Frequency Of Spontaneous Postsynaptic Currents Research Articles

Shank3 deficiency alters midbrain GABAergic neuron morphology, GABAergic markers and synaptic activity in primary striatal neurons

Abnormalities in gamma-aminobutyric acid (GABA)ergic neurotransmission play a role in the pathogenesis of autism, although the mechanisms responsible for alterations in specific brain regions remain unclear. Deficits in social motivation and interactions are core symptoms of autism, likely due to defects in dopaminergic neural pathways. Therefore, investigating the morphology and functional roles of GABAergic neurons within dopaminergic projection areas could elucidate the underlying etiology of autism. The aim of this study was to (1) compare the morphology and arborization of glutamate decarboxylase (GAD)-positive neurons from the midbrain tegmentum; (2) evaluate synaptic activity in primary neurons from the striatum; and (3) assess GABAergic postsynaptic puncta in the ventral striatum of wild-type (WT) and Shank3-deficient mice. We found a significant decrease in the number of short neurites in GAD positive primary neurons from the midbrain tegmentum in Shank3-deficient mice. The application of a specific blocker of GABAA receptors (GABAAR) revealed significantly increased frequency of spontaneous postsynaptic currents (sPSCs) in Shank3-deficient striatal neurons compared to their WT counterparts. The mean absolute amplitude of the events was significantly higher in striatal neurons from Shank3-deficient compared to WT mice. We also observed a significant reduction in gephyrin/GABAAR γ2 colocalization in the striatum of adult male Shank3-deficient mice. The gene expression of collybistin was significantly lower in the nucleus accumbens while gephyrin and GABAAR γ2 were lower in the ventral tegmental area (VTA) in male Shank3-deficient compared to WT mice. In conclusion, Shank3 deficiency leads to alterations in GABAergic neurons and impaired GABAergic function in dopaminergic brain areas. These changes may underlie autistic symptoms, and potential interventions modulating GABAergic activity in dopaminergic pathways may represent new treatment modality.

Astrocytes Are Involved in the Effects of Ketamine on Synaptic Transmission in Rat Primary Somatosensory Cortex.

Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is widely used as a general anaesthetic. However, the mechanisms of analgesic/anaesthetic effects induced by ketamine are only partially understood. Previously, studies have demonstrated that various general anaesthetics affect the primary somatosensory cortex (S1), a potential target of general anaesthetics in the central nervous system. However, it is unknown if astrocyte activities affect ketamine's effects on information transmission in S1 pyramidal neurons. The whole-cell patch-clamp technique was employed to study the role of astrocytes in ketamine-induced anaesthetic actions. The whole-cell patch-clamp method was used to record the spontaneous postsynaptic currents (SPSCs) of rat S1 pyramidal neurons. We used the glia-selective inhibitor of the aconitase enzyme fluorocitrate (FC), to test if astrocyte activities alter the effects of ketamine on S1 pyramidal neurons. Ketamine lowered the SPSCs of rat S1 pyramidal neurons in a concentration-dependent manner at clinically relevant doses. The concentration-effect curve revealed that ketamine had an EC50 value of 462.1 M for suppressing SPSCs. In rat S1 pyramidal neurons, the glia-selective metabolic inhibitor fluorocitrate (FC), which inhibits the aconitase enzyme, lowered the amplitude and frequency of SPSCs. The inhibitory impact of ketamine on the amplitude and frequency of SPSCs was significantly amplified in the presence of FC. Astrocytes impact the effects of ketamine on pre- and postsynaptic components and play a role in synaptic transmission.

5-HT7 Receptors Regulate Excitatory-Inhibitory Balance in Mouse Spinal Cord Dorsal Horn.

