Lactate as a determinant of neuronal excitability, neuroenergetics and beyond.

Over the last decades, lactate has emerged as important energy substrate for the brain fueling of neurons. A growing body of evidence now indicates that it is also a signaling molecule modulating neuronal excitability and activity as well as brain functions. In this review, we will briefly summarize how different cell types produce and release lactate. We will further describe different signaling mechanisms allowing lactate to fine-tune neuronal excitability and activity, and will finally discuss how these mechanisms could cooperate to modulate neuroenergetics and higher order brain functions both in physiological and pathological conditions.

Centrally expressed Cav3.2 T-type calcium channel is critical for the initiation and maintenance of neuropathic pain.

Cav3.2 T-type calcium channel is a major molecular actor of neuropathic pain in peripheral sensory neurons, but its involvement at the supraspinal level is almost unknown. In the anterior pretectum (APT), a hub of connectivity of the somatosensory system involved in pain perception, we show that Cav3.2 channels are expressed in a subpopulation of GABAergic neurons coexpressing parvalbumin (PV). In these PV-expressing neurons, Cav3.2 channels contribute to a high-frequency-bursting activity, which is increased in the spared nerve injury model of neuropathy. Specific deletion of Cav3.2 channels in APT neurons reduced both the initiation and maintenance of mechanical and cold allodynia. These data are a direct demonstration that centrally expressed Cav3.2 channels also play a fundamental role in pain pathophysiology.

Parvalbumin interneuron-derived tissue-type plasminogen activator shapes perineuronal net structure.

BACKGROUND: Perineuronal nets (PNNs) are specialized extracellular matrix structures mainly found around fast-spiking parvalbumin (FS-PV) interneurons. In the adult, their degradation alters FS-PV-driven functions, such as brain plasticity and memory, and altered PNN structures have been found in neurodevelopmental and central nervous system disorders such as Alzheimer's disease, leading to interest in identifying targets able to modify or participate in PNN metabolism. The serine protease tissue-type plasminogen activator (tPA) plays multifaceted roles in brain pathophysiology. However, its cellular expression profile in the brain remains unclear and a possible role in matrix plasticity through PNN remodeling has never been investigated. RESULT: By combining a GFP reporter approach, immunohistology, electrophysiology, and single-cell RT-PCR, we discovered that cortical FS-PV interneurons are a source of tPA in vivo. We found that mice specifically lacking tPA in FS-PV interneurons display denser PNNs in the somatosensory cortex, suggesting a role for tPA from FS-PV interneurons in PNN remodeling. In vitro analyses in primary cultures of mouse interneurons also showed that tPA converts plasminogen into active plasmin, which in turn, directly degrades aggrecan, a major structural chondroitin sulfate proteoglycan (CSPG) in PNNs. CONCLUSIONS: We demonstrate that tPA released from FS-PV interneurons in the central nervous system reduces PNN density through CSPG degradation. The discovery of this tPA-dependent PNN remodeling opens interesting insights into the control of brain plasticity.

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity.

Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

Regulation of perineuronal nets in the adult cortex by the activity of the cortical network.

Perineuronal net (PNN) accumulation around parvalbumin-expressing (PV) inhibitory interneurons marks the closure of critical periods of high plasticity, whereas PNN removal reinstates juvenile plasticity in the adult cortex. Using targeted chemogenetic in vivo approaches in the adult mouse visual cortex, we found that transient inhibition of PV interneurons, through metabotropic or ionotropic chemogenetic tools, induced PNN regression. Electroencephalographic recordings indicated that inhibition of PV interneurons did not elicit unbalanced network excitation. Likewise, inhibition of local excitatory neurons also induced PNN regression, whereas chemogenetic excitation of either PV or excitatory neurons did not reduce the PNN. We also observed that chemogenetically inhibited PV interneurons exhibited reduced PNN compared to their untransduced neighbors, and confirmed that single PV interneurons express multiple genes enabling individual regulation of their own PNN density. Our results indicate that PNN density is regulated in the adult cortex by local changes of network activity that can be triggered by modulation of PV interneurons. PNN regulation may provide adult cortical circuits with an activity-dependent mechanism to control their local remodeling.SIGNIFICANCE STATEMENTThe perineuronal net is an extracellular matrix, which accumulates around individual parvalbumin-expressing inhibitory neurons during postnatal development, and is seen as a barrier that prevents plasticity of neuronal circuits in the adult cerebral cortex. We found that transiently inhibiting parvalbumin-expressing or excitatory cortical neurons triggers a local decrease of perineuronal net density. Our results indicate that perineuronal nets are regulated in the adult cortex depending on the activity of local microcircuits. These findings uncover an activity-dependent mechanism by which adult cortical circuits may locally control their plasticity.

