Cannabinoids regulate an insula circuit controlling water intake.

The insular cortex, or insula, is a large brain region involved in the detection of thirst and the regulation of water intake. However, our understanding of the topographical, circuit, and molecular mechanisms for controlling water intake within the insula remains parcellated. We found that type-1 cannabinoid (CB1) receptors in the insular cortex cells participate in the regulation of water intake and deconstructed the circuit mechanisms of this control. Topographically, we revealed that the activity of excitatory neurons in both the anterior insula (aIC) and posterior insula (pIC) increases in response to water intake, yet only the specific removal of CB1 receptors in the pIC decreases water intake. Interestingly, we found that CB1 receptors are highly expressed in insula projections to the basolateral amygdala (BLA), while undetectable in the neighboring central part of the amygdala. Thus, we recorded the neurons of the aIC or pIC targeting the BLA (aIC-BLA and pIC-BLA) and found that they decreased their activity upon water drinking. Additionally, chemogenetic activation of pIC-BLA projection neurons decreased water intake. Finally, we uncovered CB1-dependent short-term synaptic plasticity (depolarization-induced suppression of excitation [DSE]) selectively in pIC-BLA, compared with aIC-BLA synapses. Altogether, our results support a model where CB1 receptor signaling promotes water intake by inhibiting the pIC-BLA pathway, thereby contributing to the fine top-down control of thirst responses.

Striatopallidal cannabinoid type-1 receptors mediate amphetamine-induced sensitization.

Repeated exposure to psychostimulants, such as amphetamine, causes a long-lasting enhancement in the behavioral responses to the drug, called behavioral sensitization.1 This phenomenon involves several neuronal systems and brain areas, among which the dorsal striatum plays a key role.2 The endocannabinoid system (ECS) has been proposed to participate in this effect, but the neuronal basis of this interaction has not been investigated.3 In the CNS, the ECS exerts its functions mainly acting through the cannabinoid type-1 (CB1) receptor, which is highly expressed at terminals of striatal medium spiny neurons (MSNs) belonging to both the direct and indirect pathways.4 In this study, we show that, although striatal CB1 receptors are not involved in the acute response to amphetamine, the behavioral sensitization and related synaptic changes require the activation of CB1 receptors specifically located at striatopallidal MSNs (indirect pathway). These results highlight a new mechanism of psychostimulant sensitization, a phenomenon that plays a key role in the health-threatening effects of these drugs.

The UPR Maintains Proteostasis and the Viability and Function of Hippocampal Neurons in Adult Mice.

The unfolded protein response (UPR), which comprises three branches: PERK, ATF6α, and IRE1, is a major mechanism for maintaining cellular proteostasis. Many studies show that the UPR is a major player in regulating neuron viability and function in various neurodegenerative diseases; however, its role in neurodegeneration is highly controversial. Moreover, while evidence suggests activation of the UPR in neurons under normal conditions, deficiency of individual branches of the UPR has no major effect on brain neurons in animals. It remains unclear whether or how the UPR participates in regulating neuronal proteostasis under normal and disease conditions. To determine the physiological role of the UPR in neurons, we generated mice with double deletion of PERK and ATF6α in neurons. We found that inactivation of PERK and ATF6α in neurons caused lysosomal dysfunction (as evidenced by decreased expression of the V0a1 subunit of v-ATPase and decreased activation of cathepsin D), impairment of autophagic flux (as evidenced by increased ratio of LC3-II/LC3-I and increased p62 level), and accumulation of p-tau and Aß42 in the hippocampus, and led to impairment of spatial memory, impairment of hippocampal LTP, and hippocampal degeneration in adult mice. These results suggest that the UPR is required for maintaining neuronal proteostasis (particularly tau and Aß homeostasis) and the viability and function of neurons in the hippocampus of adult mice.

Dysregulation of astrocytic Ca2+ signaling and gliotransmitter release in mouse models of α-synucleinopathies.