Serotonergic receptors of the 5-HT7 type (5-HT7Rs) are widely expressed in the central nervous system (CNS), where they modulate several functions, such as pain. Behavioral experiments in vivo have shown both anti- and pro-nociceptive actions of 5-HT7Rs, although an analgesic effect seems to be prevalent. In the spinal cord dorsal horn, the mechanisms involved in 5-HT7R-mediated synaptic modulation are still poorly understood, especially those regarding the control of synaptic inhibition. The present study investigated the modulation exerted by 5-HT7Rs on dorsal horn excitatory and inhibitory synaptic circuits, by performing patch-clamp recordings from lamina II neurons in mouse spinal cord slices. Our results show that applying the selective 5-HT7 agonist LP-211 facilitates glutamatergic release by enhancing the frequency of spontaneous postsynaptic currents (sEPSCs) and increasing the peak amplitude of excitatory postsynaptic currents (EPSCs) evoked by dorsal root stimulation. The effects on sEPSCs were still observed in the presence of the 5-HT1A antagonist WAY-100635, while the 5-HT7 antagonist SB-269970 blocked them. LP-211 was also able to increase the release of gamma-aminobutyric acid (GABA) and glycine, as shown by the increase of spontaneous inhibitory currents (sIPSC) frequency and evoked inhibitory postsynaptic currents (IPSC) amplitude. LP-211 was proved to be more effective in potentiating synaptic inhibition as compared to excitation: consistently, 5-HT7R activation significantly enhanced the excitability of tonic firing neurons, mainly corresponding to inhibitory interneurons. Our data bring new insights into the mechanisms of synaptic modulation mediated by 5-HT7Rs in the dorsal horn. Stronger impact on synaptic inhibition supports the hypothesis that these receptors may play an anti-nociceptive role in the spinal cord of naïve animals.

Insulin-Like Growth Factor 1 Increases GABAergic Neurotransmission to GnRH Neurons via Suppressing the Retrograde Tonic Endocannabinoid Signaling Pathway in Mice

Introduction: Hypophysiotropic gonadotropin-releasing hormone (GnRH) neurons orchestrate various physiological events that control the onset of puberty. Previous studies showed that insulin-like growth factor 1 (IGF-1) induces the secretion of GnRH and accelerates the onset of puberty, suggesting a regulatory role of this hormone upon GnRH neurons. Methods: To reveal responsiveness of GnRH neurons to IGF-1 and elucidate molecular pathways acting downstream to the IGF-1 receptor (IGF-1R), in vitro electrophysiological experiments were carried out on GnRH-GFP neurons in acute brain slices from prepubertal (23–29 days) and pubertal (50 days) male mice. Results: Administration of IGF-1 (13 nM) significantly increased the firing rate and frequency of spontaneous postsynaptic currents and that of excitatory GABAergic miniature postsynaptic currents (mPSCs). No GABAergic mPSCs were induced by IGF-1 in the presence of the GABA_A-R blocker picrotoxin. The increase in the mPSC frequency was prevented by the use of the IGF-1R antagonist, JB1 (1 µM), or the intracellularly applied PI3K blocker (LY294002, 50 µM), showing involvement of IGF-1R and PI3K in the mechanism. Blockade of the transient receptor potential vanilloid 1, an element of the tonic retrograde endocannabinoid machinery, by AMG9810 (10 µM) or antagonizing the cannabinoid receptor type-1 by AM251 (1 µM) abolished the effect. Discussion/Conclusion: These findings indicate that IGF-1 arrests the tonic retrograde endocannabinoid pathway in GnRH neurons, and this disinhibition increases the release of GABA from presynaptic terminals that, in turn, activates GnRH neurons leading to the fine-tuning of the hypothalamo-pituitary-gonadal axis.

Disruption of genes associated with Charcot-Marie-Tooth type 2 lead to common behavioural, cellular and molecular defects in Caenorhabditis elegans.

