons, which react to the noxious US, also respond to a CS after pairing with a US, suggesting that Calca neurons mediate SOC. We established an aversive SOC behavioral paradigm in mice and monitored Calca neuron activity via single-cell calcium imaging during conditioning and subsequent recall phases. These neurons were activated by both CS1 and CS2 after SOC. Chemogenetically inhibiting Calca neurons during CS1-CS2 pairing attenuated SOC. Thus, reactivation of the US pathway by a learned CS plays an important role in forming the association between the old and a new CS, promoting the formation of second-order memories.

Valence and Salience Encoding in the Central Amygdala.

The central amygdala (CeA) has emerged as an important brain region for regulating both negative (fear and anxiety) and positive (reward) affective behaviors. The CeA has been proposed to encode affective information in the form of valence (whether the stimulus is good or bad) or salience (how significant is the stimulus), but the extent to which these two types of stimulus representation occur in the CeA is not known. Here, we used single cell calcium imaging in mice during appetitive and aversive conditioning and found that majority of CeA neurons (∼65%) encode the valence of the unconditioned stimulus (US) with a smaller subset of cells (∼15%) encoding the salience of the US. Valence and salience encoding of the conditioned stimulus (CS) was also observed, albeit to a lesser extent. These findings show that the CeA is a site of convergence for encoding oppositely valenced US information.

A line attractor encoding a persistent internal state requires neuropeptide signaling

Internal states drive survival behaviors, but their neural implementation is poorly understood. Recently, we identified a line attractor in the ventromedial hypothalamus (VMH) that represents a state of aggressiveness. Line attractors can be implemented by recurrent connectivity or neuromodulatory signaling, but evidence for the latter is scant. Here, we demonstrate that neuropeptidergic signaling is necessary for line attractor dynamics in this system by using cell-type-specific CRISPR-Cas9-based gene editing combined with single-cell calcium imaging. Co-disruption of receptors for oxytocin and vasopressin in adult VMH Esr1+ neurons that control aggression diminished attack, reduced persistent neural activity, and eliminated line attractor dynamics while only slightly reducing overall neural activity and sex- or behavior-specific tuning. These data identify a requisite role for neuropeptidergic signaling in implementing a behaviorally relevant line attractor in mammals. Our approach should facilitate mechanistic studies in neuroscience that bridge different levels of biological function and abstraction.

Formation of an Enduring Ensemble of Accumbens Neurons Leads to Prepotent Seeking for Cocaine Over Natural Reward Cues.

Neuronal activity in the nucleus accumbens core (NAcore) is necessary for reward-seeking behaviors. We hypothesized that the differential encoding of natural and drug rewards in the NAcore contributes to substance use disorder. We leveraged single-cell calcium imaging of dopamine D1- and D2-receptor-expressing medium spiny neurons (MSNs) in the NAcore of mice to examine differences between sucrose and cocaine rewarded (self-administration) and unrewarded (abstinent and cue-induced) seeking. Activity was time-locked to nose-poking for reward, clustered, and compared between sucrose and cocaine. Only in cocaine-trained mice were excited D1-MSNs securely stable, capable of decoding nose-poking in all rewarded and unrewarded sessions and correlated with the intensity of nose-poking for unrewarded seeking. Furthermore, D1-MSNs formed a stable ensemble predictive of seeking behavior after extended cocaine, but not sucrose abstinence. The excited D1-MSN ensemble uniquely drives cue-induced cocaine seeking and may contribute to why drug seeking is prepotent over natural reward seeking in cocaine use disorder.

Endogenous opioids facilitate stress-induced binge eating via an insular cortex-claustrum pathway.