α-Synuclein is a major component of Lewy bodies (LB) and Lewy neurites (LN) appearing in the postmortem brain of Parkinson's disease (PD) and other α-synucleinopathies. While most studies of α-synucleinopathies have focused on neuronal and synaptic alterations as well as dysfunctions of the astrocytic homeostatic roles, whether the bidirectional astrocyte-neuronal communication is affected in these diseases remains unknown. We have investigated whether the astrocyte Ca2+ excitability and the glutamatergic gliotransmission underlying astrocyte-neuronal signaling are altered in several transgenic mouse models related to α-synucleinopathies, i.e., mice expressing high and low levels of the human A53T mutant α-synuclein (G2-3 and H5 mice, respectively) globally or selectively in neurons (iSyn mice), mice expressing human wildtype α-synuclein (I2-2 mice), and mice expressing A30P mutant α-synuclein (O2 mice). Combining astrocytic Ca2+ imaging and neuronal electrophysiological recordings in hippocampal slices of these mice, we have found that compared to non-transgenic mice, astrocytes in G2-3 mice at different ages (1-6 months) displayed a Ca2+ hyperexcitability that was independent of neurotransmitter receptor activation, suggesting that the expression of α-synuclein mutant A53T altered the intrinsic properties of astrocytes. Similar dysregulation of the astrocyte Ca2+ signal was present in H5 mice, but not in I2-2 and O2 mice, indicating α-synuclein mutant-specific effects. Moreover, astrocyte Ca2+ hyperexcitability was absent in mice expressing the α-synuclein mutant A53T selectively in neurons, indicating that the effects on astrocytes were cell-autonomous. Consistent with these effects, glutamatergic gliotransmission was enhanced in G2-3 and H5 mice, but was unaffected in I2-2, O2 and iSyn mice. These results indicate a cell-autonomous effect of pathogenic A53T expression in astrocytes that may contribute to the altered neuronal and synaptic function observed in α-synucleinopathies.

TRPV1 channels in nitric oxide-mediated signalling: insight on excitatory transmission in rat CA1 pyramidal neurons.

Nitric oxide (NO) is a fascinating signalling molecule implicated in a plethora of biological functions, especially at the synaptic level. Exploring neurotransmission in the hippocampus could be instrumental in the individuation of putative targets for nitric-oxide mediated neuromodulation, especially in terms of the potential repercussions on fundamental processes i.e. synaptic plasticity and excitability-related phenomena. Among these targets, endovanilloid signalling constitutes an object of study since Transient Receptors Vanilloid type 1 (TRPV1) channels possess a NO-sensitive gate modulating its activation. Also, NO has been referred to as a mediator for numerous endocannabinoid effects. Notwithstanding, the linkage between TRPV1 and NO systems in neuromodulation still remains elusive. To this end, we aim at investigating the involvement of TRPV1 in nitric oxide-mediated influence on hippocampal processes. Electrophysiological whole-cell recordings in CA1 pyramidal neurons were applied to evaluate excitatory neurotransmission in rat brain slices. Indeed, miniature excitatory postsynaptic currents (mEPSCs) were analysed upon pharmacological manipulation of TRPV1 and NO signalling pathways. In detail, only the administration of the specific TRPV1 exogenous agonist - capsaicin - reduced the frequency and amplitude of mEPSC similarly to the inhibitor of neuronal nitric oxide synthase (nNOS), 7-nitroindazole (7NI). In contrast, capsazepine, TRPV1 antagonist, does not influence excitatory transmission. The combined TRPV1 activation and nNOS blockade confirm the presence of a putative common mechanism. When we administered the endovanilloid-endocannabinoid ligand, i.e. anandamide, we unveiled a potentiation of neurotransmission that was selectively reverted by 7NI. Our data suggest that nitric oxide influences TRPV1 hippocampal signalling since these channels are not constitutively active, but can be "on-demand" activated to modulate excitation in CA1 pyramidal neurons, and that this effect is linked to nitric oxide production.

Reinforcing Interdisciplinary Collaborations to Unravel the Astrocyte "Calcium Code".