Charcot-Marie-Tooth (CMT) disease is an inherited peripheral motor and sensory neuropathy. The disease is divided into demyelinating (CMT1) and axonal (CMT2) neuropathies, and although we have gained molecular information into the details of CMT1 pathology, much less is known about CMT2. Due to its clinical and genetic heterogeneity, coupled with a lack of animal models, common underlying mechanisms remain elusive. In order to gain an understanding of the normal function of genes associated with CMT2, and to draw direct comparisons between them, we have studied the behavioural, cellular and molecular consequences of mutating nine different genes in the nematode Caenorhabditis elegans (lin-41/TRIM2, dyn-1/DNM2, unc-116/KIF5A, fzo-1/MFN2, osm-9/TRPV4, cua-1/ATP7A, hsp-25/HSPB1, hint-1/HINT1, nep-2/MME). We show that C. elegans defective for these genes display debilitated movement in crawling and swimming assays. Severe morphological defects in cholinergic motors neurons are also evident in two of the mutants (dyn-1 and unc-116). Furthermore, we establish methods for quantifying muscle morphology and use these to demonstrate that loss of muscle structure occurs in the majority of mutants studied. Finally, using electrophysiological recordings of neuromuscular junction (NMJ) activity, we uncover reductions in spontaneous postsynaptic current frequency in lin-41, dyn-1, unc-116 and fzo-1 mutants. By comparing the consequences of mutating numerous CMT2-related genes, this study reveals common deficits in muscle structure and function, as well as NMJ signalling when these genes are disrupted.

GABAA Receptors are Selectively Expressed in NG2 Glia of the Cerebellar White Matter

The cerebellum is involved in the coordination of movement. Its cellular composition is dominated by GABAergic neuronal types, and glial cells are known to express functional receptors. GABAergic signaling regulates cell proliferation, differentiation, and migration during neurodevelopment. However, little is known about the functional expression of GABA receptors in the cerebellar white matter (WM). Thus, the aim of this study was to test whether glial cells express functional GABA receptors during postnatal development (P7–P9) of cerebellar WM. Immunofluorescence showed that half of the astrocytes express GAD67, suggesting that glial cells synthesize GABA. Calcium imaging in cerebellar slices revealed that GABA and the GABAA agonist muscimol evoked calcium transients in sulforhodamine B negative cells, whereas the GABAB agonist baclofen failed to evoke responses in cerebellar WM. Whole-cell patch-clamp recordings of GFAP+ cells showed dye coupling and a passive current–voltage relation typical of astrocytes. Surprisingly, these cells did not respond to muscimol. Two additional populations were identified as GFAP− cells. The first population showed dye coupling, slow decaying inward and outward currents with no voltage dependence, and did not respond to GABAA agonists. The second population showed an outward-rectifying current–voltage relationship and responded to muscimol, but dye coupling was absent. These cells received synaptic input and were NG2+, but evoked calcium waves failed to modulate the frequency of spontaneous postsynaptic currents (sPSCs) or signaling into NG2 glia. We conclude that GABAA receptor-mediated signaling is selective for NG2 glia in the WM of the cerebellum.

Astrocytic rather than neuronal P2X7 receptors modulate the function of the tri-synaptic network in the rodent hippocampus

Whole-cell patch clamp recordings demonstrated that in the dentate gyrus (DG) as well as in the CA3 area of mouse hippocampal slices the prototypic P2X7 receptor (R) agonist dibenzoyl-ATP (Bz-ATP) induced inward current responses both in neurons and astrocytes. Whereas the selective P2X7R antagonist A438079 strongly inhibited both neuronal and astrocytic currents, a combination of ionotropic glutamate receptor (CNQX, AP-5) and GABAA-R (gabazine) antagonists depressed the Bz-ATP-induced current responses in the DG (granule cells) and CA3 neurons only. It was concluded that Bz-ATP activated astrocytic P2X7Rs and thereby released glutamate and GABA to stimulate nearby neurons. The residual A438079-resistant current response of astrocytes was suggested to be due to the stimulation of P2XRs of the non-P2X7-type. Further, we searched for presynaptic P2X7Rs at the axon terminals of DG and CA3 pyramidal neurons innervating CA3 and CA1 cells, respectively. Bz-ATP potentiated the frequency of spontaneous postsynaptic currents (sPSCs) in CA1 but not CA3 pyramidal cells. However, the Bz-ATP effect in CA1 cells was inhibited by gabazine or the astrocytic toxin fluorocitrate suggesting stimulation of P2X7Rs at stratum radiatum astrocytes located near to interneurons and synapsing onto CA1 neurons. Our data suggest that functional P2X7Rs are missing at neurons in the tri-synaptic network of the rodent hippocampus, but are present at nearby astrocytes indirectly regulating network activity.

Selective Targeting of Neurons with Inorganic Nanoparticles: Revealing the Crucial Role of Nanoparticle Surface Charge.

Nanoparticles (NPs) are increasingly used in biomedical applications, but the factors that influence their interactions with living cells need to be elucidated. Here, we reveal the role of NP surface charge in determining their neuronal interactions and electrical responses. We discovered that negatively charged NPs administered at low concentration (10 nM) interact with the neuronal membrane and at the synaptic cleft, whereas positively and neutrally charged NPs never localize on neurons. This effect is shape and material independent. The presence of negatively charged NPs on neuronal cell membranes influences the excitability of neurons by causing an increase in the amplitude and frequency of spontaneous postsynaptic currents at the single cell level and an increase of both the spiking activity and synchronous firing at neural network level. The negatively charged NPs exclusively bind to excitable neuronal cells, and never to nonexcitable glial cells. This specific interaction was also confirmed by manipulating the electrophysiological activity of neuronal cells. Indeed, the interaction of negatively charged NPs with neurons is either promoted or hindered by pharmacological suppression or enhancement of the neuronal activity with tetrodotoxin or bicuculline, respectively. We further support our main experimental conclusions by using numerical simulations. This study demonstrates that negatively charged NPs modulate the excitability of neurons, revealing the potential use of NPs for controlling neuron activity.

Sudden death due to paralysis and synaptic and behavioral deficits when Hip14/Zdhhc17 is deleted in adult mice.

BackgroundPalmitoylation, the addition of palmitate to proteins by palmitoyl acyltransferases (PATs), is an important regulator of synaptic protein localization and function. Many palmitoylated proteins and PATs have been implicated in neuropsychiatric diseases, including Huntington disease, schizophrenia, amyotrophic lateral sclerosis, Alzheimer disease, and X-linked intellectual disability. HIP14/DHHC17 is the most conserved PAT that palmitoylates many synaptic proteins. Hip14 hypomorphic mice have behavioral and synaptic deficits. However, the phenotype is developmental; thus, a model of post-developmental loss of Hip14 was generated to examine the role of HIP14 in synaptic function in the adult.ResultsTen weeks after Hip14 deletion (iHip14 Δ/Δ), mice die suddenly from rapidly progressive paralysis. Prior to death the mice exhibit motor deficits, increased escape response during tests of anxiety, anhedonia, a symptom indicative of depressive-like behavior, and striatal synaptic deficits, including reduced probability of transmitter release and increased amplitude but decreased frequency of spontaneous post-synaptic currents. The mice also have increased brain weight due to microgliosis and astrogliosis in the cortex.ConclusionsBehavioral changes and electrophysiological measures suggest striatal dysfunction in iHip14 Δ/Δ mice, and increased cortical volume due to astrogliosis and microgliosis suggests a novel role for HIP14 in glia. These data suggest that HIP14 is essential for maintenance of life and neuronal integrity in the adult mouse.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0333-7) contains supplementary material, which is available to authorized users.

Estrogen Receptor Beta and 2-arachidonoylglycerol Mediate the Suppressive Effects of Estradiol on Frequency of Postsynaptic Currents in Gonadotropin-Releasing Hormone Neurons of Metestrous Mice: An Acute Slice Electrophysiological Study.

Gonadotropin-releasing hormone (GnRH) neurons are controlled by 17β-estradiol (E2) contributing to the steroid feedback regulation of the reproductive axis. In rodents, E2 exerts a negative feedback effect upon GnRH neurons throughout the estrus-diestrus phase of the ovarian cycle. The present study was undertaken to reveal the role of estrogen receptor subtypes in the mediation of the E2 signal and elucidate the downstream molecular machinery of suppression. The effect of E2 administration at low physiological concentration (10 pM) on GnRH neurons in acute brain slices obtained from metestrous GnRH-green fluorescent protein (GFP) mice was studied under paradigms of blocking or activating estrogen receptor subtypes and interfering with retrograde 2-arachidonoylglycerol (2-AG) signaling. Whole-cell patch clamp recordings revealed that E2 significantly diminished the frequency of spontaneous postsynaptic currents (sPSCs) in GnRH neurons (49.62 ± 7.6%) which effect was abolished by application of the estrogen receptor (ER) α/β blocker Faslodex (1 μM). Pretreatment of the brain slices with cannabinoid receptor type 1 (CB1) inverse agonist AM251 (1 μM) and intracellularly applied endocannabinoid synthesis blocker THL (10 μM) significantly attenuated the effect of E2 on the sPSCs. E2 remained effective in the presence of tetrodotoxin (TTX) indicating a direct action of E2 on GnRH cells. The ERβ specific agonist DPN (10 pM) also significantly decreased the frequency of miniature postsynaptic currents (mPSCs) in GnRH neurons. In addition, the suppressive effect of E2 was completely blocked by the selective ERβ antagonist PHTPP (1 μM) indicating that ERβ is required for the observed rapid effect of the E2. In contrast, the ERα agonist PPT (10 pM) or the membrane-associated G protein-coupled estrogen receptor (GPR30) agonist G1 (10 pM) had no significant effect on the frequency of mPSCs in these neurons. AM251 and tetrahydrolipstatin (THL) significantly abolished the effect of E2 whereas AM251 eliminated the action of DPN on the mPSCs. These data suggest the involvement of the retrograde endocannabinoid mechanism in the rapid direct effect of E2. These results collectively indicate that estrogen receptor beta and 2-AG/CB1 signaling mechanisms are coupled and play an important role in the mediation of the negative estradiol feedback on GnRH neurons in acute slice preparation obtained from intact, metestrous mice.

Potentiation of Acetylcholine-Mediated Facilitation of Inhibitory Synaptic Transmission by an Azaindolizione Derivative, ZSET1446 (ST101), in the Rat Hippocampus.

The integrity of the hippocampal network depends on the coordination of excitatory and inhibitory signaling, which are under dynamic control by various regulatory influences such as the cholinergic systems. ZSET1446 (ST101; spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one) is a newly synthesized azaindolizinone derivative that significantly improves learning deficits in various types of Alzheimer disease (AD) models in rats. We examined the effect of ZSET1446 on the nicotinic acetylcholine (ACh) receptor (nAChR)-mediated regulation of synaptic transmission in hippocampal slices of rats. ZSET1446 significantly potentiated the facilitatory effect of nicotine and ACh on the frequency of spontaneous postsynaptic currents (sPSCs) recorded in CA1 pyramidal neurons with a maximum effect at 100 pM (tested range, 10 pM-1000 pM). The basal sPSC frequency without ACh was not affected. Such potentiation by ZSET1446 was observed in both the pharmacologic isolations of inhibitory and excitatory sPSCs and markedly reduced by blockade of either α7 or α4β2 nAChRs. ZSET1446 did not affect ACh-activated inward currents or depolarization of interneurons in the stratum radiatum and the lacunosum moleculare. These results indicate that ZSET1446 potentiates the nicotine-mediated enhancement of synaptic transmission in the hippocampal neurons without affecting nAChRs themselves, providing a novel possible mechanism of procognitive action that might improve learning deficits in clinical therapy.

Awake, but not hyperactive

Neurons that control wakefulness release both stimulating and inhibiting signals to prevent hyperactivity.

Phorbol Ester Modulation of Ca2+ Channels Mediates Nociceptive Transmission in Dorsal Horn Neurones

Phorbol esters are analogues of diacylglycerol which activate C1 domain proteins, such as protein kinase C (PKC). Phorbol ester/PKC pathways have been proposed as potential therapeutic targets for chronic pain states, potentially by phosphorylating proteins involved in nociception, such as voltage-dependent Ca2+ channels (VDCCs). In this brief report, we investigate the potential involvement of CaV2 VDCC subtypes in functional effects of the phorbol ester, phorbol 12-myristate 13-acetate (PMA) on nociceptive transmission in the spinal cord. Effects of PMA and of selective pharmacological blockers of CaV2 VDCC subtypes on nociceptive transmission at laminae II dorsal horn neurones were examined in mouse spinal cord slices. Experiments were extended to CaV2.3(−/−) mice to complement pharmacological studies. PMA increased the mean frequency of spontaneous postsynaptic currents (sPSCs) in dorsal horn neurones, without an effect on event amplitude or half-width. sPSC frequency was reduced by selective VDCC blockers, ω-agatoxin-IVA (AgTX; CaV2.1), ω-conotoxin-GVIA (CTX; CaV2.2) or SNX-482 (CaV2.3). PMA effects were attenuated in the presence of each VDCC blocker and, also, in CaV2.3(−/−) mice. These initial data demonstrate that PMA increases nociceptive transmission at dorsal horn neurones via actions on different CaV2 subtypes suggesting potential anti-nociceptive targets in this system.

Voltage-Gated Calcium Channels in Neurons Derived from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Voltage-gated calcium channels couple pre-synaptic action potentials to transmitter release in neurons. In this study, we characterized voltage-activated Ca2+ channels expressed in neurons differentiated from human embryonic stem cell (hESC) or induced pluripotent stem cell (iPSC)-derived long-term self-renewing neuroepithelial-like stem cells (lt-NES) at one to three months of differentiation in vitro. Using barium (Ba2+) as the charge carrier, activation and inactivation of voltage-dependent calcium channels were investigated by the whole-cell configuration of the patch-clamp technique. In lt-NES-derived neurons, we found both non-inactivating high-voltage-activated (HVA) and inactivating low-voltage-activated (LVA) Ca2+ currents. Pharmacological characterization of high-threshold Ca2+ currents was attempted using nifedipine (10 μM ), ω-conotoxin-GVIA (ω-CgTx GVIA, 3μM) and ω-agatoxin-IVA (ω-Aga IVA, 200 nM), which block L, N, and P/Q-type Ca2+ currents, respectively. By analysing the averaged current in response to a square depolarizing voltage step protocol from a holding potential of −110 mV to 0 mV in the presence of these specific blockers, we could show that lt-NES cell-derived neurons express N and P/Q-type high-threshold Ca2+ channels. We also observed Ba2+ currents activated at voltages below −40 mV that were sensitive to NiCl2 (100 μM), indicating that T-type Ca2+ channels also contribute to Ca2+ currents in these neurons. Furthermore, the pharmacology of calcium channels involved in synaptic transmission was analyzed with specific agonists and antagonists of different high-voltage-activated calcium channels. Bath application of ω-CgTx GVIA (3μM) or ω-Aga IVA (200 nM) reduced the frequency of spontaneous postsynaptic currents (sPSCs). Thus, neurons differentiated from lt-NES express both high-threshold and low-threshold voltage-gated Ca2+ channels, and both N and P/Q-type Ca2+ channels mediate synaptic transmission in these neurons. Correction et al.Biophysical JournalApril 16, 2013In Brief2013. Voltage-Gated Calcium Channels Exist and Function as Fragment-Channels in Neuronal Plasma Membranes. Ioannis E. Michailidis, Kathryn Abele, Wei K. Zhang, Lawrence Geyman, Bochao Lin, Yong Yu, Eftychios Pnevmatikakis, Jian Yang. Biophys. J. 104(2)s: 462a. Full-Text PDF Open Archive

Cannabinoids modulate spontaneous synaptic activity in retinal ganglion cells

The endocannabinoid (ECB) system has been found throughout the central nervous system and modulates cell excitability in various forms of short-term plasticity. ECBs and their receptors have also been localized to all retinal cells, and cannabinoid receptor activation has been shown to alter voltage-dependent conductances in several different retinal cell types, suggesting a possible role for cannabinoids in retinal processing. Their effects on synaptic transmission in the mammalian retina, however, have not been previously investigated. Here, we show that exogenous cannabinoids alter spontaneous synaptic transmission onto retinal ganglion cells (RGCs). Using whole-cell voltage-clamp recordings in whole-mount retinas, we measured spontaneous postsynaptic currents (SPSCs) in RGCs in adult and young (P14-P21) mice. We found that the addition of an exogenous cannabinoid agonist, WIN55212-2 (5 μM), caused a significant reversible reduction in the frequency of SPSCs. This change, however, did not alter the kinetics of the SPSCs, indicating a presynaptic locus of action. Using blockers to isolate inhibitory or excitatory currents, we found that cannabinoids significantly reduced the release probability of both GABA and glutamate, respectively. While the addition of cannabinoids reduced the frequency of both GABAergic and glutamatergic SPSCs in both young and adult mice, we found that the largest effect was on GABA-mediated currents in young mice. These results suggest that the ECB system may potentially be involved in the modulation of signal transmission in the retina. Furthermore, they suggest that it might play a role in the developmental maturation of synaptic circuits, and that exogenous cannabinoids are likely able to disrupt retinal processing and consequently alter vision.

Astrocytes control GABAergic inhibition of neurons in the mouse barrel cortex

Astrocytes in the barrel cortex respond with a transient Ca2+ increase to neuronal stimulation and this response is restricted to the stimulated barrel field. In the present study we suppressed the astrocyte response by dialysing these cells with the Ca2+ chelator BAPTA. Electrical stimulation triggered a depolarization in stellate or pyramidal ‘regular spiking' neurons from cortex layer 4 and 2/3 and this response was augmented in amplitude and duration after astrocytes were dialysed with BAPTA. Combined blockade of GABAA and GABAB receptors mimicked the effect of BAPTA dialysis, while glutamate receptor blockers had no effect. Moreover, the frequency of spontaneous postsynaptic currents was increased after BAPTA dialysis. Outside the range of BAPTA dialysis astrocytes responded with a Ca2+ increase, but in contrast to control, the response was no longer restricted to one barrel field. Our findings indicate that astrocytes control neuronal inhibition in the barrel cortex.

Long-term dark rearing induces permanent reorganization in retinal circuitry

Recent data challenged the assumption that light has little effect on retina development. Here, we report evidence that dark rearing permanently changes the synaptic input to GCs. A reduced spontaneous postsynaptic currents (SPSCs) frequency was found in retinal GCs from rats born and raised in the dark for three months. Glutamate antagonists (CNQX and AP-5) reversibly reduced SPSCs frequency in control and dark-reared (DR) retinae. The GABA antagonist picrotoxin (PTX) reduced SPSCs frequency in control retinas, but increased SPSCs frequency in DR, mainly by presynaptic action on excitatory currents. In DR animals exposed to normal cyclic light for 3 months, SPSCs frequency remained lower then in control rats and increased following PTX, suggesting that long-term dark rearing induces permanent modifications of the retinal circuitry. Our results strongly support the idea that light stimulation plays a role in establishing normal synaptic input to GCs.

Acute treatment with 17β‐estradiol attenuates astrocyte–astrocyte and astrocyte–neuron communication

Astrocytes are now recognized as dynamic signaling elements in the brain. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. The ovarian steroid hormone, 17beta-estradiol, in addition to its rapid actions on neuronal electrical activity can rapidly alter astrocyte intracellular calcium concentration ([Ca2+]i) through a membrane-associated estrogen receptor. Using calcium imaging and electrophysiological techniques, we investigated the functional consequences of acute treatment with estradiol on astrocyte-astrocyte and astrocyte-neuron communication in mixed hippocampal cultures. Mechanical stimulation of an astrocyte evoked a [Ca2+]i rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a [Ca2+]i wave. Following acute treatment with estradiol, the amplitude of the [Ca2+]i elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the [Ca2+]i rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. Mechanical stimulation of astrocytes induced [Ca2+]i elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated [Ca2+]i rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in the presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings may indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol-mediated hormonal control.

Reactive Oxygen Species Mediate the Potentiating Effects of ATP on GABAergic Synaptic Transmission in the Immature Hippocampus

Reactive oxygen species (ROS) constitute important signaling molecules in the central nervous system. They regulate a number of different functions both under physiological conditions and under pathological conditions. Here we tested the hypothesis that in the immature hippocampus ATP, the most diffuse neurotransmitter in the brain, modulates synaptic transmission via ROS. We show that ATP, acting on metabotropic P2Y1 receptors, increased the frequency of GABA(A)-mediated spontaneous postsynaptic currents (SPSCs) in CA3 principal cells, an effect that was prevented by the antioxidant N-acetyl-cysteine or by catalase, an enzyme that breaks down H2O2. The effect of ATP on SPSCs was mimicked by H2O2 or by the pro-oxidant, Fe2+, which, through the Fentol reaction, catalyzes the conversion of H2O2 into highly reactive hydroxyl radicals. MRS-2179, a P2Y1 receptor antagonist, removed the facilitatory action of Fe2+ on SPSCs, suggesting that endogenous ATP acting on P2Y1 receptors is involved in Fe2+-induced modulation of synaptic transmission. Imaging ROS with the H2O2-sensitive dye DCF revealed that ATP induces generation of peroxide in astrocytes via activation of P2Y1 receptors coupled to intracellular calcium rise. Neither N-acetyl-cysteine nor catalase prevented Ca2+ transients induced by ATP in astrocytes. Since a single hippocampal astrocyte can contact many neurons, ATP-induced ROS signaling may control thousands of synapses. This may be crucial for information processing in the immature brain when GABAergic activity is essential for the proper wiring of the hippocampal network.

Purinergic modulation of synaptic input to Purkinje neurons in rat cerebellar brain slices.

Adenosine triphosphate (ATP) is a cotransmitter and an extracellular neuromodulator in nervous systems, and it influences neural circuits and synaptic strength. We have studied a stimulating effect of ATP (100 micro m) on the synaptic input of Purkinje neurons in acute cerebellar brain slices of juvenile rats (p14-19). Bath application of ATP increased the frequency of spontaneous postsynaptic currents (sPSCs) almost twofold, and increased their amplitude. These effects were fully suppressed by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid (PPADS; 10 microm), or after blocking action potentials with tetrodotoxin (TTX; 0.5 microm), but were not impaired by inhibiting ionotropic, non-NMDA glutamate receptors with 2,3-dioxo-6-nitro-1,2,3,4,-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX; 5 microm). The frequency of sPSCs was reduced by 35% by PPADS and increased by 50% after inhibiting ectonucleotidase with ARL67156 (50 microm), suggesting intrinsic, 'tonic', stimulation of synaptic activity via P2 receptors. The pharmacological profile indicated that the ATP effect was mediated by both P2X and P2Y receptors, most probably of the P2X5- and P2Y(2,4)-like subtypes. The action potential frequency in the inhibitory basket cells was increased by 65%, and decreased in Purkinje neurons by 25%, in the presence of ATP. Our results suggest that ATP continuously modulates the cerebellar circuit by increasing the activity of inhibitory input to Purkinje neurons, and thus decreasing the main cerebellar output activity, which contributes to locomotor coordination.