Play and tickling responses map to the lateral columns of the rat periaqueductal gray.

The persistence of play after decortication points to a subcortical mechanism of play control. We found that global blockade of the rat periaqueductal gray with either muscimol or lidocaine interfered with ticklishness and play. We recorded vocalizations and neural activity from the periaqueductal gray of young, playful rats during interspecific touch, play, and tickling. Rats vocalized weakly to touch and more strongly to play and tickling. Periaqueductal gray units showed diverse but strong modulation to tickling and play. Hierarchical clustering based on neuronal responses to play and tickling revealed functional clusters mapping to different periaqueductal gray columns. Specifically, we observed play-neutral/tickling-inhibited and tickling/play-neutral units in dorsolateral and dorsomedial periaqueductal gray columns. In contrast, strongly play/tickling-excited units mapped to the lateral columns and were suppressed by anxiogenic conditions. Optogenetic inactivation of lateral periaqueductal columns disrupted ticklishness and play. We conclude that the lateral periaqueductal gray columns are decisive for play and laughter.

Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons.

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGluu that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.

Uncoupling the Excitatory Amino Acid Transporter 2 From Its C-Terminal Interactome Restores Synaptic Glutamate Clearance at Corticostriatal Synapses and Alleviates Mutant Huntingtin-Induced Hypokinesia.

Rapid removal of glutamate from the sites of glutamate release is an essential step in excitatory synaptic transmission. However, despite many years of research, the molecular mechanisms underlying the intracellular regulation of glutamate transport at tripartite synapses have not been fully uncovered. This limits the options for pharmacological treatment of glutamate-related motor disorders, including Huntington's disease (HD). We therefore investigated the possible binding partners of transgenic EAAT2 and their alterations under the influence of mutant huntingtin (mHTT). Mass spectrometry analysis after pull-down of striatal YFP-EAAT2 from wild-type (WT) mice and heterozygote (HET) Q175 mHTT-knock-in mice identified a total of 148 significant (FDR < 0.05) binders to full-length EAAT2. Of them 58 proteins exhibited mHTT-related differences. Most important, in 26 of the 58 mHTT-sensitive cases, protein abundance changed back toward WT levels when the mice expressed a C-terminal-truncated instead of full-length variant of EAAT2. These findings motivated new attempts to clarify the role of astrocytic EAAT2 regulation in cortico-basal movement control. Striatal astrocytes of Q175 HET mice were targeted by a PHP.B vector encoding EAAT2 with different degree of C-terminal modification, i.e., EAAT2-S506X (truncation at S506), EAAT2-4KR (4 lysine to arginine substitutions) or EAAT2 (full-length). The results were compared to HET and WT injected with a tag-only vector (CTRL). It was found that the presence of a C-terminal-modified EAAT2 transgene (i) increased the level of native EAAT2 protein in striatal lysates and perisynaptic astrocyte processes, (ii) enhanced the glutamate uptake of transduced astrocytes, (iii) stimulated glutamate clearance at individual corticostriatal synapses, (iv) increased the glutamate uptake of striatal astrocytes and (iv) alleviated the mHTT-related hypokinesia (open field indicators of movement initiation). In contrast, over-expression of full-length EAAT2 neither facilitated glutamate uptake nor locomotion. Together, our results support the new hypothesis that preventing abnormal protein-protein interactions at the C-terminal of EAAT2 could eliminate the mHTT-related deficits in corticostriatal synaptic glutamate clearance and movement initiation.

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice.

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington's disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

The mechanisms of potentiation and inhibition of GABAA receptors by non-steroidal anti-inflammatory drugs, mefenamic and niflumic acids.

Fenamates mefanamic and niflumic acids (MFA and NFA) induced dual potentiating and inhibitory effects on GABA currents recorded in isolated cerebellar Purkinje cells using the whole-cell patch-clamp and fast-application techniques. Regardless of the concentration, both drugs induced a pronounced prolongation of the current response. We demonstrated that the same concentration of drugs can produce both potentiating and inhibitory effects, depending on the GABA concentration, which indicates that both processes take place simultaneously and the net effect depends on the concentrations of both the agonist and fenamate. We found that the NFA-induced block is strongly voltage-dependent. The Woodhull analysis of the block suggests that NFA has two binding sites in the pore - shallow and deep. We built a homology model of the open GABAAR based on the cryo-EM structure of the open α1 GlyR and applied Monte-Carlo energy minimization to optimize the ligand-receptor complexes. A systematic search for MFA/NFA binding sites in the GABAAR pore revealed the existence of two sites, the location of which coincides well with predictions of the Woodhull model. In silico docking suggests that two fenamate molecules are necessary to occlude the pore. We showed that MFA, acting as a PAM, competes with an intravenous anesthetic etomidate for a common binding site. We built structural models of MFA and NFA binding at the transmembrane ß(+)/α(-) intersubunit interface. We suggested a hypothesis on the molecular mechanism underlying the prolongation of the receptor lifetime in open state after MFA/NFA binding and ß subunit specificity of the fenamate potentiation.

Single Synapse Indicators of Impaired Glutamate Clearance Derived from Fast iGlu u Imaging of Cortical Afferents in the Striatum of Normal and Huntington (Q175) Mice.

Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single-synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGlu u We report findings from individual corticostriatal synapses in acute slices prepared from mice of either sex that were >1 year of age. Contrasting patterns of short-term plasticity and a size criterion identified two classes of terminals, presumably corresponding to the previously defined IT (intratelencephalic) and PT (pyramidal tract) synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGlu u single-synapse study in Q175 mice, a model of Huntington's disease (HD). In HD mice, the decay time constant of the perisynaptic Glu concentration (TauD), as an indicator of uptake, and the peak iGlu u amplitude, as an indicator of release, were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nm) mimicked the TauD changes in homozygotes. Considering the largest TauD values encountered in WT, ∼40% of PT synapses tested in Q175 heterozygotes can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGlu u Finally, EAAT2 (excitatory amino acid transport protein 2) immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention.SIGNIFICANCE STATEMENT Alterations in astrocytic Glu uptake can play a role in synaptic plasticity and neurodegeneration. Until now, the sensitivity of synaptic responses to pharmacological transport block and the resulting activation of NMDA receptors were regarded as reliable evidence for a mismatch between synaptic uptake and release. But the latter parameters are interdependent. Using a new genetically encoded sensor to monitor extracellular glutamate concentration ([Glu]) at individual corticostriatal synapses, we can now quantify the time constant of perisynaptic [Glu] decay (as an indicator of uptake) and the maximal [Glu] elevation next to the active zone (as an indicator of Glu release). The results provide a positive answer to the hitherto unresolved question of whether neurodegeneration (e.g., Huntington's disease) associates with a glutamate uptake deficit at tripartite excitatory synapses.

Astrocytes and presynaptic plasticity in the striatum: Evidence and unanswered questions.

One of the main functions of astrocytes is to ensure glutamate homeostasis by glutamate uptake and glutamine synthesis. However, during the past ten years it has become clear that astrocytes may also induce changes in synaptic glutamate release when respective pathways must cope with the consequences of brain damage or other alterations in their functional requirements. The loss of glutamatergic synapses in Parkinson's and Huntington's disease is likely to associate with a continuous redistribution of presynaptic activity within the pool of surviving synapses, and astrocytes may have a role in the maintenance of independent control at individual glutamate release sites. The rodent striatum should be a good model structure to analyse astrocyte-synapse interactions underlying disease-related plasticity, because it does not itself contain any glutamatergic neurons. Here we examine recent results that may shed light on the mechanisms underlying pathway-specific alterations in the corticostriatal or thalamostriatal synaptic transmission with a possible involvement of astrocytic release or uptake of glutamate. The conclusions emphasize the need of exploring the highly compartmentalised and presumably heterogeneous synapse astrocyte-interactions at a single synapse level.

Systemic application of AAV vectors targeting GFAP-expressing astrocytes in Z-Q175-KI Huntington's disease mice.

Huntington's disease (HD) affects both neurons and astrocytes. To target the latter and to ensure brain-wide transgene expression, adeno-associated viral (AAV) vectors can be administered intravenously, as AAV vectors cross the blood-brain barrier (BBB) and enable preferential transduction of astrocytes due to their close association with blood vessels. However, there is a possibility that the subclass of GFAP-expressing astrocytes performs a distinct role in HD and reacts differently to therapeutic measures than the rest of the astrocytes. The gfaABC1D promoter allows specific targeting of the GFAP-expressing astrocytes (~25% of S100ß-expressing astrocytes). We have examined the expression of three different transgenes (GCaMP6f, Kir4.1 and GLT1) and tested the effects of the AAV serotypes 9 and rh8. The AAV vectors were injected into the tail vein of 1-year-old homozygous Z-Q175-KI HD mice and their wild-type (WT) littermates. At this age, HD mice exhibit motor symptoms, including pronounced hypokinesia and circling behaviour. The expression times ranged from 3 to 6weeks. The target cell population was defined as the cells expressing S100ß in addition to GFAP. Viewfields in the dorsal striatum and the overlaying cortex were evaluated and the transduction rate was defined as the percentage of target cells that expressed the reporter transgene (enhanced green fluorescent protein, EGFP, or Tomato). In all cases, the transduction rate was higher in the cortex than in the striatum. AAV9 was more efficient than AAVrh8. One of the injected constructs (AAV9-gfaABC1D-GLT1-Tomato) was tested for the first time. GLT1, the principal astrocytic glutamate transporter, is deficient in HD and therefore considered as a potential target for gene therapy. At a dose of 1.86×1011 vector genome (vg) per animal, the fraction of GLT1-Tomato+ cells in the striatum and the cortex amounted to 30% and 49%, respectively. In individual Tomato+ HD astrocytes, treatment with the GLT1 vector increased the level of GLT1 immunofluorescence by 21% compared to the HD control. The described approach offers new and interesting opportunities to examine the pathophysiological consequences of brain-wide transgene expression in a specific astrocyte subpopulation.

Functional Indicators of Glutamate Transport in Single Striatal Astrocytes and the Influence of Kir4.1 in Normal and Huntington Mice.

UNLABELLED: This study evaluates single-cell indicators of glutamate transport in sulforhodamine 101-positive astrocytes of Q175 mice, a knock-in model of Huntington's disease (HD). Transport-related fluorescent ratio signals obtained with sodium-binding benzofuran isophtalate (SBFI) AM from unperturbed or voltage-clamped astrocytes and respective glutamate transporter currents (GTCs) were induced by photolytic or synaptic glutamate release and isolated pharmacologically. The HD-induced deficit ranged from -27% (GTC maximum at -100 mV in Ba(2+)) to -41% (sodium transients in astrocytes after loading SBFI-AM). Our specific aim was to clarify the mechanism(s) by which Kir4.1 channels can influence glutamate transport, as determined by either Na(+) imaging or transport-associated electrical signals. A decrease of Kir4.1 conductance was mimicked with Ba(2+) (200 µm), and an increase of Kir4.1 expression was obtained by intravenous administration of AAV9-gfaABC1D-Kir4.1-EGFP. The decrease of Kir4.1 conductance reduced the sodium transients but increased the amplitudes of somatic GTCs. Accordingly, after genetic upregulation of Kir4.1, somatic GTCs were found to be decreased. In individual cells, there was a negative correlation between Kir4.1 currents and GTCs. The relative effect of the Kir4.1 conductance was higher in the astrocyte periphery. These and other results suggest that the Kir4.1 conductance affects glutamate transporter activity in a dual manner: (1) by providing the driving force (voltage dependency of the transport itself) and (2) by limiting the lateral charge transfer (thereby reducing the interference with other electrogenic transporter functions). This leads to the testable prediction that restoring the high conductance state of passive astrocytes will not only normalize glutamate uptake but also restore other astrocytic transporter activities afflicted with HD. SIGNIFICANCE STATEMENT: Insufficiency of astrocytic glutamate uptake is a major element in the pathophysiology of neurodegenerative diseases. Considering the heterogeneity of astrocytes and their differential susceptibility to therapeutic interventions, it becomes necessary to evaluate the determinants of transport activity in individual astroglial cells. We have examined intracellular Na(+) transients and glutamate transporter currents as the most telling indicators of glutamate clearance after synaptic or photolytic release of glutamate in striatal slices. The results show that, in Huntington's disease, glutamate uptake activity critically depends on Kir4.1. These channels enable the high conductance state of the astrocytic plasma membrane, which ensures the driving force for glutamate transport and dumps the transport-associated depolarization along the astrocyte processes. This has significant implications for developing therapeutic targets.

High frequency stimulation of the subthalamic nucleus leads to presynaptic GABA(B)-dependent depression of subthalamo-nigral afferents.

Patients with akinesia benefit from chronic high frequency stimulation (HFS) of the subthalamic nucleus (STN). Among the mechanisms contributing to the therapeutic success of HFS-STN might be a suppression of activity in the output region of the basal ganglia. Indeed, recordings in the substantia nigra pars reticulata (SNr) of fully adult mice revealed that HFS-STN consistently produced a reduction of compound glutamatergic excitatory postsynaptic currents at a time when the tetrodotoxin-sensitive components of the local field potentials had already recovered after the high frequency activation. These observations suggest that HFS-STN not only alters action potential conduction on the way towards the SNr but also modifies synaptic transmission within the SNr. A classical conditioning-test paradigm was then designed to better separate the causes from the indicators of synaptic depression. A bipolar platinum-iridium macroelectrode delivered conditioning HFS trains to a larger group of fibers in the STN, while a separate high-ohmic glass micropipette in the rostral SNr provided test stimuli at minimal intensity to single fibers. The conditioning-test interval was set to 100 ms, i.e. the time required to recover the excitability of subthalamo-nigral axons after HFS-STN. The continuity of STN axons passing from the conditioning to the test sites was examined by an action potential occlusion test. About two thirds of the subthalamo-nigral afferents were occlusion-negative, i.e. they were not among the fibers directly activated by the conditioning STN stimulation. Nonetheless, occlusion-negative afferents exhibited signs of presynaptic depression that could be eliminated by blocking GABA(B) receptors with CGP55845 (1 µM). Further analysis of single fiber-activated responses supported the proposal that the heterosynaptic depression of synaptic glutamate release during and after HFS-STN is mainly caused by the tonic release of GABA from co-activated striato-nigral afferents to the SNr. This mechanism would be consistent with a gain-of-function hypothesis of DBS.

Reduced tonic inhibition in striatal output neurons from Huntington mice due to loss of astrocytic GABA release through GAT-3.

The extracellular concentration of the two main neurotransmitters glutamate and GABA is low but not negligible which enables a number of tonic actions. The effects of ambient GABA vary in a region-, cell-type, and age-dependent manner and can serve as indicators of disease-related alterations. Here we explored the tonic inhibitory actions of GABA in Huntington's disease (HD). HD is a devastating neurodegenerative disorder caused by a mutation in the huntingtin gene. Whole cell patch clamp recordings from striatal output neurons (SONs) in slices from adult wild type mice and two mouse models of HD (Z_Q175_KI homozygotes or R6/2 heterozygotes) revealed an HD-related reduction of the GABA(A) receptor-mediated tonic chloride current (I(Tonic(GABA))) along with signs of reduced GABA(B) receptor-mediated presynaptic depression of synaptic GABA release. About half of I(Tonic(GABA)) depended on tetrodotoxin-sensitive synaptic GABA release, but the remaining current was still lower in HD. Both in WT and HD, I(Tonic(GABA)) was more prominent during the first 4 h after preparing the slices, when astrocytes but not neurons exhibited a transient depolarization. All further tests were performed within 1-4 h in vitro. Experiments with SNAP5114, a blocker of the astrocytic GABA transporter GAT-3, suggest that in WT but not HD GAT-3 operated in the releasing mode. Application of a transportable substrate for glutamate transporters (D-aspartate 0.1-1 mM) restored the non-synaptic GABA release in slices from HD mice. I(Tonic(GABA)) was also rescued by applying the hyperagonist gaboxadol (0.33 µM). The results lead to the hypothesis that lesion-induced astrocyte depolarization facilitates non-synaptic release of GABA through GAT-3. However, the capacity of depolarized astrocytes to provide GABA for tonic inhibition is strongly reduced in HD.

Transporter-mediated replacement of extracellular glutamate for GABA in the developing murine neocortex.

During early development, cortical neurons migrate from their places of origin to their final destinations where they differentiate and establish synaptic connections. During corticogenesis, radially migrating cells move from deeper zone to the marginal zone, but they do not invade the latter. This "stop" function of the marginal zone is mediated by a number of factors, including glutamate and γ-aminobutyric acid (GABA), two main neurotransmitters in the central nervous system. In the marginal zone, GABA has been shown to be released via GABA transporters (GAT)-2/3, whereas glutamate transporters (EAATs) operate in the uptake mode. In this study, GABAergic postsynaptic currents (GPSCs) were recorded from Cajal-Retzius cells in the marginal zone of murine neonatal neocortex using a whole-cell patch-clamp technique. Minimal electrical stimulation was applied to elicit evoked GPSCs using a paired-pulse protocol. EAAT blockade with dl-threo-b-benzyloxyaspartic acid (dl-TBOA), a specific non-transportable EAAT antagonist, abolishes constitutive GAT-2/3-mediated GABA release. In contrast to dl-TBOA, d-aspartate, an EAAT substrate, fails to block GAT-2/3-mediated GABA release. SNAP-5114, a specific GAT-2/3 antagonist, induced an elevation of intracellular sodium concentration ([Na(+) ]i ) under resting conditions and in the presence of d-aspartate, indicating that GAT-2/3 operates in reverse mode. In the presence of dl-TBOA, however, SNAP-5114 elicited a [Na(+) ]i decrease, demonstrating that GAT-2/3 operates in uptake mode. We conclude that EAATs via intracellular Na(+) signaling and/or cell depolarization can govern the strength/direction of GAT-mediated GABA transport.

TNF-α provokes electrical abnormalities in rat atrial myocardium via a NO-dependent mechanism.

Stretch-induced depolarizations of cardiomyocytes, which are related to activity of mechano-gated cation channels (MGCs), can lead to serious arrhythmias. However, signaling pathways leading to activation of mechano-gated channels by stretch remain almost unexplored. Using standard sharp microelectrodes, the present study addresses the hypothesis that tumor necrosis factor-alpha (TNF-α) modulates stretch-induced electrophysiological abnormalities in rat atrial myocardium by a mechanism involving nitric oxide (NO)-dependent pathways. TNF-α (50 ng/ml) produced a marked prolongation of action potential, subsequently transforming into humplike depolarizations and, finally, leading to occurrence of arrhythmias. These effects developed slowly during 25 min of TNF-α application. Similar electrical effects were induced by stretching the preparations. A blocker of MGCs, Gd(3+) (40 µM), completely abolished action potential (AP) prolongations and electrical abnormalities caused by TNF-α or stretch. Further, a donor of exogenous NO, S-nitroso-N-acetylpenicillamine SNAP (300 µM), evoked the same electrical abnormalities as TNF-α and tissue stretch. Both TNF-α and stretch failed to produce their typical effects after pretreatment of the preparations with the NO-synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) (100 µM). Thus, the present study shows (i) that TNF-α and the NO-donor SNAP evoke MGC-mediated electrical abnormalities in rat atrial myocardium in the absence of stretch that is very similar to stretch-evoked electrical events and (ii) that the TNF-α-induced electrical abnormalities are mediated by NO synthase. In conclusion, our data suggest that NO is an endogenous modulator of MGCs and mediates proarrhythmic effects of TNF-α in mammalian organism.

Tonic mGluR5/CB1-dependent suppression of inhibition as a pathophysiological hallmark in the striatum of mice carrying a mutant form of huntingtin.

Changes in the activity of striatal output neurons (SONs) have been implicated in the pathogenesis of Huntington's disease (HD). In this inherited polyglutamine disorder, accumulation of intracellular toxins causes a variety of deficits, including synaptic dysfunction, but it is still unclear to what extent striatal GABA release is afflicted as well. Two murine HD models were used, a recently created knock-in mouse (Z_Q175_KI) and an established model of HD (R6/2). In sagittal slices with relatively well-preserved glutamatergic connections throughout the basal ganglia, we have characterized the following: (i) the excitability of SONs; (ii) their spontaneous action potential-dependent GABAergic synaptic activity; (iii) the capacity of exogenous GABA to inhibit spontaneous action potential generation; and (iv) the properties of GABAergic unitary evoked responses (eIPSCs) in response to intrastriatal minimal stimulation at low and high frequency. The HD SONs exhibited enhanced intrisic excitability and higher levels of GABAergic spontaneous activity without presenting evidence for homeostatic upregulation of endogenous or exogenous GABA actions. Unitary eIPSC amplitudes were reduced, with a clear deficit in the probability of release, as indicated by a higher paired-pulse ratio, failure rate and coefficient of variation. In conditions of high-frequency activation, GABAergic connections of HD SONs were prone to asynchronous release and delayed IPSC generation at the expense of synchronized release. Both in wild-type and in HD SONs, GABA was inhibitory. Our results support the conclusion that the enhanced spontaneous synaptic activity in the HD striatum reflects disinhibition. Pharmacological tests identified the HD-related tonic suppression of synaptic inhibition as a glutamate- and endocannabinoid-dependent process.

Glutamate transporters and presynaptic metabotropic glutamate receptors protect neocortical Cajal-Retzius cells against over-excitation.

Cajal-Retzius (CR) cells, early generated neurons in the marginal zone of developing neocortex, are reported to be highly vulnerable to excitotoxic damage. Because extracellular glutamate concentration in the central nervous system is mainly controlled by glutamate transporters (EAATs), we studied the effects of EAAT blockade on CR cells. DL: -TBOA, a specific EAAT antagonist, induced NMDA receptor-dependent bursting discharges in layer 2/3 pyramidal neurons, indicating that EAATs operate in the uptake mode and their blockade leads to elevation of extracellular glutamate concentration. In CR cells, however, DL: -TBOA failed to change either the membrane resistance or holding current, and moreover, it reduced the frequency of spontaneous GABAergic postsynaptic currents. DL: -TBOA decreased the mean amplitude and increased paired-pulse ratio of evoked GABAergic postsynaptic currents, indicating the presynaptic locus of its action. Indeed, LY379268, a specific agonist of group II metabotropic glutamate receptors (mGluR-II), mimicked the DL: -TBOA-mediated effects, and LY341495, an unspecific mGluR antagonist, eliminated the DL: -TBOA-induced effects. As dihydrokainic acid, a specific EAAT2 blocker, failed to affect evoked GABAergic postsynaptic currents, whereas TFB-TBOA, a selective blocker of EAAT1 and EAAT2, produced effects similar to that of DL: -TBOA, extracellular glutamate concentration in the marginal zone is mainly controlled by EAAT1 (GLAST). Thus, even though CR cells are highly vulnerable to excitotoxic damage, a number of mechanisms serve to protect them against excessive extracellular glutamate concentration including a lack of functional glutamatergic synapses, Mg(2+) blockade of NMDA receptors, and presynaptic mGluRs that inhibit transmission at GABAergic synapses.

Estimation of ambient GABA levels in layer I of the mouse neonatal cortex in brain slices.

GABAergic synapses on Cajal-Retzius neurons in layer I of the murine neocortex experience GABA(B) receptor (GABA(B)R)-mediated tonic inhibition. Extracellular GABA concentration ([GABA](o)) that determines the strength of GABA(B)R-mediated inhibition is controlled by GABA transporters (GATs). In this study, we hypothesized that the strength of presynaptic GABA(B)R activation reflects [GABA](o) in the vicinity of synaptic contacts. Slices obtained from two age groups were used, namely postnatal days (P)2-3 and P5-7. GABAergic postsynaptic currents (IPSCs) were recorded using the whole-cell patch-clamp technique. Minimal electrical stimulation in layer I was applied to elicit evoked IPSCs (eIPSCs) using a paired-pulse protocol. Three parameters were selected for comparison: the mean eIPSC amplitude, paired-pulse ratio, and failure rate. When GAT-1 and GAT-2/3 were blocked by NO-711 (10 microM) and SNAP-5114 (40 microM), respectively, no tonic GABA(B)R-mediated inhibition was observed. In order to restore the control levels of GABA(B)R-mediated inhibition, 250 and 125 nm exogenous GABA was required at P2-3 and P5-7, respectively. Addition of 3-mercaptopropionic acid, a glutamate decarboxylase inhibitor, did not significantly change the obtained values arguing against the suggestion that a mechanism different from GATs contributes to [GABA](o) control. We conclude that juxtasynaptic [GABA](o) is higher (about 250 nM) at P2-3 than at P5-7 (about 125 nM). As both radial cell migration and corticogenesis in general are strongly dependent on [GABA](o) and the formation of the last layer 2/3 is finished by P4 in rodents, the observed [GABA](o) reduction in layer I might reflect this crucial event in the cortical development.

GABA transporter 1 tunes GABAergic synaptic transmission at output neurons of the mouse neostriatum.

GABAergic medium-sized striatal output neurons (SONs) provide the principal output for the neostriatum. In vitro and in vivo data indicate that spike discharge of SONs is tightly controlled by effective synaptic inhibition. Although phasic GABAergic transmission critically depends on ambient GABA levels, the role of GABA transporters (GATs) in neostriatal GABAergic synaptic transmission is largely unknown. In the present study we aimed at elucidating the role of GAT-1 in the developing mouse neostriatum (postnatal day (P) 7-34). We recorded GABAergic postsynaptic currents (PSCs) using the whole-cell patch-clamp technique. Based on the effects of NO-711, a specific GAT-1 blocker, we demonstrate that GAT-1 is operative at this age and influences GABAergic synaptic transmission by presynaptic and postsynaptic mechanisms. Presynaptic GABA(B)R-mediated suppression of GABA release was found to be functional at all ages tested; however, there was no evidence for persistent GABA(B)R activity under control conditions, unless GAT-1 was blocked (P12-34). In addition, whereas no tonic GABA(A)R-mediated conductances were detected in SONs until P14, application of a specific GABA(A)R antagonist caused distinct tonic outward currents later in development (P19-34). In the presence of NO-711, tonic GABA(A)R-mediated currents were also observed at P7-14 and were dramatically increased at more mature stages. Furthermore, GAT-1 block reduced the median amplitude of GABAergic miniature PSCs indicating a decrease in quantal size. We conclude that in the murine neostriatum GAT-1 operates in a net uptake mode. It prevents the persistent activation of presynaptic GABA(B)Rs (P12-34) and prevents (P7-14) or reduces (P19-34) tonic postsynaptic GABA(A)R activity.

Postsynaptically different inhibitory postsynaptic currents in Cajal-Retzius cells in the developing neocortex.

Fast and slowly rising inhibitory postsynaptic currents (IPSCs, IPSCF and IPSCS) in neocortical Cajal-Retzius cells are observed. In this study, zolpidem, a benzodiazepine agonist that specifically modulates gamma-aminobutyric acid type A receptors (GABAARs) containing gamma2 subunit, was used to characterize GABAARs mediating IPSCF and IPSCS. One-hundred-nanomolar zolpidem prolonged IPSCS, increased evoked IPSCS (eIPSCS) amplitude, and decreased paired-pulse ratio (PPR) of eIPSCS. Two micromolar zolpidem prolonged both IPSCF and IPSCS, increased miniature IPSCF and eIPSCF amplitudes, increased eIPSCS amplitude but not miniature IPSCS amplitude, decreased PPR of eIPSCS, but failed to affect PPR of eIPSCF. We conclude that IPSCF are mediated by alpha2/3-containing GABAARs, which are not saturated by synaptic GABA release, whereas IPSCS are mediated by alpha1-containing and alpha2/3-containing GABAARs, which are saturated by quantal GABA release.

Developmental downregulation of excitatory GABAergic transmission in neocortical layer I via presynaptic adenosine A(1) receptors.

Layer I of the developing cortex contains a dense GABAergic fiber plexus. These fibers provide excitatory inputs to Cajal-Retzius (CR) cells, the early born neurons in layer I. CR cells possess an extensive axonal projection and form synaptic contacts with excitatory, presumably pyramidal, neurons before birth. Interestingly, activity of CR cells declines during the first postnatal week, but mechanism(s) underlying this phenomenon is not yet known. Here we recorded inhibitory postsynaptic currents (IPSCs) in CR cells at postnatal day (P) 1-2 and P5-7. Blockade of adenosine A(1) receptors (A(1)Rs) increased the amplitude of evoked IPSCs (eIPSCs) and decreased paired-pulse ratio at P5-7 but not at P1-2. A(1)R activation decreased the mean eIPSC amplitude at P5-7, but failed to affect eIPSCs at P1-2. Ecto-adenosine triphosphatase (ATPase) inhibition completely abolished the A(1)R-mediated effects suggesting that extracellular ATP is the main source of adenosine. Because A(1)R blockade did not affect the median miniature IPSC amplitude, our results demonstrate that adenosine reduces gamma-aminiobutyric acid (GABA) release probability via presynaptic A(1)Rs at P5-7. As neuronal activity in layer I can depolarize pyramidal neurons influencing thereby glutamatergic synaptogenesis in the lower cortical layers, postnatal weakening of GABAergic transmission by adenosinergic system might reflect a developmental downregulation of this excitatory drive when glutamatergic synapses are formed.

Cajal Retzius cells in the mouse neocortex receive two types of pre- and postsynaptically distinct GABAergic inputs.

Cajal-Retzius (CR) cells are principal cells of layer I in the developing neocortex. They are able to generate action potentials, make synaptic contacts in layer I and receive excitatory GABAergic inputs before birth. Although CR cells participate in neuronal network activity in layer I, the properties of their synaptic inputs are not yet characterized. We recorded miniature (mIPSCs) and evoked (eIPSCs) postsynaptic currents using the whole-cell patch-clamp technique. Most of CR cells displayed two types of mIPSCs, namely those with fast (mIPSC(F)) and slow (mIPSC(S)) rise kinetics. The mIPSC(F) mean amplitude was significantly larger than that of mIPSC(S), while their decay rates were not different. Peak-scaled non-stationary noise analysis revealed that mIPSC(S) and mIPSC(F) differed in their weighted single-channel conductance. In addition, zolpidem (100 nm), a modulator of alpha(1) subunit-containing GABA(A) receptors, selectively affected mIPSC(S) suggesting that different postsynaptic GABA(A) receptors mediate mIPSC(F) and mIPSC(S). eIPSCs also split into two populations with different rise kinetics. Fast eIPSCs (eIPSC(F)) displayed higher paired-pulse ratio (PPR) and lower GABA release probability than slowly rising eIPSCs (eIPSC(S)). As CGP55845, a GABA(B) receptor antagonist, eliminated the observed difference in PPR, the lower release probability at IPSC(F) connections probably reflects a stronger tonic GABA(B) receptor-mediated inhibition of IPSC(F) synapses. At low (0.1 Hz) stimulation frequency both inputs can effectively convert presynaptic action potentials into postsynaptic ones; however, only IPSC(F) connections reliably transfer the presynaptic activity patterns at higher stimulation rates. Thus, CR cells receive two GABAergic inputs, which differ in the quantal amplitude, the probability of GABA release and the frequency dependence of signal transfer.