Extracellular ATP Neurotransmission and Nicotine Sex-Specifically Modulate Habenular Neuronal Activity in Adolescence.

The recent increase in the use of nicotine products by teenagers has revealed an urgent need to better understand the impact of nicotine on the adolescent brain. Here, we sought to examine the actions of extracellular ATP as a neurotransmitter and to investigate whether ATP and nicotinic signaling interact during adolescence. With the GRABATP (G-protein-coupled receptor activation-based ATP sensor), we first demonstrated that nicotine induces extracellular ATP release in the medial habenula, a brain region involved in nicotine aversion and withdrawal. Using patch-clamp electrophysiology, we then demonstrated that activation of the ATP receptors P2X or P2Y1 increases the neuronal firing of cholinergic neurons. Surprisingly, contrasting interactive effects were observed with nicotine exposure. For the P2X receptor, activation had no observable effect on acute nicotine-mediated activity, but during abstinence after 10 d of nicotine exposure, coexposure to nicotine and the P2X agonist potentiated neuronal activity in female, but not male, neurons. For P2Y1 signaling, a potentiated effect of the agonist and nicotine was observed with acute exposure, but not following extended nicotine exposure. These data reveal a complex interactive effect between nicotinic and ATP signaling in the adolescent brain and provide mechanistic insights into extracellular ATP signaling with sex-specific alterations of neuronal responses based on prior drug exposure.SIGNIFICANCE STATEMENT In these studies, it was discovered that nicotine induces extracellular ATP release in the medial habenula and subsequent activation of the ATP purinergic receptors increases habenular cholinergic neuronal firing in the adolescent brain. Interestingly, following extended nicotine exposure, nicotine was found to alter the interplay between purinergic and nicotinic signaling in a sex-specific manner. Together, these studies provide a novel understanding for the role of extracellular ATP in mediating habenular activity and reveal how nicotine exposure during adolescence alters these signaling mechanisms, which has important implications given the high incidence of e-cigarette/vape use by youth.

The Importance of Accounting for Movement When Relating Neuronal Activity to Sensory and Cognitive Processes.

A surprising finding of recent studies in mouse is the dominance of widespread movement-related activity throughout the brain, including in early sensory areas. In awake subjects, failing to account for movement risks misattributing movement-related activity to other (e.g., sensory or cognitive) processes. In this article, we (1) review task designs for separating task-related and movement-related activity, (2) review three "case studies" in which not considering movement would have resulted in critically different interpretations of neuronal function, and (3) discuss functional couplings that may prevent us from ever fully isolating sensory, motor, and cognitive-related activity. Our main thesis is that neural signals related to movement are ubiquitous, and therefore ought to be considered first and foremost when attempting to correlate neuronal activity with task-related processes.

Simultaneous mesoscopic and two-photon imaging of neuronal activity in cortical circuits.

Spontaneous and sensory-evoked activity propagates across varying spatial scales in the mammalian cortex, but technical challenges have limited conceptual links between the function of local neuronal circuits and brain-wide network dynamics. We present a method for simultaneous cellular-resolution two-photon calcium imaging of a local microcircuit and mesoscopic widefield calcium imaging of the entire cortical mantle in awake mice. Our multi-scale approach involves a microscope with an orthogonal axis design where the mesoscopic objective is oriented above the brain and the two-photon objective is oriented horizontally, with imaging performed through a microprism. We also introduce a viral transduction method for robust and widespread gene delivery in the mouse brain. These approaches allow us to identify the behavioral state-dependent functional connectivity of pyramidal neurons and vasoactive intestinal peptide-expressing interneurons with long-range cortical networks. Our imaging system provides a powerful strategy for investigating cortical architecture across a wide range of spatial scales.

Repeated Exposure to Multiple Concurrent Stresses Induce Circuit Specific Loss of Inputs to the Posterior Parietal Cortex.