Stress has been shown to promote the development and persistence of binge eating behaviors. However, the neural circuit mechanisms for stress-induced binge-eating behaviors are largely unreported. The endogenous dynorphin (dyn)/kappa opioid receptor (KOR) opioid neuropeptide system has been well established to be a crucial mediator of the anhedonic component of stress. Here, we aimed to dissect the basis of dynorphinergic control of stress-induced binge-like eating behavior. We first established a mouse behavioral model for stress-induced binge-like eating behaviors. We found that mice exposed to stress increased their food intake of familiar palatable food (high fat, high sugar, HPD) compared to non-stressed mice. Following a brain-wide analysis, we isolated robust cFos-positive cells in the Claustrum (CLA), a subcortical structure with highly abundant KOR expression, following stress-induced binge-eating behavior. We report that KOR signaling in CLA is necessary for this elevated stress-induced binge eating behavior using local pharmacology and local deletion of KOR. In vivo calcium recordings using fiber photometry revealed a disinhibition circuit structure in the CLA during the initiation of HPD feeding bouts. We further established the dynamics of endogenous dynorphinergic control of this behavior using a genetically encoded dynorphin biosensor, Klight. Combined with 1-photon single-cell calcium imaging, we report significant heterogeneity with the CLA population during stress-induced binge eating and such behavior attenuates local dynorphin tone. Furthermore, we isolate the anterior Insular cortex (aIC) as the potential source of endogenous dynorphin afferents in the CLA. By characterizing neural circuits and peptidergic mechanisms within the CLA, we uncover a pathway that implicates endogenous opioid regulation stress-induced binge eating.

Coupling of Sharp Wave Events between Zebrafish Hippocampal and Amygdala Homologs.

The mammalian hippocampus exhibits spontaneous sharp wave events (1-30 Hz) with an often-present superimposed fast ripple oscillation (120-220 Hz) to form a sharp wave ripple (SWR) complex. During slow-wave sleep or quiet restfulness, SWRs result from the sequential spiking of hippocampal cell assemblies initially activated during learned or imagined experiences. Additional cortical/subcortical areas exhibit SWR events that are coupled to hippocampal SWRs, and studies in mammals suggest that coupling may be critical for the consolidation and recall of specific memories. In the present study, we have examined juvenile male and female zebrafish and show that SWR events are intrinsically generated and maintained within the telencephalon and that their hippocampal homolog, the anterodorsolateral lobe (ADL), exhibits SW events with ∼9% containing an embedded ripple (SWR). Single-cell calcium imaging coupled to local field potential recordings revealed that ∼10% of active cells in the dorsal telencephalon participate in any given SW event. Furthermore, fluctuations in cholinergic tone modulate SW events consistent with mammalian studies. Moreover, the basolateral amygdala (BLA) homolog exhibits SW events with ∼5% containing an embedded ripple. Computing the SW peak coincidence difference between the ADL and BLA showed bidirectional communication. Simultaneous coupling occurred more frequently within the same hemisphere, and in coupled events across hemispheres, the ADL more commonly preceded BLA. Together, these data suggest conserved mechanisms across species by which SW and SWR events are modulated, and memories may be transferred and consolidated through regional coupling.

Cisplatin Provokes Peripheral Nociception and Neuronal Features of Therapy-Induced Senescence and Calcium Dysregulation in Rats.

Therapy-Induced Senescence (TIS) is a form of senescence that is typically described in malignant cells in response to the exposure of cancer chemotherapy or radiation but can also be precipitated in non-malignant cells. TIS has been shown to contribute to the development of several cancer therapy-related adverse effects; however, evidence on its role in mediating chemotherapy-induced neurotoxicity, such as Chemotherapy-induced Peripheral Neuropathy (CIPN), is limited. We hereshow that cisplatin treatment over two cycles (cumulative dose of 23 mg/kg) provoked mechanical allodynia and thermal hyperalgesia in Sprague-Dawley rats. Isolation of dorsal root ganglia (DRG) from the cisplatin-treated rats demonstrated robust SA-β-gal upregulation at both day 8 (after the first cycle) and day 18 (after the second cycle), decreased lmnb1 expression, increased expression of cdkn1a and cdkn2a, and of several factors of the Senescence-associated Secretory Phenotype (SASP) (Il6, Il1b, and mmp9). Moreover, single-cell calcium imaging of cultured DRGs revealed a significant increase in terms of the magnitude of KCl-evoked calcium responses in cisplatin-treated rats compared to vehicle-treated rats. No significant change was observed in terms of the magnitude of capsaicin-evoked calcium responses in cisplatin-treated rats compared to vehicle-treated rats but with decreased area under the curve of the responses in cisplatin-treated rats. Further evidence to support the contribution of TIS to therapy adverse effects is required but should encourage the use of senescence-modulating agents (senotherapeutics) as novel palliative approaches to mitigate chemotherapy-induced neurotoxicity.

