Astroglial Ca2+-Dependent Hyperexcitability Requires P2Y1 Purinergic Receptors and Pannexin-1 Channel Activation in a Chronic Model of Epilepsy

Astrocytes from the hippocampus of chronic epileptic rats exhibit an abnormal pattern of intracellular calcium oscillations, characterized by an augmented frequency of long lasting spontaneous Ca2+ transients, which are sensitive to purinergic receptor antagonists but resistant to tetrodotoxin. The above suggests that alterations in astroglial Ca2+-dependent excitability observed in the epileptic tissue could arise from changes in astrocyte-to-astrocyte signaling, which is mainly mediated by purines in physiological and pathological conditions. In spite of that, how purinergic signaling contributes to astrocyte dysfunction in epilepsy remains unclear. Here, we assessed the possible contribution of P2Y1R as well as pannexin1 and connexin43 hemichannels—both candidates for non-vesicular ATP-release—by performing astroglial Ca2+ imaging and dye uptake experiments in hippocampal slices from control and fully kindled rats. P2Y1R blockade with MRS2179 decreased the mean duration of astroglial Ca2+ oscillations by reducing the frequency of slow Ca2+ transients, and thereby restoring the balance between slow (ST) and fast transients (FT) in the kindled group. The potential contribution of astroglial pannexin1 and connexin43 hemichannels as pathways for purine release (e.g., ATP) was assessed through dye uptake experiments. Astrocytes from kindled hippocampi exhibit three-fold more EtBr uptake than controls, whereby pannexin1 hemichannels (Panx1 HCs) accounts for almost all dye uptake with only a slight contribution from connexin43 hemichannels (Cx43 HCs). Confirming its functional involvement, Panx1 HCs inhibition decreased the mean duration of astroglial Ca2+ transients and the frequency of slow oscillations in kindled slices, but had no noticeable effects on the control group. As expected, Cx43 HCs blockade did not have any effects over the mean duration of astroglial Ca2+ oscillations. These findings suggest that P2Y1R and Panx1 HCs play a pivotal role in astroglial pathophysiology, which would explain the upregulation of glutamatergic neurotransmission in the epileptic brain and thus represents a new potential pharmacological target for the treatment of drug-refractory epilepsy

Novel tool to quantify with single-cell resolution the number of incoming AAV genomes co-expressed in the mouse nervous system.

Adeno-associated viral (AAV) vectors are an established and safe gene delivery tool to target the nervous system. However, the payload capacity of <4.9 kb limits the transfer of large or multiple genes. Oversized payloads could be delivered by fragmenting the transgenes into separate AAV capsids that are then mixed. This strategy could increase the AAV cargo capacity to treat monogenic, polygenic diseases and comorbidities only if controlled co-expression of multiple AAV capsids is achieved on each transduced cell. We developed a tool to quantify the number of incoming AAV genomes that are co-expressed in the nervous system with single-cell resolution. By using an isogenic mix of three AAVs each expressing single fluorescent reporters, we determined that expression of much greater than 31 AAV genomes per neuron in vitro and 20 genomes per neuron in vivo is obtained across different brain regions including anterior cingulate, prefrontal, somatomotor and somatosensory cortex areas, and cerebellar lobule VI. Our results demonstrate that multiple AAV vectors containing different transgenes or transgene fragments, can efficiently co-express in the same neuron. This tool can be used to design and improve AAV-based interrogation of neuronal circuits, map brain connectivity, and treat genetic diseases affecting the nervous system

Restraint stress increases hemichannel activity in hippocampal glial cells and neurons