Severe loss of excitatory synapses in key brain regions is thought to be one of the major mechanisms underlying stress-induced cognitive impairment. To date, however, the identity of the affected circuits remains elusive. Here we examined the effect of exposure to repeated multiple concurrent stressors (RMS) on the connectivity of the posterior parietal cortex (PPC) in adolescent male mice. We found that RMS led to layer-specific elimination of excitatory synapses with the most pronounced loss observed in deeper cortical layers. Quantitative analysis of cortical projections to the PPC revealed a significant loss of sensory and retrosplenial inputs to the PPC while contralateral and frontal projections were preserved. These results were confirmed by decreased synaptic strength from sensory, but not from contralateral, projections in stress-exposed animals. Functionally, RMS disrupted visuospatial working memory performance, implicating disrupted higher-order visual processing. These effects were not observed in mice subjected to restraint-only stress for an identical period of time. The PPC is considered to be a cortical hub for multisensory integration, working memory, and perceptual decision-making. Our data suggest that sensory information streams targeting the PPC may be impacted by recurring stress, likely contributing to stress-induced cognitive impairment.SIGNIFICANCE STATEMENT Repeated exposure to stress profoundly impairs cognitive functions like memory, attention, or decision-making. There is emerging evidence that stress not only impacts high-order regions of the brain, but may affect earlier stages of cognitive processing. Our work focuses on the posterior parietal cortex, a brain region supporting short-term memory, multisensory integration, and decision-making. We show evidence that repeated stress specifically damages sensory inputs to this region. This disruption of synaptic connectivity is linked to working memory impairment and is specific to repeated exposure to multiple stressors. Altogether, our data provide a potential alternative explanation to ailments previously attributed to downstream, cognitive brain structures.

New Aspects of the Contribution of ER to SOCE Regulation: The Role of the ER and ER-Plasma Membrane Junctions in the Regulation of SOCE.

The junctions between the endoplasmic reticulum and the plasma membrane are essential platforms for the activation of store-operated Ca2+ influx. These junctions have specific dimensions and are nonuniformly distributed in polarized cells. The mechanisms involved in the formation of the junctions are currently undergoing vigorous investigation, and significant progress was attained in this research area during the last 10 years. Some cell types display stationary junctions, while in other cells, new junctions can form rapidly following cytosolic Ca2+ signals and/or the reduction of the Ca2+ concentration in the lumen of the endoplasmic reticulum; furthermore, in moving cells, junctions can undergo saltatory formation, long distance sliding, and dissolution. The proteins involved in the activation of the Ca2+ influx could be also involved in the formation of the junctions. The architecture, dynamics, and localization of the junctions are important for the regulation of Ca2+ signaling cascades and their downstream events.

Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca2+ release through IP3 receptors.

Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, which is the initiating step for alcohol-induced pancreatitis. We used intact or two-photon permeabilized acinar cells isolated from wild-type mice or mice in which inositol trisphosphate receptors of type 2 or types 2 and 3 were knocked out. In permeabilized pancreatic acinar cells even a relatively low ethanol concentration elicited calcium release from intracellular stores and intracellular trypsinogen activation. The calcium sensor calmodulin (at a normal intracellular concentration) markedly reduced ethanol-induced calcium release and trypsinogen activation in permeabilized cells, effects prevented by the calmodulin inhibitor peptide. A calmodulin activator virtually abolished the modest ethanol effects in intact cells. Both ethanol-elicited calcium liberation and trypsinogen activation were significantly reduced in cells from type 2 inositol trisphosphate receptor knockout mice. More profound reductions were seen in cells from double inositol trisphosphate receptor (types 2 and 3) knockout mice. The inositol trisphosphate receptors, required for normal pancreatic stimulus-secretion coupling, are also responsible for the toxic ethanol action. Calmodulin protects by reducing calcium release sensitivity.

Repeated exposure to multiple concurrent stressors alters visual processing in the adult posterior parietal cortex.