The Potential Role of Thyroid Hormone Therapy in Neural Progenitor Cell Differentiation and Its Impact on Neurodevelopmental Disorders.

Thyroid hormone (T3) plays a vital role in brain development and its dysregulation can impact behavior, nervous system function, and cognitive development. Large case-cohort studies have associated abnormal maternal T3 during early pregnancy to epilepsy, autism, and attention deficit hyperactivity disorder (ADHD) in children. Recent experimental findings have also shown T3's influence on the fate of neural precursor cells and raise the question of its convergence with embryonic neural progenitors. Our objective was to investigate how T3 treatment affects neuronal development and functionality at the cellular level. In vitro experiments using neural precursor cells (NPCs) measured cell growth and numbers after exposure to varying T3 concentrations. Time points included week 0 (W0) representing NPCs treated with 100nM T3 for 5days, and differentiated cortical neurons assessed at weeks 3 (W3), 6 (W6), and 8 (W8). Techniques such as single-cell calcium imaging and whole-cell patch clamp were utilized to evaluate neuronal activity and function. IHC staining detected mature neuron markers, and RNA sequencing enabled molecular profiling. W6 and W8 neurons exhibited higher action potential frequencies, with W6 showing increased peak amplitudes and shortened inter-spike intervals by 50%, indicating enhanced activity. Transcriptomic analysis revealed that W6 T3-treated neurons formed a distinct cluster, suggesting accelerated maturation. Comparison with the whole transcriptome further unveiled a correlation between W6 neurons treated with T3 and neuronal regulatory elements associated with autism and ADHD. These findings provide insights into T3's impact on neuronal development and potential mechanisms of T3 dysregulation and neurodevelopmental disorders.

Molecular Mechanism Analysis of STIM1 Thermal Sensation.

STIM1 has been identified as a new warm sensor, but the exact molecular mechanism remains unclear. In this study, a variety of mutants of STIM1, Orai1 and Orai3 were generated. The single-cell calcium imaging and confocal analysis were used to evaluate the thermal sensitivity of the resulting STIM mutants and the interaction between STIM1 and Orai mutants in response to temperature. Our results suggested that the CC1-SOAR of STIM1 was a direct activation domain of temperature, leading to subsequent STIM1 activation, and the transmembrane (TM) region and K domain but not EF-SAM were needed for this process. Furthermore, both the TM and SOAR domains exhibited similarities and differences between STIM1-mediated thermal sensation and store-operated calcium entry (SOCE), and the key sites of Orai1 showed similar roles in these two responses. Additionally, the TM23 (comprising TM2, loop2, and TM3) region of Orai1 was identified as the key domain determining the STIM1/Orai1 thermal response pattern, while the temperature reactive mode of STIM1/Orai3 seemed to result from a combined effect of Orai3. These findings provide important support for the specific molecular mechanism of STIM1-induced thermal response, as well as the interaction mechanism of STIM1 with Orai1 and Orai3 after being activated by temperature.