In this review article, we present the major insights from and challenges faced in the acquisition, analysis and modeling of astrocyte calcium activity, aiming at bridging the gap between those fields to crack the complex astrocyte "Calcium Code". We then propose strategies to reinforce interdisciplinary collaborative projects to unravel astrocyte function in health and disease.

Astroglial ER-mitochondria calcium transfer mediates endocannabinoid-dependent synaptic integration.

Intracellular calcium signaling underlies the astroglial control of synaptic transmission and plasticity. Mitochondria-endoplasmic reticulum contacts (MERCs) are key determinants of calcium dynamics, but their functional impact on astroglial regulation of brain information processing is unexplored. We found that the activation of astrocyte mitochondrial-associated type-1 cannabinoid (mtCB1) receptors determines MERC-dependent intracellular calcium signaling and synaptic integration. The stimulation of mtCB1 receptors promotes calcium transfer from the endoplasmic reticulum to mitochondria through a specific molecular cascade, involving the mitochondrial calcium uniporter (MCU). Physiologically, mtCB1-dependent mitochondrial calcium uptake determines the dynamics of cytosolic calcium events in astrocytes upon endocannabinoid mobilization. Accordingly, electrophysiological recordings in hippocampal slices showed that conditional genetic exclusion of mtCB1 receptors or dominant-negative MCU expression in astrocytes blocks lateral synaptic potentiation, through which astrocytes integrate the activity of distant synapses. Altogether, these data reveal an endocannabinoid link between astroglial MERCs and the regulation of brain network functions.

CB1R-dependent regulation of astrocyte physiology and astrocyte-neuron interactions.

The endocannabinoid system (ECS) is involved in a variety of brain functions, mainly through the activation of the type-1 cannabinoid receptors (CB1R). CB1R are highly expressed throughout the brain at different structural, cellular and subcellular locations and its activity and expression levels have a direct impact in synaptic activity and behavior. In the last few decades, astrocytes have arisen as active players of brain physiology through their participation in the tripartite synapse and through their metabolic interaction with neurons. Here, we discuss some of the mechanisms by which astroglial CB1R at different subcellular locations, regulate astrocyte calcium signals and have an impact on gliotransmission and metabolic regulation. In addition, we discuss evidence pointing at astrocytes as potential important sources of endocannabinoid synthesis and release. Thus, we summarize recent findings that add further complexity and establish that the ECS is a fundamental effector of astrocyte functions in the brain. This article is part of the special issue on 'Cannabinoids'.

A Novel Cortical Mechanism for Top-Down Control of Water Intake.

Water intake is crucial for maintaining body fluid homeostasis and animals' survival [1-4]. In the brain, complex processes trigger thirst and drinking behavior [1-5]. The anterior wall of the third ventricle formed by the subfornical organ (SFO), the median preoptic nucleus, and the organum vasculosum of the lamina terminalis (OVLT) constitute the primary structures sensing thirst signals and modulating water intake [6-10]. These subcortical regions are connected with the neocortex [11]. In particular, insular and anterior cingulate cortices (IC and ACC, respectively) have been shown to receive indirect innervations from the SFO and OVLT in rats [11] and to be involved in the control of water intake [12-15]. Type-1 cannabinoid receptors (CB1) modulate consummatory behaviors, such as feeding [16-26]. However, the role of CB1 receptors in the control of water intake is still a matter of debate [27-31]. Here, we show that endogenous activation of CB1 in cortical glutamatergic neurons of the ACC promotes water intake. Notably, presynaptic CB1 receptors of ACC glutamatergic neurons are abundantly located in the basolateral amygdala (BLA), a key area in the regulation of water intake. The selective expression of CB1 receptors in the ACC-to-BLA-projecting neurons is sufficient to stimulate drinking behavior. Moreover, chemogenetic stimulation of these projecting neurons suppresses drinking behavior, further supporting the role of this neuronal population in the control of water intake. Altogether, these data reveal a novel cortico-amygdalar mechanism involved in the regulation of drinking behavior.

Dopamine-Evoked Synaptic Regulation in the Nucleus Accumbens Requires Astrocyte Activity.