Astrocytes respond to a neurotoxic Aß fragment with state-dependent Ca2+ alteration and multiphasic transmitter release.

Excessive amounts of amyloid ß (Aß) peptide have been suggested to dysregulate synaptic transmission in Alzheimer's disease (AD). As a major type of glial cell in the mammalian brain, astrocytes regulate neuronal function and undergo activity alterations upon Aß exposure. Yet the mechanistic steps underlying astrocytic responses to Aß peptide remain to be elucidated. Here by fluorescence imaging of signaling pathways, we dissected astrocytic responses to Aß25-35 peptide, a neurotoxic Aß fragment present in AD patients. In native health astrocytes, Aß25-35 evoked Ca2+ elevations via purinergic receptors, being also dependent on the opening of connexin (CX) hemichannels. Aß25-35, however, induced a Ca2+ diminution in Aß-preconditioned astrocytes as a result of the potentiation of the plasma membrane Ca2+ ATPase (PMCA). The PMCA and CX protein expression was observed with immunostaining in the brain tissue of hAPPJ20 AD mouse model. We also observed both Ca2+-independent and Ca2+-dependent glutamate release upon astrocytic Aß exposure, with the former mediated by CX hemichannel and the latter by both anion channels and lysosome exocytosis. Our results suggest that Aß peptide causes state-dependent responses in astrocytes, in association with a multiphasic release of signaling molecules. This study therefore helps to understand astrocyte engagement in AD-related amyloidopathy.

Mapping astrocyte activity domains by light sheet imaging and spatio-temporal correlation screening.

Astrocytes are a major type of glial cell in the mammalian brain, essentially regulating neuronal development and function. Quantitative imaging represents an important approach to study astrocytic signaling in neural circuits. Focusing on astrocytic Ca2+ activity, a key pathway implicated in astrocye-neuron interaction, we here report a strategy combining fast light sheet fluorescence microscopy (LSFM) and correlative screening-based time series analysis, to map activity domains in astrocytes in living mammalian nerve tissue. Light sheet of micron-scale thickness enables wide-field optical sectioning to image astrocytes in acute mouse brain slices. Using both chemical and genetically encoded Ca2+ indicators, we demonstrate the complementary advantages of LSFM in mapping Ca2+ domains in astrocyte populations as compared to epifluorescence and two-photon microscopy. Our approach then revealed distinct kinetics of Ca2+ signals between cortical and hypothalamic astrocytes in resting conditions and following the activation of adrenergic G protein coupled receptor (GPCR). This observation highlights the activity heterogeneity across regionally distinct astrocyte populations, and indicates the potential of our method for investigating dynamic signals in astrocytes.

Impairment of Glycolysis-Derived l-Serine Production in Astrocytes Contributes to Cognitive Deficits in Alzheimer's Disease.

Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer's disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD.

Gene Expression Analysis by Multiplex Single-Cell RT-PCR.

Brain circuit assemblies comprise different cellular subpopulations that exhibit morphological, electrophysiological, and molecular diversity. Here we describe a protocol which, combined with whole-cell patch-clamp recording and morphological reconstruction, allows the transcriptomic analysis of the recorded cell. This protocol provides recipes on how to detect simultaneously the expression of 24 genes/markers at the single-cell level using polymerase chain reaction (PCR), how to design gene-specific probes, and how to validate them. This technique provides multiplexed expression data that cannot be easily obtained by other approaches such as immunological co-labeling.

Bioluminescence calcium imaging of network dynamics and their cholinergic modulation in slices of cerebral cortex from male rats.