Chronic stress is well known to erode cognitive functions. Yet, our understanding of how repeated stress exposure impacts one of the fundamental bases of cognition: sensory processing, remains limited. The posterior parietal cortex (PPC) is a high order visual region, known for its role in visually guided decision making, multimodal integration, attention, and working memory. Here, we used functional measures to determine how repeated exposure to multiple concurrent stressors (RMS) affects sensory processing in the PPC in adult male mice. A longitudinal experimental design, repeatedly surveying the same population of neurons using in vivo two-photon imaging, revealed that RMS disrupts the balanced turnover of visually responsive cells in layer 2/3 of the PPC. Across the population, RMS-induced changes in visual responsiveness followed a bimodal distribution suggesting idiosyncratic stress effects. In cells that maintained their responsiveness across recording sessions, we found that stress reduced visual response magnitudes and feature selectivity. While we did not observe stress-induced elimination of excitatory synapses, noise correlation statistics indicated that RMS altered visual input to the neuronal population. The impact of RMS was restricted to visually evoked responses and was not evident in neuronal activity associated with locomotion onset. Together, our results indicate that despite no apparent synaptic reorganization, stress exposure in adulthood can disrupt sensory processing in the PPC, with the effects showing remarkable individual variation.

Practical considerations in an era of multicolor optogenetics.

The ability to control synaptic communication is indispensable to modern neuroscience. Until recently, only single-pathway manipulations were possible due to limited availability of opsins activated by distinct wavelengths. However, extensive protein engineering and screening efforts have drastically expanded the optogenetic toolkit, ushering in an era of multicolor approaches for studying neural circuits. Nonetheless, opsins with truly discrete spectra are scarce. Experimenters must therefore take care to avoid unintended cross-activation of optogenetic tools (crosstalk). Here, we demonstrate the multidimensional nature of crosstalk in a single model synaptic pathway, testing stimulus wavelength, irradiance, duration, and opsin choice. We then propose a "lookup table" method for maximizing the dynamic range of opsin responses on an experiment-by-experiment basis.

A Comprehensive, Affordable, Open-Source Hardware-Software Solution for Flexible Implementation of Complex Behaviors in Head-Fixed Mice.

Experiments that take advantage of head-fixed behavioral tasks have been a staple of systems neuroscience research for half a century. More recently, rodents came to the forefront of these efforts, primarily because of the rich experimental possibilities afforded by modern genetic tools. There is, however, a considerable barrier to entering this field, requiring expertise in engineering, hardware and software development, and significant time and financial commitment. Here, we present a comprehensive, open-source hardware and software solution to implement a head-fixed environment for rodent behaviors (HERBs). Our solution provides access to three frequently used experimental frameworks (two-alternative forced choice, Go-NoGo, or passive sensory stimulus presentation) in a single package. The required hardware can be built at a relatively low cost compared with commercially available solutions, from off-the-shelf components. Our graphical user interface-based software provides great experimental flexibility and requires no programming experience for either installation or use. Furthermore, an HERBs takes advantage of motorized components that allow the precise, temporal separation of behavioral phases (stimulus presentation, delays, response window and reward). Overall, we present a solution that will allow laboratories to join the growing community of systems neuroscience research at a substantially lower cost of entry.

Cellular geography of IP3 receptors, STIM and Orai: a lesson from secretory epithelial cells.

Pancreatic acinar cells exhibit a remarkable polarization of Ca2+ release and Ca2+ influx mechanisms. In the present brief review, we discuss the localization of channels responsible for Ca2+ release [mainly IP3 (inositol 1,4,5-trisphosphate) receptors] and proteins responsible for SOCE (store-operated Ca2+ entry). We also place these Ca2+-transporting mechanisms on the map of cellular organelles in pancreatic acinar cells, and discuss the physiological implications of the cellular geography of Ca2+ signalling. Finally, we highlight some unresolved questions stemming from recent observations of co-localization and co-immunoprecipitation of IP3 receptors with Orai channels in the apical (secretory) region of pancreatic acinar cells.

