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Cortical malformations such as focal cortical dysplasia type II (FCDII) are associated with pediatric drug-resistant epilepsy that necessitates neurosurgery. FCDII results from somatic mosaicism due to post-zygotic mutations in genes of the PI3K-AKT-mTOR pathway, which produce a subset of dysmorphic cells clustered within healthy brain tissue. Here we show a correlation between epileptiform activity in acute cortical slices obtained from human surgical FCDII brain tissues and the density of dysmorphic neurons. We uncovered multiple signatures of cellular senescence in these pathological cells, including p53/p16 expression, SASP expression and senescence-associated β-galactosidase activity. We also show that administration of senolytic drugs (dasatinib/quercetin) decreases the load of senescent cells and reduces seizure frequency in an Mtor S2215F FCDII preclinical mouse model, providing proof of concept that senotherapy may be a useful approach to control seizures. These findings pave the way for therapeutic strategies selectively targeting mutated senescent cells in FCDII brain tissue., (© 2024. The Author(s).)

Objective: Mesial Temporal Lobe Epilepsy-associated Hippocampal Sclerosis (MTLE-HS) is a syndrome associated with various aetiologies. We previously identified CD34-positive extravascular stellate cells (CD34+ cells) possibly related to BRAF V600E oncogenic variant in a subset of MTLE-HS. We aimed to identify the BRAF V600E oncogenic variants and characterise the CD34+ cells., Methods: We analysed BRAF V600E oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLE-HS samples (25 with CD34+ cells) and nine non-expansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multi-electrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAF wildtype MTLE-HS and BRAF V600E mutant non-expansive lesion of hippocampus and/or neocortex., Results: We identified a BRAF V600E oncogenic variant in five MTLE-HS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAF V600E mutant samples. The co-expression of the oncogene-induced senescence marker p16 INK4A and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions., Interpretation: BRAF V600E underlies a subset of MTLE-HS and epileptogenic non-expansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies., (© 2023 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Delayed upregulation of the neuronal chloride extruder KCC2 underlies the progressive shift in GABA signaling polarity during development. Conversely, KCC2 downregulation is observed in a variety of neurological and psychiatric disorders often associated with cognitive impairment. Reduced KCC2 expression and function in mature networks may disrupt GABA signaling and promote anomalous network activities underlying these disorders. However, the causal link between KCC2 downregulation, altered brain rhythmogenesis, and cognitive function remains elusive. Here, by combining behavioral exploration with in vivo electrophysiology we assessed the impact of chronic KCC2 downregulation in mouse dorsal hippocampus and showed it compromises both spatial and contextual memory. This was associated with altered hippocampal rhythmogenesis and neuronal hyperexcitability, with increased burst firing in CA1 neurons during non-REM sleep. Reducing neuronal excitability with terbinafine, a specific Task-3 leak potassium channel opener, occluded the impairment of contextual memory upon KCC2 knockdown. Our results establish a causal relationship between KCC2 expression and cognitive performance and suggest that non-epileptiform rhythmopathies and neuronal hyperexcitability are central to the deficits caused by KCC2 downregulation in the adult mouse brain., (© 2022. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Focal neuronal lipofuscinosis (FNL) is an uncommon epileptic disorder related to an excess of lipofuscin accumulation within dysmorphic-appearing neurons (DANs), whose epileptogenic mechanisms are still poorly understood. It shares some clinical and neuroimaging similarities with focal cortical dysplasia of type IIb (FCDIIb), but it represents a different pathological entity. Here, we identified two patients with FNL among a 10-year cohort of 323 patients who underwent neurosurgery for a focal pharmacoresistant epilepsy. We describe the electroclinical, metabolic and neuropathological features of both patients with FNL who benefited from a comprehensive presurgical investigation. While the previous reports showed frontal lobe localization of the lesion, FNL was identified in the temporal lobe, in one of our patients. EEG investigations in both patients showed striking focal and rich interictal activity resembling that described in FCDIIb. Besides focal intraneuronal lipofuscin accumulation, the neuropathological analysis demonstrated that somata of DANs were surrounded by a large amount of GABAergic presynaptic buttons, suggesting the involvement of interneurons in the epileptogenicity of FNL. To further explore the role of GABAergic transmission in the generation of epileptiform activity in FNL, we performed in vitro multi-electrode array recordings on the post-surgery tissue from one patient. Spontaneous interictal-like discharges (IILDs) were identified only in the restricted area displaying the highest density of lipofuscin-containing DANs, suggesting a close correlation between the density of lipofuscin-containing neurons and epileptogenicity. Moreover, IILDs were blocked by the GABAA receptor antagonist gabazine. All together, these findings showed how GABA signaling may contribute to the generation of interictal-like activity in FNL tissue., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

The K + -Cl - cotransporter KCC2, encoded by the Slc12a5 gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABA A receptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons. SIGNIFICANCE STATEMENT Fast synaptic inhibition in the brain is mediated by chloride-permeable GABA A receptors (GABA A Rs) and therefore relies on transmembrane chloride gradients. In neurons, these gradients are primarily maintained by the K/Cl cotransporter KCC2. Therefore, understanding the mechanisms controlling KCC2 expression and function is crucial to understand its physiological regulation and rescue its function in the pathology. KCC2 function depends on its membrane expression and clustering, but the underlying mechanisms remain unknown. We describe the interaction between KCC2 and gephyrin, the main scaffolding protein at inhibitory synapses. We show that gephyrin controls plasmalemmal KCC2 clustering and that loss of gephyrin compromises KCC2 function. Our data suggest functional units comprising GABA A Rs, gephyrin, and KCC2 act to regulate synaptic GABA signaling., (Copyright © 2022 the authors.)