The activity of neuronal ensembles was monitored in neocortical slices from male rats using wide-field bioluminescence imaging of a calcium sensor formed with the fusion of green fluorescent protein and aequorin (GA) and expressed through viral transfer. GA expression was restricted to pyramidal neurons and did not conspicuously alter neuronal morphology or neocortical cytoarchitecture. Removal of extracellular magnesium or addition of GABA receptor antagonists triggered epileptiform flashes of variable amplitude and spatial extent, indicating that the excitatory and inhibitory networks were functionally preserved in GA-expressing slices. We found that agonists of muscarinic acetylcholine receptors largely increased the peak bioluminescence response to local electrical stimulation in layer I or white matter, and gave rise to a slowly decaying response persisting for tens of seconds. The peak increase involved layers II/III and V and did not result in marked alteration of response spatial properties. The persistent response involved essentially layer V and followed the time course of the muscarinic afterdischarge depolarizing plateau in layer V pyramidal cells. This plateau potential triggered spike firing in layer V, but not layer II/III pyramidal cells, and was accompanied by recurrent synaptic excitation in layer V. Our results indicate that wide-field imaging of GA bioluminescence is well suited to monitor local and global network activity patterns, involving different mechanisms of intracellular calcium increase, and occurring on various timescales.

Excitation of Cortical nNOS/NK1R Neurons by Hypocretin 1 is Independent of Sleep Homeostasis.

We have proposed that cortical nNOS/NK1R interneurons have a role in sleep homeostasis. The hypocretins (orexins) are wake-promoting neuropeptides and hypocretin/orexin (Hcrt) neurons project to the cortex. Hcrt peptides affect deep layer cortical neurons, and Hcrt receptor 1 (Hcrtr1; Ox1r) mRNA is expressed in cortical nNOS/NK1R cells. Therefore, we investigated whether Hcrt neuron stimulation affects cingulate cortex nNOS/NK1R neurons. Bath application of HCRT1/orexin-A evoked an inward current and membrane depolarization in most nNOS/NK1R cells which persisted in tetrodotoxin; optogenetic stimulation of Hcrt terminals expressing channelrhodopsin-2 confirmed these results, and pharmacological studies determined that HCRTR1 mediated these responses. Single-cell RT-PCR found Hcrtr1 mRNA in 31% of nNOS/NK1R cells without any Hcrtr2 mRNA expression; immunohistochemical studies of Hcrtr1-EGFP mice confirmed that a minority of nNOS/NK1R cells express HCRTR1. When Hcrt neurons degenerated in orexin-tTA;TetO DTA mice, the increased EEG delta power during NREM sleep produced in response to 4 h sleep deprivation and c-FOS expression in cortical nNOS/NK1R cells during recovery sleep were indistinguishable from that of controls. We conclude that Hcrt excitatory input to these deep layer cells is mediated through HCRTR1 but is unlikely to be involved in the putative role of cortical nNOS/NK1R neurons in sleep homeostasis.

Supragranular Pyramidal Cells Exhibit Early Metabolic Alterations in the 3xTg-AD Mouse Model of Alzheimer's Disease.

The impairment of cerebral glucose utilization is an early and predictive biomarker of Alzheimer's disease (AD) that is likely to contribute to memory and cognition disorders during the progression of the pathology. Yet, the cellular and molecular mechanisms underlying these metabolic alterations remain poorly understood. Here we studied the glucose metabolism of supragranular pyramidal cells at an early presymptomatic developmental stage in non-transgenic (non-Tg) and 3xTg-AD mice, a mouse model of AD replicating numerous hallmarks of the disease. We performed both intracellular glucose imaging with a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose biosensor and transcriptomic profiling of key molecular elements of glucose metabolism with single-cell multiplex RT-PCR (scRT-mPCR). We found that juvenile pyramidal cells exhibit active glycolysis and pentose phosphate pathway at rest that are respectively enhanced and impaired in 3xTg-AD mice without alteration of neuronal glucose uptake or transcriptional modification. Given the importance of glucose metabolism for neuronal survival, these early alterations could initiate or at least contribute to the later neuronal dysfunction of pyramidal cells in AD.

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

Brain Perfusion and Astrocytes.

How can blood rapidly and precisely reach active neurons at a given time and location has remained enigmatic for a long time. A 2003 paper by Zonta et al. suggested key roles for astrocytes in the signaling between neurons and blood vessels. While a consensus on the specific intermediary roles of astrocytes in this process is still evolving, research in the past 15 years has led to a deeper and more refined understanding of the neuro-glio-vascular unit.

Cortical nNOS/NK1 Receptor Neurons are Regulated by Cholinergic Projections From the Basal Forebrain.