InsP3receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences.

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca²âº entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP3Rs (InsP3 receptors). The co-localization between Orai1 and IP3Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca²âº store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca²âº store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP3Rs and a basolateral pool that interacts with STIM1 following the Ca²âº store depletion. Experiments on IP3R knockout animals demonstrated that the apical Orai1 localization does not require IP3Rs and that IP3Rs are not necessary for the activation of SOCE. However, the InsP3-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP3Rs could have an inhibitory effect on this Ca²âº entry mechanism.

Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors.

Toxic alcohol effects on pancreatic acinar cells, causing the often fatal human disease acute pancreatitis, are principally mediated by fatty acid ethyl esters (non-oxidative products of alcohol and fatty acids), emptying internal stores of Ca(2+). This excessive Ca(2+) liberation induces Ca(2+)-dependent necrosis due to intracellular trypsin activation. Our aim was to identify the specific source of the Ca(2+) release linked to the fatal intracellular protease activation. In 2-photon permeabilized mouse pancreatic acinar cells, we monitored changes in the Ca(2+) concentration in the thapsigargin-sensitive endoplasmic reticulum (ER) as well as in a bafilomycin-sensitive acid compartment, localized exclusively in the apical granular pole. We also assessed trypsin activity in the apical granular region. Palmitoleic acid ethyl ester (POAEE) elicited Ca(2+) release from both the ER as well as the acid pool, but trypsin activation depended predominantly on Ca(2+) release from the acid pool, that was mainly mediated by functional inositol 1,4,5- trisphosphate receptors (IP(3)Rs) of types 2 and 3. POAEE evoked very little Ca(2+) release and trypsin activation when IP(3)Rs of both types 2 and 3 were knocked out. Antibodies against IP(3)Rs of types 2 and 3, but not type 1, markedly inhibited POAEE-elicited Ca(2+) release and trypsin activation. We conclude that Ca(2+) release through IP(3)Rs of types 2 and 3 in the acid granular Ca(2+) store induces intracellular protease activation, and propose that this is a critical process in the initiation of alcohol-related acute pancreatitis.

To deconvolve, or not to deconvolve: Inferences of neuronal activities using calcium imaging data.

BACKGROUND: With the increasing popularity of calcium imaging in neuroscience research, choosing the right methods to analyze calcium imaging data is critical to address various scientific questions. Unlike spike trains measured using electrodes, fluorescence intensity traces provide an indirect and noisy measurement of the underlying neuronal activities. The observed calcium traces are either analyzed directly or deconvolved to spike trains to infer neuronal activities. When both approaches are applicable, it is unclear whether deconvolving calcium traces is a necessary step. METHODS: In this article, we compare the performance of using calcium traces or their deconvolved spike trains for three common analyses: clustering, principal component analysis (PCA), and population decoding. RESULTS: We found that (1) the two approaches lead to diverging results; (2) estimated spike trains, when smoothed or binned appropriately, usually lead to satisfactory performances, such as more accurate estimation of cluster membership; (3) although estimate spike train produce results more similar to true spike data than trace data, we found that the PCA results from trace data might better reflect the underlying neuronal ensembles (clusters); and (4) for both approaches, decobability can be improved by using denoising or smoothing methods. COMPARISON WITH EXISTING METHODS: Our simulations and applications to real data suggest that estimated spike data outperform trace data in cluster analysis and give comparable results for population decoding. In addition, the decobability of estimated spike data can be slightly better than that of calcium trace data with appropriate filtering / smoothing methods. CONCLUSION: We conclude that spike detection might be a useful pre-processing step for certain problems such as clustering; however, the continuous nature of calcium imaging data provides a natural smoothness that might be helpful for problems such as dimensional reduction.

Cell-type-specific integration of feedforward and feedback synaptic inputs in the posterior parietal cortex.