Unravelling a novel role for cannabidivarin in the modulation of subventricular zone postnatal neurogenesis

Postnatal neurogenesis has been shown to rely on the endocannabinoid system. Here we aimed at unravelling the role of Cannabidivarin (CBDV), a non-psychoactive cannabinoid, with high affinity for the non-classical cannabinoid receptor TRPV1, on subventricular zone (SVZ) postnatal neurogenesis. Using the neurosphere assay, SVZ-derived neural stem/progenitor cells (NSPCs) were incubated with CBDV and/or 5′-Iodoresinferotoxin (TRPV1 antagonist), and their role on cell viability, proliferation, and differentiation were dissected. CBDV was able to promote, through a TRPV1-dependent mechanism, cell survival, cell proliferation and neuronal differentiation. Furthermore, pulse-chase experiments revealed that CBDV-induced neuronal differentiation was a result of cell cycle exit of NSPCs. Regarding oligodendrocyte differentiation, CBDV inhibited oligodendrocyte differentiation and maturation. Since our data suggested that the CBDV-induced modulation of NSPCs acted via TRPV1, a sodium-calcium channel, and that intracellular calcium levels are known regulators of NSPCs fate and neuronal maturation, single cell calcium imaging was performed to evaluate the functional response of SVZ-derived cells. We observed that CBDV-responsive cells displayed a two-phase calcium influx profile, being the initial phase dependent on TRPV1 activation. Taken together, this work unveiled a novel and untapped neurogenic potential of CBDV via TRPV1 modulation. These findings pave the way to future neural stem cell biological studies and repair strategies by repurposing this non-psychoactive cannabinoid as a valuable therapeutic target.

Deletion of TRPC6, an Autism Risk Gene, Induces Hyperexcitability in Cortical Neurons Derived from Human Pluripotent Stem Cells

Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder linked to numerous rare, inherited, and arising de novo genetic variants. ASD often co-occurs with attention-deficit hyperactivity disorder and epilepsy, which are associated with hyperexcitability of neurons. However, the physiological and molecular mechanisms underlying hyperexcitability in ASD remain poorly understood. Transient receptor potential canonical-6 (TRPC6) is a Ca2+-permeable cation channel that regulates store-operated calcium entry (SOCE) and is a candidate risk gene for ASD. Using human pluripotent stem cell (hPSC)–derived cortical neurons, single-cell calcium imaging, and electrophysiological recording, we show that TRPC6 knockout (KO) reduces SOCE signaling and leads to hyperexcitability of neurons by increasing action potential frequency and network burst frequency. Our data provide evidence that reduction of SOCE by TRPC6 KO results in neuronal hyperexcitability, which we hypothesize is an important contributor to the cellular pathophysiology underlying hyperactivity in some ASD.

Impact of cryopreservation on viability, gene expression and function of enteric nervous system derived neurospheres.

Introduction: Impairment of both the central and peripheral nervous system is a major cause of mortality and disability. It varies from an affection of the brain to various types of enteric dysganglionosis. Congenital enteric dysganglionosis is characterized by the local absence of intrinsic innervation due to deficits in either migration, proliferation or differentiation of neural stem cells. Despite surgery, children's quality of life is reduced. Neural stem cell transplantation seems a promising therapeutic approach, requiring huge amounts of cells and multiple approaches to fully colonize the diseased areas completely. A combination of successful expansion and storage of neural stem cells is needed until a sufficient amount of cells is generated. This must be combined with suitable cell transplantation strategies, that cover all the area affected. Cryopreservation provides the possibility to store cells for long time, unfortunately with side effects, i.e., upon vitality. Methods: In this study we investigate the impact of different freezing and thawing protocols (M1-M4) upon enteric neural stem cell survival, protein and gene expression, and cell function. Results: Freezing enteric nervous system derived neurospheres (ENSdN) following slow-freezing protocols (M1-3) resulted in higher survival rates than flash-freezing (M4). RNA expression profiles were least affected by freezing protocols M1/2, whereas the protein expression of ENSdN remained unchanged after treatment with protocol M1 only. Cells treated with the most promising freezing protocol (M1, slow freezing in fetal calf serum plus 10% DMSO) were subsequently investigated using single-cell calcium imaging. Freezing of ENSdN did not alter the increase in intracellular calcium in response to a specific set of stimuli. Single cells could be assigned to functional subgroups according to response patterns and a significant shift towards cells responding to nicotine was observed after freezing. Discussion: The results demonstrate that cryopreservation of ENSdN is possible with reduced viability, only slight changes in protein/gene expression patterns and without an impact on the neuronal function of different enteric nervous system cell subtypes, with the exception of a subtle upregulation of cells expressing nicotinergic acetylcholine receptors. In summary, cryopreservation presents a good method to store sufficient amounts of enteric neural stem cells without neuronal impairment, in order to enable subsequent transplantation of cells into compromised tissues.