Dopamine is involved in physiological processes like learning and memory, motor control and reward, and pathological conditions such as Parkinson's disease and addiction. In contrast to the extensive studies on neurons, astrocyte involvement in dopaminergic signaling remains largely unknown. Using transgenic mice, optogenetics, and pharmacogenetics, we studied the role of astrocytes on the dopaminergic system. We show that in freely behaving mice, astrocytes in the nucleus accumbens (NAc), a key reward center in the brain, respond with Ca2+ elevations to synaptically released dopamine, a phenomenon enhanced by amphetamine. In brain slices, synaptically released dopamine increases astrocyte Ca2+, stimulates ATP/adenosine release, and depresses excitatory synaptic transmission through activation of presynaptic A1 receptors. Amphetamine depresses neurotransmission through stimulation of astrocytes and the consequent A1 receptor activation. Furthermore, astrocytes modulate the acute behavioral psychomotor effects of amphetamine. Therefore, astrocytes mediate the dopamine- and amphetamine-induced synaptic regulation, revealing a novel cellular pathway in the brain reward system.

In vivo knockdown of astroglial glutamate transporters GLT-1 and GLAST increases excitatory neurotransmission in mouse infralimbic cortex: Relevance for depressive-like phenotypes.

Alterations of energy metabolism and of astrocyte number/function in ventral anterior cingulate cortex (vACC) have been reported in major depressive disorder (MDD) patients and may contribute to MDD pathophysiology. We recently developed a mouse model of MDD mimicking these alterations. We knocked down the astroglial glutamate transporters GLAST and GLT-1 in infralimbic cortex (IL, rodent equivalent of vACC) using small interfering RNA (siRNA). GLAST and GLT-1 siRNA microinfusion in IL evoked a depressive-like phenotype, associated with a reduced serotonergic function and reduced forebrain BDNF expression. Neither effect occurred after siRNA application in the adjacent prelimbic cortex (PrL), thus emphasizing the critical role of vACC/IL in MDD pathogenesis. Here we examined the cellular/network basis of the changes induced in IL using intracellular recordings of layer V pyramidal neurons from mice microinjected with siRNA 24 h before. We analyzed (i) the electrophysiological characteristics of neurons; (ii) the synaptic transmission properties, by monitoring miniature, spontaneous and evoked EPSCs, and (iii) the gliotransmission, by monitoring slow inward currents (SICs), mediated by astrocytic glutamate release and activation of extra-synaptic NMDA receptors. GLT-1 and GLAST knockdown led to a more depolarized membrane potential and increased action potential firing rate of layer V pyramidal neurons, and enhanced excitatory synaptic transmission, as shown by the enhanced amplitude/frequency of spontaneous EPSCs. Gliotransmission was also increased, as indicated by the enhanced SIC amplitude/frequency. Hence, the depressive-like phenotype is associated with IL hyperactivity, likely leading to an excessive top-down inhibitory control of serotonergic activity through IL-midbrain descending pathways.

Tau is required for progressive synaptic and memory deficits in a transgenic mouse model of α-synucleinopathy.

Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) are clinically and neuropathologically highly related α-synucleinopathies that collectively constitute the second leading cause of neurodegenerative dementias. Genetic and neuropathological studies directly implicate α-synuclein (αS) abnormalities in PDD and DLB pathogenesis. However, it is currently unknown how αS abnormalities contribute to memory loss, particularly since forebrain neuronal loss in PDD and DLB is less severe than in Alzheimer's disease. Previously, we found that familial Parkinson's disease-linked human mutant A53T αS causes aberrant localization of the microtubule-associated protein tau to postsynaptic spines in neurons, leading to postsynaptic deficits. Thus, we directly tested if the synaptic and memory deficits in a mouse model of α-synucleinopathy (TgA53T) are mediated by tau. TgA53T mice exhibit progressive memory deficits associated with postsynaptic deficits in the absence of obvious neuropathological and neurodegenerative changes in the hippocampus. Significantly, removal of endogenous mouse tau expression in TgA53T mice (TgA53T/mTau-/-), achieved by mating TgA53T mice to mouse tau-knockout mice, completely ameliorates cognitive dysfunction and concurrent synaptic deficits without affecting αS expression or accumulation of selected toxic αS oligomers. Among the known tau-dependent effects, memory deficits in TgA53T mice were associated with hippocampal circuit remodeling linked to chronic network hyperexcitability. This remodeling was absent in TgA53T/mTau-/- mice, indicating that postsynaptic deficits, aberrant network hyperactivity, and memory deficits are mechanistically linked. Our results directly implicate tau as a mediator of specific human mutant A53T αS-mediated abnormalities related to deficits in hippocampal neurotransmission and suggest a mechanism for memory impairment that occurs as a consequence of synaptic dysfunction rather than synaptic or neuronal loss. We hypothesize that these initial synaptic deficits contribute to network hyperexcitability which, in turn, exacerbate cognitive dysfunction. Our results indicate that these synaptic changes present potential therapeutic targets for amelioration of memory deficits in α-synucleinopathies.