Cholinergic (ACh) basal forebrain (BF) neurons are active during wakefulness and rapid eye movement (REM) sleep and are involved in sleep homeostasis. We have previously shown in adult animals that cortical neurons that express neuronal nitric oxide synthase (nNOS) and the receptor for Substance P (NK1R) are activated during non-REM (NREM) sleep in proportion to homeostatic sleep drive. Here, we show that BF neurons modulate cortical nNOS/NK1R cells. In vitro optogenetic stimulation of BF terminals both activated and inhibited nNOS/NK1R neurons. Pharmacological studies revealed cholinergic responses mediated by postsynaptic activation of muscarinic receptors (mAChRs; M3R > M2/4R > M1R) and that presynaptic M3R and M2R activation reduced glutamatergic input onto nNOS/NK1R neurons whereas nicotinic receptor (nAChR)-mediated responses of nNOS/NK1R neurons were mixed. Cholinergic responses of nNOS/NK1R neurons were largely unaffected by prolonged wakefulness. ACh release, including from BF cells, appears to largely excite cortical nNOS/NK1R cells while reducing glutamatergic inputs onto these neurons. We propose that cholinergic signaling onto cortical nNOS/NK1R neurons may contribute to the regulation of cortical activity across arousal states, but that this response is likely independent of the role of these neurons in sleep homeostasis.

Cyclooxygenase-2-Derived Prostaglandins Mediate Cerebral Microcirculation in a Juvenile Ischemic Rat Model.

BACKGROUND AND PURPOSE: We previously showed that the selective neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) increases cerebral microcirculation in a juvenile ischemic rat model. We address the roles of cyclooxygenase (COX)-elaborated prostaglandins in collateral recruitment and blood supply. METHODS: Fourteen-day-old rats were subjected to ischemia-reperfusion and treated with either PBS or 7-NI (25 mg/kg) at the reperfusion onset. Six-keto-prostaglandin F1α was measured using ELISA. COX-1 and COX-2 and prostaglandin terminal synthesizing enzymes were evaluated using reverse-transcriptase polymerase chain reaction and immunofluorescence. Microvascular blood flow indexes (artery diameter and capillaries number) were measured using sidestream dark-field videomicroscopy in PBS- and 7-NI-treated ischemic rats in the absence or presence of the COX-2 inhibitor NS-398 (5 mg/kg). Cell death was measured with the TUNEL (terminal transferase dUTP nick end labeling) assay and cleaved-caspase-3 immunostaining. RESULTS: Six-keto-prostaglandin F1α and COX-2, associated with a prostaglandin E synthase, were significantly increased in PBS- and 7-NI-treated animals 15 minutes and 1 hour after ischemia-reperfusion, respectively. In contrast and as compared with PBS, 7-NI significantly decreased prostacyclin synthase and cytosolic prostaglandins E synthase mRNA. Selective COX-2 inhibition significantly decreased blood flow indexes and significantly reversed the effects of 7-NI, including the number of TUNEL+- and cleaved-caspase-3+-nuclei. CONCLUSIONS: These results show that the juvenile rat brains mostly respond to ischemia by a COX-2-dependent prostaglandins production and suggest that the transcriptional responses observed under 7-NI facilitate and reorient COX-2-dependent prostaglandins production.

Comment on "Principles of connectivity among morphologically defined cell types in adult neocortex".

Jiang et al (Research Article, 27 November 2015, aac9462) describe detailed experiments that substantially add to the knowledge of cortical microcircuitry and are unique in the number of connections reported and the quality of interneuron reconstruction. The work appeals to experts and laypersons because of the notion that it unveils new principles and provides a complete description of cortical circuits. We provide a counterbalance to the authors' claims to give those less familiar with the minutiae of cortical circuits a better sense of the contributions and the limitations of this study.

Multiscale single-cell analysis reveals unique phenotypes of raphe 5-HT neurons projecting to the forebrain.