Excitatory and Inhibitory Descending Commissural Interneurons Differentially Integrate Supraspinal and Segmental Sensory Signals.

The limited information about how descending inputs from the brain and sensory inputs from the periphery use spinal cord interneurons (INs) is a major barrier to understanding how these inputs may contribute to motor functions under normal and pathologic conditions. Commissural interneurons (CINs) are a heterogeneous population of spinal INs that has been implicated in crossed motor responses and bilateral motor coordination (ability to use the right and left side of the body in a coordinated manner) and, therefore, are likely involved in many types of movement (e.g., dynamic posture stabilization, jumping, kicking, walking). In this study, we incorporate mouse genetics, anatomy, electrophysiology, and single-cell calcium imaging to investigate how a subset of CINs, those with descending axons called dCINs, are recruited by descending reticulospinal and segmental sensory signals independently and in combination. We focus on two groups of dCINs set apart by their principal neurotransmitter (glutamate and GABA) and identified as VGluT2+ dCINs and GAD2+ dCINs. We show that VGluT2+ and GAD2+ dCINs are both extensively recruited by reticulospinal and sensory input alone but that VGluT2+ and GAD2+ dCINs integrate these inputs differently. Critically, we find that when recruitment depends on the combined action of reticulospinal and sensory inputs (subthreshold inputs), VGluT2+ dCINs, but not GAD2+ dCINs, are recruited. This difference in the integrative capacity of VGluT2+ and GAD2+ dCINs represents a circuit mechanism that the reticulospinal and segmental sensory systems may avail themselves of to regulate motor behaviors both normally and after injury.SIGNIFICANCE STATEMENT The way supraspinal and peripheral sensory inputs use spinal cord interneurons is fundamental to defining how motor functions are supported both in health and disease. This study, which focuses on dCINs, a heterogeneous population of spinal interneurons critical for crossed motor responses and bilateral motor coordination, shows that both glutamatergic (excitatory) and GABAergic (inhibitory) dCINs can be recruited by supraspinal (reticulospinal) or peripheral sensory inputs. Additionally, the study demonstrates that in conditions where the recruitment of dCINs depends on the combined action of reticulospinal and sensory inputs, only excitatory dCINs are recruited. The study uncovers a circuit mechanism that the reticulospinal and segmental sensory systems may avail themselves of to regulate motor behaviors both normally and after injury.

Reprogramming cultured human fungiform (HBO) taste cells into neuron-like cells through in vitro induction.

Human taste cells are a heterogeneous population of specialized epithelial cells that are constantly generated from progenitor taste cells. Type I and type III taste cells express some neural markers, and studies have reported that direct innervation by neurons is not required for taste cell development. To our knowledge, no previous study has demonstrated that taste cells can differentiate into neuron-like cells or any other non-taste cell type. Here, for the first time, we describe a simple in vitro method that uses a serum-free neural induction medium to differentiate cultured physiologically functional primary human taste (HBO) cells into neuron-like cells in 2-3 wk with high efficiency. We verified neural attributes of these HBO-derived neuron-like with immunocytochemistry, single-cell calcium imaging, and DiI staining and examined cell morphology using transmission electron microscopy. Induced neuron-like cells demonstrated neuron-specific proteins, dendritic and axonal morphology, and networking behaviors. This technique will open new avenues for translational medicine, autologous cell therapy, regenerative medicine, therapy for neurodegenerative disorders, and drug screening.