Gi/o protein-coupled receptors inhibit neurons but activate astrocytes and stimulate gliotransmission.

G protein-coupled receptors (GPCRs) play key roles in intercellular signaling in the brain. Their effects on cellular function have been largely studied in neurons, but their functional consequences on astrocytes are less known. Using both endogenous and chemogenetic approaches with DREADDs, we have investigated the effects of Gq and Gi/o GPCR activation on astroglial Ca2+ -based activity, gliotransmitter release, and the functional consequences on neuronal electrical activity. We found that while Gq GPCR activation led to cellular activation in both neurons and astrocytes, Gi/o GPCR activation led to cellular inhibition in neurons and cellular activation in astrocytes. Astroglial activation by either Gq or Gi/o protein-mediated signaling stimulated gliotransmitter release, which increased neuronal excitability. Additionally, activation of Gq and Gi/o DREADDs in vivo increased astrocyte Ca2+ activity and modified neuronal network electrical activity. Present results reveal additional complexity of the signaling consequences of excitatory and inhibitory neurotransmitters in astroglia-neuron network operation and brain function.

A53T Mutant Alpha-Synuclein Induces Tau-Dependent Postsynaptic Impairment Independently of Neurodegenerative Changes.

Abnormalities in α-synuclein are implicated in the pathogenesis of Parkinson's disease (PD). Because α-synuclein is highly concentrated within presynaptic terminals, presynaptic dysfunction has been proposed as a potential pathogenic mechanism. Here, we report novel, tau-dependent, postsynaptic deficits caused by A53T mutant α-synuclein, which is linked to familial PD. We analyzed synaptic activity in hippocampal slices and cultured hippocampal neurons from transgenic mice of either sex expressing human WT, A53T, and A30P α-synuclein. Increased α-synuclein expression leads to decreased spontaneous synaptic vesicle release regardless of genotype. However, only those neurons expressing A53T α-synuclein exhibit postsynaptic dysfunction, including decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. We also found that long-term potentiation and spatial learning were impaired by A53T α-synuclein expression. Mechanistically, postsynaptic dysfunction requires glycogen synthase kinase 3ß-mediated tau phosphorylation, tau mislocalization to dendritic spines, and calcineurin-dependent AMPA receptor internalization. Previous studies reveal that human A53T α-synuclein has a high aggregation potential, which may explain the mutation's unique capacity to induce postsynaptic deficits. However, patients with sporadic PD with severe tau pathology are also more likely to have early onset cognitive decline. Our results here show a novel, functional role for tau: mediating the effects of α-synuclein on postsynaptic signaling. Therefore, the unraveled tau-mediated signaling cascade may contribute to the pathogenesis of dementia in A53T α-synuclein-linked familial PD cases, as well as some subgroups of PD cases with extensive tau pathology.SIGNIFICANCE STATEMENT Here, we report mutation-specific postsynaptic deficits that are caused by A53T mutant α-synuclein, which is linked to familial Parkinson's disease (PD). The overexpression of WT, A53T, or A30P human α-synuclein leads to decreased spontaneous synaptic vesicle release. However, only those neurons expressing A53T α-synuclein exhibit tau phosphorylation-dependent postsynaptic dysfunction, which is characterized by decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. The mutation-specific postsynaptic effects caused by human A53T α-synuclein will help us better understand the neurobiological basis of this specific form of familial PD. The differential effects of exogenous human WT, A53T, A30P, and E46K α-synuclein on glutamatergic synaptic responses will help to explain the clinical heterogeneity of sporadic and familial PD.