Stress affects brain areas involved in learning and emotional responses, which may contribute in the development of cognitive deficits associated with major depression. These effects have been linked to glial cell activation, glutamate release and changes in neuronal plasticity and survival including atrophy of hippocampal apical dendrites, loss of synapses and neuronal death. Under neuro-inflammatory conditions we recently unveiled a sequential activation of glial cells that release ATP and glutamate via hemichannels inducing neuronal death due to activation of neuronal NMDA/P2X7 receptors and pannexin1 hemichannels. In the present work, we studied if stress-induced glia activation is associated to changes in hemichannel activity. To this end, we compared hemichannel activity of brain cells after acute or chronic restraint stress in mice. Dye uptake experiments in hippocampal slices revealed that acute stress induces opening of both Cx43 and Panx1 hemichannels in astrocytes, which were further increased by chronic stress; whereas enhanced Panx1 hemichannel activity was detected in microglia and neurons after acute/chronic and chronic stress, respectively. Moreover, inhibition of NMDA/P2X7 receptors reduced the chronic stress-induced hemichannel opening, whereas blockade of Cx43 and Panx1 hemichannels fully reduced ATP and glutamate release in hippocampal slices from stressed mice. Thus, we propose that gliotransmitter release through hemichannels may participate in the pathogenesis of stress-associated psychiatric disorders and possibly depression

Heavy Alcohol Exposure Activates Astroglial Hemichannels and Pannexons in the Hippocampus of Adolescent Rats: Effects on Neuroinflammation and Astrocyte Arborization

A mounting body of evidence indicates that adolescents are specially more susceptible to alcohol influence than adults. However, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity and recently, the opening of hemichannels and pannexons has been found to participate in both processes. Here, we evaluated whether adolescent rats exposed to ethanol exhibit changes in the activity of astrocyte hemichannels and pannexons in the hippocampus, as well as alterations in astrocyte arborization and cytokine levels. Adolescent rats were subjected to ethanol (3.0 g/kg) for two successive days at 48-h periods over 14 days. The opening of hemichannels and pannexons was examined in hippocampal slices by dye uptake, whereas hippocampal cytokine levels and astroglial arborization were determined by ELISA and Sholl analysis, respectively. We found that adolescent ethanol exposure increased the opening of connexin 43 (Cx43) hemichannels and pannexin-1 (Panx1) channels in astrocytes. Blockade of p38 mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS) and cyclooxygenases (COXs), as well as chelation of intracellular Ca2+, drastically reduced the ethanol-induced channel opening in astrocytes. Importantly, ethanol-induced Cx43 hemichannel and Panx1 channel activity was correlated with increased levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6 in the hippocampus, as well as with profound alterations in astrocyte arbor complexity. Thus, we propose that uncontrolled opening of astrocyte hemichannels and pannexons may contribute not only to the glial dysfunction and neurotoxicity caused by adolescent alcohol consumption, but also to the pathogenesis of alcohol use disorders in the adulthood

Mouse footpad inoculation model to study viral-induced neuroinflammatory responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viralinduced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development

Possible Involvement of TLRs and Hemichannels in Stress-Induced CNS Dysfunction via Mastocytes, and Glia Activation

In the central nervous system (CNS), mastocytes and glial cells (microglia, astrocytes and oligodendrocytes) function as sensors of neuroinflammatory conditions, responding to stress triggers or becoming sensitized to subsequent proinflammatory challenges. The corticotropin-releasing hormone and glucocorticoids are critical players in stress-induced mastocyte degranulation and potentiation of glial inflammatory responses, respectively. Mastocytes and glial cells express different toll-like receptor (TLR) family members, and their activation via proinflammatory molecules can increase the expression of connexin hemichannels and pannexin channels in glial cells. These membrane pores are oligohexamers of the corresponding protein subunits located in the cell surface. They allow ATP release and Ca2+ influx, which are two important elements of inflammation. Consequently, activated microglia and astrocytes release ATP and glutamate, affecting myelinization, neuronal development, and survival. Binding of ligands to TLRs induces a cascade of intracellular events leading to activation of several transcription factors that regulate the expression of many genes involved in inflammation. During pregnancy, the previous responses promoted by viral infections and other proinflammatory conditions are common and might predispose the offspring to develop psychiatric disorders and neurological diseases. Such disorders could eventually be potentiated by stress and might be part of the etiopathogenesis of CNS dysfunctions including autism spectrum disorders and schizophrenia

Astroglial Ca2+-Dependent Hyperexcitability Requires P2Y1 Purinergic Receptors and Pannexin-1 Channel Activation in a Chronic Model of Epilepsy