Abstract P2118: Modeling Arrhythmia Risk In Human Stem Cell Derived Cardiomyocytes

In older (45+ years) populations, sudden cardiac death (SCD) is primarily caused by ischemic heart disease while in younger populations, SCD is often due to genetic causes. Despite recent advances in risk stratification based on clinical evaluation and genetic testing, our ability to predict SCD risk in the younger population remains inadequate. Implantable cardioverter defibrillators (ICDs) have been an essential intervention to reduce the number of out-of-hospital cardiac arrests in patients determined to be at risk of SCD. Nevertheless, one third of patients with an ICD receive inappropriate shocks and are exposed to infection without clear benefit. In addition, some children and young adults receive an ICD that will never deliver a shock even after decades of follow-up. No clear guidelines have been established to detect SCD-predisposing conditions due to cost, sensitivity, and specificity of testing modalities. Human iPSC-derived cardiomyocytes (hiPSC-CMs) are a widely recognized and reliable in vitro platform to study clinical phenotypes but have never been used in the context of SCD risk prediction. We hypothesize that we can establish an hiPSC-CMs cellular model to predict SCD risk. Sixty individuals were recruited and classified in one of five categories, ranging from healthy controls with no known cardiac disease (category 1) to individuals that show the highest SCD risk (category 5). These 5 categories are defined by 3 main parameters: 1) absence or presence of ICD, 2) whether the ICD was placed as primary or secondary prevention and 3) the number of shocks received after ICD implantation. hiPSCs were reprogrammed using blood samples and then differentiated into cardiomyocytes. Molecular and electrophysiology measurements were performed for the different risk categories using high-throughput systems. hiPSC-CMs were assayed by single-cell calcium imaging, microelectrode array and automated action potential and current recordings. In vitro measurements revealed that specific parameters such as field potential duration correlate with the SCD risk and outcome. In conclusion, this study will provide a cellular-based tool to determine patient-specific SCD risk in order to advance medical care for at-risk patients and their family members.

Sleep-controlling neurons are sensitive and vulnerable to multiple forms of α-synuclein: implications for the early appearance of sleeping disorders in α-synucleinopathies.

Parkinson's disease, Multiple System Atrophy, and Lewy Body Dementia are incurable diseases called α-synucleinopathies as they are mechanistically linked to the protein, α-synuclein (α-syn). α-syn exists in different structural forms which have been linked to clinical disease distinctions. However, sleeping disorders (SDs) are common in the prodromal phase of all three α-synucleinopathies, which suggests that sleep-controlling neurons are affected by multiple forms of α-syn. To determine whether a structure-independent neuronal impact of α-syn exists, we compared and contrasted the cellular effect of three different α-syn forms on neurotransmitter-defined cells of two sleep-controlling nuclei located in the brainstem: the laterodorsal tegmental nucleus and the pedunculopontine tegmental nucleus. We utilized size exclusion chromatography, fluorescence spectroscopy, circular dichroism spectroscopy and transmission electron microscopy to precisely characterize ​​timepoints in the α-syn aggregation process with three different dominating forms of this protein (monomeric, oligomeric and fibril) and we conducted an in-depth investigation of the underlying neuronal mechanism behind cellular effects of the different forms of the protein using electrophysiology, multiple-cell calcium imaging, single-cell calcium imaging and live-location tracking with fluorescently-tagged α-syn. Interestingly, α-syn altered membrane currents, enhanced firing, increased intracellular calcium and facilitated cell death in a structure-independent manner in sleep-controlling nuclei, and postsynaptic actions involved a G-protein-mediated mechanism. These data are novel as the sleep-controlling nuclei are the first brain regions reported to be affected by α-syn inthisstructure-independent manner. These regions may represent highly important targets for future neuroprotective therapy to modify or delay disease progression in α-synucleinopathies.