The integration of feedforward (sensory) and feedback (top-down) neuronal signals is a principal function of the neocortex. Yet, we have limited insight into how these information streams are combined by individual neurons. Using a two-color optogenetic strategy, we found that layer 5 pyramidal neurons in the posterior parietal cortex receive monosynaptic dual innervation, combining sensory inputs with top-down signals. Subclasses of layer 5 pyramidal neurons integrated these synapses with distinct temporal dynamics. Specifically, regular spiking cells exhibited supralinear enhancement of delayed-but not coincident-inputs, while intrinsic burst-firing neurons selectively boosted coincident synaptic events. These subthreshold integration characteristics translated to a nonlinear summation of action potential firing. Complementing electrophysiology with computational modeling, we found that distinct integration profiles arose from a cell-type-specific interaction of ionic mechanisms and feedforward inhibition. These data provide insight into the cellular properties that guide the nonlinear interaction of distinct long-range afferents in the neocortex.

Sex-dependent long-term effects of prepubescent stress on the posterior parietal cortex.

Adolescence is a time of intense cortical development and a period of heightened sensitivity to insult. To determine how sex affects the short- and long-term outcomes of early-adolescent stress exposure, we subjected prepubescent (postnatal day 30) male and female mice to repeated multiple concurrent stressors (RMS). In the posterior parietal cortex (PPC), RMS caused the elimination of excitatory synapses in deeper layers while inhibitory synapse density was predominantly diminished in superficial layers. These short-term effects coincided with reduced visuo-spatial working memory and were similar in both sexes. The loss of excitatory synapses and impaired working memory persisted in males past a 30-day recovery period. In contrast, we observed a remarkable recovery of excitatory transmission and behavioral performance in females. Inhibitory synapse density recovered in both sexes. We have also observed a late onset anxiety phenotype in RMS exposed females that was absent in males. Overall, our results indicate that there are marked sex differences in the long-term effects of prepubescent stress on cortical synapses and behavior.

Multiple Simultaneous Acute Stresses in Mice: Single or Repeated Induction.

Stress is crucial to the survival of an organism, but excessive stress can lead to psychological disorders including depression, anxiety, substance abuse, and suicidality. The prevailing notion is that chronic stress promotes adverse outcomes on brain and body health, whereas acute stressors are generally benign. Notably, acute events such mass shootings or natural disasters are now emerging as significant sources of cognitive and emotional problems including post-traumatic stress disorder (PTSD). These events are characterized by the simultaneous occurrence of physical, emotional, and social stresses, which last minutes to hours. Hence, there is a need to model such multiple concurrent acute stresses (MAS) to uncover the mechanisms by which they lead to profound adverse outcomes. The MAS paradigm described here involves simultaneously exposing a rodent to several different stressors including restraint, crowding, and jostling alongside peers in a brightly lit and very noisy environment. Moreover, the MAS paradigm can be used once or imposed repeatedly to emulate complex, repeated modern life stresses, advancing our mechanistic understanding of consequent mental and cognitive impairments.

Ketamine disrupts neuromodulatory control of glutamatergic synaptic transmission.

A growing body of literature has demonstrated the potential for ketamine in the treatment of major depression. Sub-anesthetic doses produce rapid and sustained changes in depressive behavior, both in patients and rodent models, associated with reorganization of glutamatergic synapses in the prefrontal cortex (PFC). While ketamine is known to regulate N-methyl-D-aspartate (NMDA) -type glutamate receptors (NMDARs), the full complement of downstream cellular consequences for ketamine administration are not well understood. Here, we combine electrophysiology with 2-photon imaging and glutamate uncaging in acute slices of mouse PFC to further examine how ketamine alters glutamatergic synaptic transmission. We find that four hours after ketamine treatment, glutamatergic synapses themselves are not significantly affected. However, levels of the neuromodulatory Regulator of G-protein Signaling (RGS4) are dramatically reduced. This loss of RGS4 activity is associated with disruption of the normal compartmentalization of synaptic neuromodulation. Thus, under control conditions, α2 adrenergic receptors and type B γ-aminobutyric acid (GABAB) receptors selectively inhibit α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) -type glutamate receptors (AMPARs) and NMDARs, respectively. After ketamine administration and reduction in RGS4 activity, this selectivity is lost, with both modulatory systems broadly inhibiting glutamatergic transmission. These results suggest a novel mechanism by which ketamine may influence synaptic signaling and provide new avenues for the exploration of therapeutics directed at treating neuropsychiatric disorders, such as depression.

Evans Blue fluorescence permits the rapid visualization of non-intact cells in the perilesional rim of cold-injured rat brain.

A focal cold lesion-induced injury, i.e., a model of focal vasogenic brain edema, enhances the permeability of the blood-brain barrier and cell membrane in the perilesional rim. However, non-intact cells can be detected, e.g. by markers of apoptosis, only hours or even days after the injury. The early membrane dysfunction allows extravasated serum proteins to enter the injured cells, which can be readily visualized if the plasma albumin was previously bound to fluorescent tracers, such as Evans Blue (EB). The aim of this study was to demonstrate injured cells that take up the EB/albumin conjugate in the perilesional rim. This tracer was administered 3.5 h after the induction of the injury and the animals were sacrificed 30 min later. With an excitation wavelength of 530-550 nm, the EB-positive cells emitted bright-red fluorescence at > 590 nm and were easy to count. No positive cells were observed in the controls. This method provides more information than the classical 2,3,5-triphenyltetrazolium chloride reaction, because it permits an assessment of the density and distribution of cells with non-intact cell membranes in the perilesional area following cerebrocortical injury.

Developmental Dysfunction of VIP Interneurons Impairs Cortical Circuits.

GABAergic interneurons play important roles in cortical circuit development. However, there are multiple populations of interneurons and their respective developmental contributions remain poorly explored. Neuregulin 1 (NRG1) and its interneuron-specific receptor ERBB4 are critical genes for interneuron maturation. Using a conditional ErbB4 deletion, we tested the role of vasoactive intestinal peptide (VIP)-expressing interneurons in the postnatal maturation of cortical circuits in vivo. ErbB4 removal from VIP interneurons during development leads to changes in their activity, along with severe dysregulation of cortical temporal organization and state dependence. These alterations emerge during adolescence, and mature animals in which VIP interneurons lack ErbB4 exhibit reduced cortical responses to sensory stimuli and impaired sensory learning. Our data support a key role for VIP interneurons in cortical circuit development and suggest a possible contribution to pathophysiology in neurodevelopmental disorders. These findings provide a new perspective on the role of GABAergic interneuron diversity in cortical development. VIDEO ABSTRACT.

Hippocampal (CA1) activities in Wistar rats from different vendors. Fundamental differences in acute ischemia.

Two-vessel occlusion, a frequently used model of global cerebral ischemia in rats, results in a dysfunction predominantly within the CA1 field of the hippocampus; it induces many processes with different time-scales. However, the great divergence in the results of the studies reported in the literature suggests valuable differences in response to hypoperfusion-induced ischemia among the laboratory rats used in these studies. In the present work, the acute effects of two-carotid occlusion-induced global ischemia (2VO) on the CA3 stimulation-evoked population spike activity in the CA1 region of Wistar rats from different suppliers (Charles-River and Harlan) were compared. In the acute electrophysiological experiments, the hippocampal CA1 responses revealed that the Charles-River rats immediately compensated the 2VO much better than did the Harlan rats. However, 3 days later, no difference could be observed between the CA1 activities of these rats. The presented data show that the Wistar rats from different vendors represent an important source of variability in the results of acute experiments on the hippocampal ischemia. These observations draw attention to the importance of the careful choice of the laboratory rats (both strains and breeds) used in such experiments.