Serotonergic neurons of the raphe nuclei exhibit anatomical, neurochemical and elecrophysiological heterogeneity that likely underpins their specific role in multiple behaviors. However, the precise organization of serotonin (5-HT) neurons to orchestrate 5-HT release patterns throughout the brain is not well understood. We compared the electrophysiological and neurochemical properties of dorsal and median raphe 5-HT neurons projecting to the medial prefrontal cortex (mPFC), amygdala (BLA) and dorsal hippocampus (dHP), combining retrograde tract tracing with brain slice electrophysiology and single-cell RT-PCR in Pet1-EGFP mice. Our results show that 5-HT neurons projecting to the dHP and the mPFC and the BLA form largely non-overlapping populations and that BLA-projecting neurons have characteristic excitability and membrane properties. In addition, using an unbiased clustering method that correlates anatomical, molecular and electrophysiological phenotypes, we find that 5-HT neurons with projections to the mPFC and the dHP segregate from those projecting to the BLA. Single-cell gene profiling showed a restricted expression of the peptide galanin in the population of 5-HT neurons projecting to the mPFC. Finally, cluster analysis allowed identifying an atypical subtype of 5-HT neuron with low excitability, long firing delays and preferential expression of the vesicular glutamate transporter type 3. Overall, these findings allow to define correlated anatomical and physiological identities of serotonin raphe neurons that help understanding how discrete raphe cells subpopulations account for the heterogeneous activities of the midbrain serotonergic system.

Tissue Plasminogen Activator Expression Is Restricted to Subsets of Excitatory Pyramidal Glutamatergic Neurons.

Although the extracellular serine protease tissue plasminogen activator (tPA) is involved in pathophysiological processes such as learning and memory, anxiety, epilepsy, stroke, and Alzheimer's disease, information about its regional, cellular, and subcellular distribution in vivo is lacking. In the present study, we observed, in healthy mice and rats, the presence of tPA in endothelial cells, oligodendrocytes, mastocytes, and ependymocytes, but not in pericytes, microglial cells, and astrocytes. Moreover, blockage of the axo-dendritic transport unmasked tPA expression in neurons of cortical and hippocampal areas. Interestingly, combined electrophysiological recordings, single-cell reverse transcription polymerase chain reaction (RT-PCR), and immunohistological analyses revealed that the presence of tPA is restricted to subsets of excitatory pyramidal glutamatergic neurons. We further evidenced that tPA is stored in synaptobrevin-2-positive glutamatergic synaptic vesicles. Based on all these data, we propose the existence of tPA-ergic neurons in the mature brain.

COX-2-Derived Prostaglandin E2 Produced by Pyramidal Neurons Contributes to Neurovascular Coupling in the Rodent Cerebral Cortex.

Vasodilatory prostaglandins play a key role in neurovascular coupling (NVC), the tight link between neuronal activity and local cerebral blood flow, but their precise identity, cellular origin and the receptors involved remain unclear. Here we show in rats that NMDA-induced vasodilation and hemodynamic responses evoked by whisker stimulation involve cyclooxygenase-2 (COX-2) activity and activation of the prostaglandin E2 (PgE2) receptors EP2 and EP4. Using liquid chromatography-electrospray ionization-tandem mass spectrometry, we demonstrate that PgE2 is released by NMDA in cortical slices. The characterization of PgE2 producing cells by immunohistochemistry and single-cell reverse transcriptase-PCR revealed that pyramidal cells and not astrocytes are the main cell type equipped for PgE2 synthesis, one third expressing COX-2 systematically associated with a PgE2 synthase. Consistent with their central role in NVC, in vivo optogenetic stimulation of pyramidal cells evoked COX-2-dependent hyperemic responses in mice. These observations identify PgE2 as the main prostaglandin mediating sensory-evoked NVC, pyramidal cells as their principal source and vasodilatory EP2 and EP4 receptors as their targets. SIGNIFICANCE STATEMENT: Brain function critically depends on a permanent spatiotemporal match between neuronal activity and blood supply, known as NVC. In the cerebral cortex, prostaglandins are major contributors to NVC. However, their biochemical identity remains elusive and their cellular origins are still under debate. Although astrocytes can induce vasodilations through the release of prostaglandins, the recruitment of this pathway during sensory stimulation is questioned. Using multidisciplinary approaches from single-cell reverse transcriptase-PCR, mass spectrometry, to ex vivo and in vivo pharmacology and optogenetics, we provide compelling evidence identifying PgE2 as the main prostaglandin in NVC, pyramidal neurons as their main cellular source and the vasodilatory EP2 and EP4 receptors as their main targets. These original findings will certainly change the current view of NVC.