P618. To Bite or Not to Bite: Isolating a Cellular Activity Signature for Stress-Induced Aggression in Mice

Patients with stress-induced psychopathologies like posttraumatic stress disorder experience debilitating changes in emotional reactivity that can lead to aggression. The central amygdala (CeA) serves as a point of intersection for neural circuits that regulate both stress and aggression. Here, we leveraged mouse models of naturalistic agonistic behavior to examine the neural bases of stress-induced aggression. We used single-cell calcium imaging to evaluate CeA neurons that express the stress-signaling neuropeptide, corticotropin releasing hormone (Crh).

TRPV4 Stimulation Level Regulates Ca2+-Dependent Control of Human Corneal Endothelial Cell Viability and Survival.

The functional contribution of transient receptor potential vanilloid 4 (TRPV4) expression in maintaining human corneal endothelial cells (HCEC) homeostasis is unclear. Accordingly, we determined the effects of TRPV4 gene and protein overexpression on responses modulating the viability and survival of HCEC. Q-PCR, Western blot, FACS analyses and fluorescence single-cell calcium imaging confirmed TRPV4 gene and protein overexpression in lentivirally transduced 12V4 cells derived from their parent HCEC-12 line. Although TRPV4 overexpression did not alter the baseline transendothelial electrical resistance (TEER), its cellular capacitance (Ccl) was larger than that in its parent. Scanning electron microscopy revealed that only the 12V4 cells developed densely packed villus-like protrusions. Stimulation of TRPV4 activity with GSK1016790A (GSK101, 10 µmol/L) induced larger Ca2+ transients in the 12V4 cells than those in the parental HCEC-12. One to ten nmol/L GSK101 decreased 12V4 viability, increased cell death rates and reduced the TEER, whereas 1 µmol/L GSK101 was required to induce similar effects in the HCEC-12. However, the TRPV4 channel blocker RN1734 (1 to 30 µmol/L) failed to alter HCEC-12 and 12V4 morphology, cell viability and metabolic activity. Taken together, TRPV4 overexpression altered both the HCEC morphology and markedly lowered the GSK101 dosages required to stimulate its channel activity.

Dynamic dichotomy of accumbal population activity underlies cocaine sensitization.

Locomotor sensitization (LS) is an early behavioral adaptation to addictive drugs, driven by the increase of dopamine in the Nucleus Accumbens (NAc). However, the effect on accumbal population activity remains elusive. Here, we used single-cell calcium imaging in mice to record the activity of dopamine-1-receptor (D1R) and dopamine-2-receptor (D2R) expressing spiny projection neurons (SPNs) during cocaine LS. Acute exposure to cocaine elevated D1R SPN activity and reduced D2R SPN activity, albeit with high variability between neurons. During LS, the number of D1R and D2R neurons responding in opposite directions increased. Moreover, preventing LS by inhibition of the ERK signaling pathway decreased the number of cocaine responsive D1R SPNs, but had little effect on D2R SPNs. These results indicate that accumbal population dichotomy is dynamic and contains a subgroup of D1R SPNs that eventually drives LS. Insights into the drug-related activity dynamics provides a foundation for understanding the circuit-level addiction pathogenesis.

Maximally selective single-cell target for circuit control in epilepsy models

Neurological and psychiatric disorders are associated with pathological neural dynamics. The fundamental connectivity patterns of cell-cell communication networks that enable pathological dynamics to emerge remain unknown. Here, we studied epileptic circuits using a newly developed computational pipeline that leveraged single-cell calcium imaging of larval zebrafish and chronically epileptic mice, biologically constrained effective connectivity modeling, and higher-order motif-focused network analysis. We uncovered a novel functional cell type that preferentially emerged in the preseizure state, the superhub, that was unusually richly connected to the rest of the network through feedforward motifs, critically enhancing downstream excitation. Perturbation simulations indicated that disconnecting superhubs was significantly more effective in stabilizing epileptic circuits than disconnecting hub cells that were defined traditionally by connection count. In the dentate gyrus of chronically epileptic mice, superhubs were predominately modeled adult-born granule cells. Collectively, these results predict a new maximally selective and minimally invasive cellular target for seizure control.