<p>Astrocytes from the hippocampus of chronic epileptic rats exhibit an abnormal pattern of intracellular calcium oscillations, characterized by an augmented frequency of long lasting spontaneous Ca²⁺ transients, which are sensitive to purinergic receptor antagonists but resistant to tetrodotoxin. The above suggests that alterations in astroglial Ca²⁺-dependent excitability observed in the epileptic tissue could arise from changes in astrocyte-to-astrocyte signaling, which is mainly mediated by purines in physiological and pathological conditions. In spite of that, how purinergic signaling contributes to astrocyte dysfunction in epilepsy remains unclear. Here, we assessed the possible contribution of P2Y₁R as well as pannexin1 and connexin43 hemichannels—both candidates for non-vesicular ATP-release—by performing astroglial Ca²⁺ imaging and dye uptake experiments in hippocampal slices from control and fully kindled rats. P2Y₁R blockade with MRS2179 decreased the mean duration of astroglial Ca²⁺ oscillations by reducing the frequency of slow Ca²⁺ transients, and thereby restoring the balance between slow (ST) and fast transients (FT) in the kindled group. The potential contribution of astroglial pannexin1 and connexin43 hemichannels as pathways for purine release (e.g., ATP) was assessed through dye uptake experiments. Astrocytes from kindled hippocampi exhibit three-fold more EtBr uptake than controls, whereby pannexin1 hemichannels (Panx1 HCs) accounts for almost all dye uptake with only a slight contribution from connexin43 hemichannels (Cx43 HCs). Confirming its functional involvement, Panx1 HCs inhibition decreased the mean duration of astroglial Ca²⁺ transients and the frequency of slow oscillations in kindled slices, but had no noticeable effects on the control group. As expected, Cx43 HCs blockade did not have any effects over the mean duration of astroglial Ca²⁺ oscillations. These findings suggest that P2Y₁R and Panx1 HCs play a pivotal role in astroglial pathophysiology, which would explain the upregulation of glutamatergic neurotransmission in the epileptic brain and thus represents a new potential pharmacological target for the treatment of drug-refractory epilepsy.</p

Small Alphaherpesvirus Latency-Associated Promoters Drive Efficient and Long-Term Transgene Expression in the CNS

Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy to identify three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp), and LAP1_2 (880 bp). They drive chronic transcription of the virus-encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1α promoter (1,264 bp). Our data suggest that the LAPs are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration

NMDA and P2X7 Receptors Require Pannexin 1 Activation to Initiate and Maintain Nociceptive Signaling in the Spinal Cord of Neuropathic Rats

Pannexin 1 (Panx1) is involved in the spinal central sensitization process in rats with neuropathic pain, but its interaction with well-known, pain-related, ligand-dependent receptors, such as NMDA receptors (NMDAR) and P2X7 purinoceptors (P2X7R), remains largely unexplored. Here, we studied whether NMDAR- and P2X7R-dependent nociceptive signaling in neuropathic rats require the activation of Panx1 channels to generate spinal central sensitization, as assessed by behavioral (mechanical hyperalgesia) and electrophysiological (C-reflex wind-up potentiation) indexes. Administration of either a selective NMDAR agonist i.t. (NMDA, 2 mM) or a P2X7R agonist (BzATP, 150 &mu;M) significantly increased both the mechanical hyperalgesia and the C-reflex wind-up potentiation, effects that were rapidly reversed (minutes) by i.t. administration of a selective pannexin 1 antagonist (10panx peptide, 300 &mu;M), with the scores even reaching values of rats without neuropathy. Accordingly, 300 &mu;M 10panx completely prevented the effects of NMDA and BzATP administered 1 h later, on mechanical hyperalgesia and C-reflex wind-up potentiation. Confocal immunofluorescence imaging revealed coexpression of Panx1 with NeuN protein in intrinsic dorsal horn neurons of neuropathic rats. The results indicate that both NMDAR- and P2X7R-mediated increases in mechanical hyperalgesia and C-reflex wind-up potentiation require neuronal Panx1 channel activation to initiate and maintain nociceptive signaling in neuropathic rats