Stimulating Astrocytes to Remember.

Adamsky et al. show that stimulation of astrocytes potentiates synaptic transmission and enhances behavioral performance in memory tasks. These results demonstrate that astrocytes are actively involved in synaptic physiology and brain function and lend further support to the idea that animal behavior results from the coordinated activity of neurons and astrocytes.

Neuronal activity determines distinct gliotransmitter release from a single astrocyte.

Accumulating evidence indicates that astrocytes are actively involved in brain function by regulating synaptic activity and plasticity. Different gliotransmitters, such as glutamate, ATP, GABA or D-serine, released form astrocytes have been shown to induce different forms of synaptic regulation. However, whether a single astrocyte may release different gliotransmitters is unknown. Here we show that mouse hippocampal astrocytes activated by endogenous (neuron-released endocannabinoids or GABA) or exogenous (single astrocyte Ca2+ uncaging) stimuli modulate putative single CA3-CA1 hippocampal synapses. The astrocyte-mediated synaptic modulation was biphasic and consisted of an initial glutamate-mediated potentiation followed by a purinergic-mediated depression of neurotransmitter release. The temporal dynamic properties of this biphasic synaptic regulation depended on the firing frequency and duration of the neuronal activity that stimulated astrocytes. Present results indicate that single astrocytes can decode neuronal activity and, in response, release distinct gliotransmitters to differentially regulate neurotransmission at putative single synapses.

Synapse-Specific Regulation Revealed at Single Synapses Is Concealed When Recording Multiple Synapses.

Synaptic transmission and its activity-dependent modulation, known as synaptic plasticity, are fundamental processes in nervous system function. Neurons may receive thousands of synaptic contacts, but synaptic regulation may occur only at individual or discrete subsets of synapses, which may have important consequences on the spatial extension of the modulation of synaptic information. Moreover, while several electrophysiological methods are used to assess synaptic transmission at different levels of observation, i.e., through local field potential and individual whole-cell recordings, their experimental limitations to detect synapse-specific modulation is poorly defined. We have investigated how well-known synapse-specific short-term plasticity, where some synapses are regulated and others left unregulated, mediated by astrocytes and endocannabinoid (eCB) signaling can be assessed at different observational levels. Using hippocampal slices, we have combined local field potential and whole-cell recordings of CA3-CA1 synaptic activity evoked by Schaffer collateral stimulation of either multiple or single synapses through bulk or minimal stimulation, respectively, to test the ability to detect short-term synaptic changes induced by eCB signaling. We also developed a mathematical model assuming a bimodal distribution of regulated and unregulated synapses based on realistic experimental data to simulate physiological results and to predict the experimental requirements of the different recording methods to detect discrete changes in subsets of synapses. We show that eCB-induced depolarization-induced suppression of excitation (DSE) and astrocyte-mediated synaptic potentiation can be observed when monitoring single or few synapses, but are statistically concealed when recording the activity of a large number of synapses. These results indicate that the electrophysiological methodology is critical to properly assess synaptic changes occurring in subsets of synapses, and they suggest that relevant synapse-specific regulatory phenomena may be experimentally undetected but may have important implications in the spatial extension of synaptic plasticity phenomena.

Activity-dependent switch of GABAergic inhibition into glutamatergic excitation in astrocyte-neuron networks.

Interneurons are critical for proper neural network function and can activate Ca2+ signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, we found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and determined the sign (inhibition vs potentiation) of the GABA-mediated effects. While synaptic inhibition was mediated by GABAA receptors, potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1) knockout mice, we confirmed the glial source of the interneuron-induced potentiation, and demonstrated the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay.