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.

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.

Unravelling and Exploiting Astrocyte Dysfunction in Huntington's Disease.

Astrocytes are abundant within mature neural circuits and are involved in brain disorders. Here, we summarize our current understanding of astrocytes and Huntington's disease (HD), with a focus on correlative and causative dysfunctions of ion homeostasis, calcium signaling, and neurotransmitter clearance, as well as on the use of transplanted astrocytes to produce therapeutic benefit in mouse models of HD. Overall, the data suggest that astrocyte dysfunction is an important contributor to the onset and progression of some HD symptoms in mice. Additional exploration of astrocytes in HD mouse models and humans is needed and may provide new therapeutic opportunities to explore in conjunction with neuronal rescue and repair strategies.

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.

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.

Functional hallmarks of GABAergic synapse maturation and the diverse roles of neurotrophins.

Functional impairment of the adult brain can result from deficits in the ontogeny of GABAergic synaptic transmission. Gene defects underlying autism spectrum disorders, Rett's syndrome or some forms of epilepsy, but also a diverse set of syndromes accompanying perinatal trauma, hormonal imbalances, intake of sleep-inducing or mood-improving drugs or, quite common, alcohol intake during pregnancy can alter GABA signaling early in life. The search for therapeutically relevant endogenous molecules or exogenous compounds able to alleviate the consequences of dysfunction of GABAergic transmission in the embryonic or postnatal brain requires a clear understanding of its site- and state-dependent development. At the level of single synapses, it is necessary to discriminate between presynaptic and postsynaptic alterations, and to define parameters that can be regarded as both suitable and accessible for the quantification of developmental changes. Here we focus on the performance of GABAergic synapses in two brain structures, the hippocampus and the superior colliculus, describe some novel aspects of neurotrophin effects during the development of GABAergic synaptic transmission and examine the applicability of the following rules: (1) synaptic transmission starts with GABA, (2) nascent/immature GABAergic synapses operate in a ballistic mode (multivesicular release), (3) immature synaptic terminals release vesicles with higher probability than mature synapses, (4) immature GABAergic synapses are prone to paired pulse and tetanic depression, (5) synapse maturation is characterized by an increasing dominance of synchronous over asynchronous release, (6) in immature neurons GABA acts as a depolarizing transmitter, (7) synapse maturation implies inhibitory postsynaptic current shortening due to an increase in alpha1 subunit expression, (8) extrasynaptic (tonic) conductances can inhibit the development of synaptic (phasic) GABA actions.

Role of GABA transporter 3 in GABAergic synaptic transmission at striatal output neurons.

Striatal GABAergic signaling has been shown to be essential for basal ganglia output and proper motor performance. In the mouse neostriatum GABA transporter 1 (GAT-1) was previously found to assist in the clearance of GABA from the extracellular space and influence both phasic and tonic GABAergic inhibition of medium-sized striatal output neurons (SONs). It currently remains unknown whether GAT subtypes other than GAT-1 participate in the modulation of GABAergic transmission in this brain structure. In this study, we aimed at assessing the role of GAT-3 in the mouse neostriatum. To this end, we recorded GABAergic inhibitory postsynaptic currents (IPSCs) from SONs in brain slices at different developmental stages (postnatal days (P) 7-9, 12-14, and 28-34) using the whole-cell patch-clamp technique. When applied under control conditions, SNAP-5114 (40 microM), a specific GAT-3 blocker, did not affect miniature or evoked IPSCs (m/eIPSCs) and produced no significant effect on tonic GABAA receptor-mediated conductances in SONs. However, in the presence of NO-711 (10 microM), a specific GAT-1 blocker, SNAP-5114 reduced mIPSC frequencies without affecting mIPSC amplitudes or kinetics. In addition, SNAP-5114 reduced the mean amplitude of eIPSCs and increased the paired-pulse ratio. These effects were entirely abolished by CGP55845 (1 microM), a specific GABAB receptor blocker, indicating that they were mediated by presynaptic GABAB receptors. Similar results were obtained from all age groups. We conclude that GAT-3 is functionally expressed in the mouse neostriatum. Whereas an acute block of GAT-3 under resting conditions is fully compensated for by GAT-1, GAT-3 might provide an additional uptake capacity when neuronal activity and GABA release are increased.

A 29-amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation.

Small GTPases of the Rho family play versatile roles in the formation and development of axons and dendrites, effects often studied by the Rho-inactivating C3 transferase (C3bot) from Clostridium botulinum. Recently, we reported that transferase-deficient C3bot also exerted axonotrophic activity. Using overlapping peptides from the C3bot sequence, we identified a small peptide of 29 amino acids (covering residues 154-182) from the C-terminal region of C3bot that promotes both axonal and dendritic growth, as well as branching of hippocampal neurons, at submicromolar concentrations. Several C3bot constructs, including the short peptide, enhanced the number of axonal segments from mid- to higher-order segments. C3bot(154-182) also increased the number of synaptophysin-expressing terminals, up-regulated various synaptic proteins, and functionally increased the glutamate uptake. Staining against the vesicular glutamate and GABA transporters further revealed that the effect was attributable to a higher number of glutamatergic and GABAergic inputs on proximal dendrites of enhanced green fluorescent protein (EGFP)-transfected neurons. Using organotypical slice cultures, we also detected trophic effects of C3bot(154-182) on length and density of outgrowing fibers from the entorhinal cortex that were comparable to the effects elicited by full-length C3bot. In addition, an enhanced reinnervation was observed in a hippocampal-entorhinal lesion model. In summary, the neurotrophic effect of C3bot is executed by a C-terminal peptide fragment covering aa 154-182 of C3; it triggers dendritic and axonal growth and branching as well as increased synaptic connectivity. In contrast to full-length C3, this C3 peptide selectively acts on neurons but not on glial cells.

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.

Glycinergic tonic inhibition of hippocampal neurons with depolarizing GABAergic transmission elicits histopathological signs of temporal lobe epilepsy.

An increasing number of epilepsy patients are afflicted with drug-resistant temporal lobe epilepsy (TLE) and require alternative therapeutic approaches. High-affinity glycine receptors (haGlyRs) are functionally adapted to tonic inhibition due to their response to hippocampal ambient glycine, and their synthesis is activity-dependent. Therefore, in our study, we scanned TLE hippocampectomies for expression of haGlyRs and characterized the effects mediated by these receptors using primary hippocampal neurons. Increased haGlyR expression occurred in TLE hippocampi obtained from patients with a severe course of disease. Furthermore, in TLE patients, haGlyR and potassium chloride cotransporter 2 (KCC2) expressions were inversely regulated. To examine this potential causal relationship with respect to TLE histopathology, we established a hippocampal cell culture system utilising tonic inhibition mediated by haGlyRs in response to hippocam-pal ambient glycine and in the context of a high Cl equilibrium potential, as is the case in TLE hippocampal neurons. We showed that hypoactive neurons increase their ratio between glutamatergic and GABAergic synapses, reduce their dendrite length and finally undergo excitotoxicity. Pharmacological dissection of the underlying processes revealed ionotropic glutamate and TrkB receptors as critical mediators between neuronal hypoactivity and the emergence of these TLE-characteristic histopathological signs. Moreover, our results indicate a beneficial role for KCC2, because decreasing the Cl- equilibrium potential by KCC2 expression also rescued hypoactive hippocampal neurons. Thus, our data support a causal relationship between increased haGlyR expression and the emergence of histopathological TLE-characteristic signs, and they establish a pathophysiological role for neuronal hypoactivity in the context of a high Cl- equilibrium potential.

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.

Cortical efferent control of subcortical sensory neurons by synaptic disinhibition.

A long-standing hypothesis predicts that pyramidal neurons of the cerebral cortex control the influx of sensory information at the level of primary sensory representations areas. Yet little is known about the cellular mechanisms governing selective attention to behaviorally relevant objects in space. Neurons in the superficial layers of the superior colliculus are notably involved in this process, and they are directly targeted by retinal and cortical afferents. To study long-term and short-term effects of the visual cortex (VC) on subcortical visual neurons we established an in vitro model of the developing cortico-tectal projection. To this end, cortical explants expressing Green Fluorescent Protein were allowed to form connections with non-labeled dissociated tectal neurons. The presence of VC explants led to an enhancement of tectal activity by 2 mechanisms. First, glutamatergic input was increased. Second, intrinsic GABAergic inhibition was suppressed. The latter effect was shown to be acute and mediated through postsynaptic metabotropic glutamate receptor activation, G-protein acitivity, and endocannabinoid receptor activation. The VC-induced disinhibition was readily reversed by application of an mGluR antagonist. However, high-frequency activation of the glutamatergic cortico-tectal input turned the labile disinhibition into a persistent suppression of inhibition.

Notch and NGF/p75NTR control dendrite morphology and the balance of excitatory/inhibitory synaptic input to hippocampal neurones through Neurogenin 3.

We have previously shown that dendrite morphology of cultured hippocampal neurones is controlled by Notch receptor activation or binding of nerve growth factor (NGF) to its low affinity receptor p75NTR, i.e. processes that up-regulate the expression of the Homologue of enhancer of split 1 and 5. Thus, the increased expression of these genes decreases the number of dendrites, whereas abrogation of Homologue of enhancer of split 1/5 activity stimulates the outgrowth of new dendrites. Here, we show that Neurogenin 3 is a proneural gene that is negatively regulated by Homologue of enhancer of split 1/5. It also influences dendrite morphology. Hence, a deficit of Notch or NGF/p75NTR activation can lead to the production of high levels of Neurogenin 3, which stimulates the outgrowth of new dendrites. Conversely, activation of either Notch or p75NTR depressed Neurogenin 3 expression, which not only decreased the number of dendrites but also favoured inhibitory (GABAergic) synaptogenesis, thereby diminishing the ratios of excitatory/inhibitory inputs. NGF also augmented the levels of mRNA encoding the vesicular inhibitory amino acid transporter, but it did not affect the fraction of GAD65/67-positive neurones. Conversely, overexpression of Neurogenin 3 largely reduced the number of inhibitory synaptic contacts and, consequently, produced a strong increase in the ratios of excitatory/inhibitory synaptic terminals. Our results reveal a hitherto unknown contribution of NGF/p75NTR to dendritic and synaptic plasticity through Neurogenin 3 signalling.

Developmental downregulation of low-voltage-activated Ca2+ channels in Cajal-Retzius cells of the mouse visual cortex.

Cajal-Retzius (CR) cells have been demonstrated to fulfil an important secretory function in the developing neocortex. On the other hand, the contribution of CR cell electrical activity during cortex development is still unclear. Using the whole-cell patch-clamp technique, we studied low-voltage-activated (LVA) Ca(2+) channels in CR cells in the layer I of the mouse visual cortex. CR cells were found to display a transient Ca(2+) current (I(T)) in response to a depolarization step from -100 mV to -40 mV. I(T) showed: (i) typical for LVA Ca(2+) channels voltage dependence of activation (half-activation at -55 mV) and inactivation (half-inactivation at -76 mV); (ii) fast activation and inactivation kinetics, with time constants of 1.4 and 28 ms, respectively, at -40 mV; (iii) fast recovery from steady-state inactivation (time constant: 290 ms); (iv) a complete block by 1 microm mibefradil; and (v) a partial block (to 55%) by 100 microm Ni(2+). The density of I(T) dramatically decreased between postnatal day (P) 1 and P9. Immunostaining demonstrated the presence and postnatal downregulation of the alpha(1G)-subunit of LVA Ca(2+) channels in CR cells. Experiments performed in the current-clamp mode revealed that mibefradil delayed an action potential generation in response to a suprathreshold depolarizing current at P1, but not at P8-9. We suggest that LVA Ca(2+) channels might influence CR cell excitability during the first postnatal week and thereby contribute to the shaping of synaptic connectivity in the neocortical layer I.