The nucleoredoxin gene NXNL2 encodes for two products through alternative splicing, rod-derived cone viability factor-2 (RdCVF2) that mediates neuronal survival and the thioredoxin-related protein (RdCVF2L), an enzyme that regulates the phosphorylation of TAU. To investigate the link between NXNL2 and tauopathies, we studied the Nxnl2 knockout mouse (Nxnl2 -/- ). We established the expression pattern of the Nxnl2 gene in the brain using a Nxnl2 reporter mouse line, and characterized the behavior of the Nxnl2 -/- mouse at 2 months of age. Additionally, long term potentiation and metabolomic from hippocampal specimens were collected at 2 months of age. We studied TAU oligomerization, phosphorylation and aggregation in Nxnl2 -/- brain at 18 months of age. Finally, newborn Nxnl2 -/- mice were treated with adeno-associated viral vectors encoding for RdCVF2, RdCVF2L or both and measured the effect of this therapy on long-term potential, glucose metabolism and late-onset tauopathy. Nxnl2 -/- mice at 2 months of age showed severe behavioral deficiency in fear, pain sensitivity, coordination, learning and memory. The Nxnl2 -/- also showed deficits in long-term potentiation, demonstrating that the Nxnl2 gene is involved in regulating brain functions. Dual delivery of RdCVF2 and RdCVF2L in newborn Nxnl2 -/- mice fully correct long-term potentiation through their synergistic action. The expression pattern of the Nxnl2 gene in the brain shows a predominant expression in circumventricular organs, such as the area postrema. Glucose metabolism of the hippocampus of Nxnl2 -/- mice at 2 months of age was reduced, and was not corrected by gene therapy. At 18-month-old Nxnl2 -/- mice showed brain stigmas of tauopathy, such as oligomerization, phosphorylation and aggregation of TAU. This late-onset tauopathy can be prevented, albeit with modest efficacy, by recombinant AAVs administrated to newborn mice. The Nxnl2 -/- mice have memory dysfunction at 2-months that resembles mild-cognitive impairment and at 18-months exhibit tauopathy, resembling to the progression of Alzheimer's disease. We propose the Nxnl2 -/- mouse is a model to study multistage aged related neurodegenerative diseases. The NXNL2 metabolic and redox signaling is a new area of therapeutic research in neurodegenerative diseases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

KCC2, best known as the neuron-specific chloride-extruder that sets the strength and polarity of GABAergic currents during neuronal maturation, is a multifunctional molecule that can regulate cytoskeletal dynamics via its C-terminal domain (CTD). We describe the molecular and cellular mechanisms involved in the multiple functions of KCC2 and its splice variants, ranging from developmental apoptosis and the control of early network events to the formation and plasticity of cortical dendritic spines. The versatility of KCC2 actions at the cellular and subcellular levels is also evident in mature neurons during plasticity, disease, and aging. Thus, KCC2 has emerged as one of the most important molecules that shape the overall neuronal phenotype., Competing Interests: Declaration of Interests The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Neuronal Cl - homeostasis is regulated by the activity of two cation chloride co-transporters (CCCs), the K + -Cl - cotransporter KCC2 and the Na + -K + -Cl - cotransporter NKCC1, which are primarily extruding and importing chloride in neurons, respectively. Several neurological and psychiatric disorders including epilepsy, neuropathic pain, schizophrenia and autism are associated with altered neuronal chloride (Cl - ) homeostasis. A current view is that the accumulation of intracellular Cl - in neurons as a result of KCC2 down-regulation and/or NKCC1 up-regulation may weaken inhibitory GABA signaling and thereby promote the development of pathological activities. CCC activity is determined mainly by their level of expression in the plasma membrane. Furthermore, CCCs undergo "diffusion-trapping" in the membrane, a mechanism that is rapidly adjusted by activity-dependent post-translational modifications i.e. phosphorylation/dephosphorylation of key serine and threonine residues. This represents probably the most rapid cellular mechanism for adapting CCC function to changes in neuronal activity. Therefore, interfering with these mechanisms may help restoring Cl - homeostasis and inhibition under pathological conditions. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Key Points: Cation-chloride cotransporters (CCCs) play a critical role in controlling the efficacy and polarity of GABA A receptor (GABA A R)-mediated transmission in the brain, yet their expression and function in GABAergic interneurons has been overlooked. We compared the polarity of GABA signalling and the function of CCCs in mouse hippocampal pyramidal neurons and parvalbumin-expressing interneurons. Under resting conditions, GABA A R activation was mostly depolarizing and yet inhibitory in both cell types. KCC2 blockade further depolarized the reversal potential of GABA A R-mediated currents often above action potential threshold. However, during repetitive GABA A R activation, the postsynaptic response declined independently of the ion flux direction or KCC2 function, suggesting intracellular chloride build-up is not responsible for this form of plasticity. Our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal pyramidal neurons and parvalbumin interneurons., Abstract: Transmembrane chloride gradients govern the efficacy and polarity of GABA signalling in neurons and are usually maintained by the activity of cation-chloride cotransporters, such as KCC2 and NKCC1. Whereas their role is well established in cortical principal neurons, it remains poorly documented in GABAergic interneurons. We used complementary electrophysiological approaches to compare the effects of GABA A receptor (GABA A R) activation in adult mouse hippocampal parvalbumin interneurons (PV-INs) and pyramidal cells (PCs). Loose cell-attached, tight-seal and gramicidin-perforated patch recordings all show GABA A R-mediated transmission is slightly depolarizing and yet inhibitory in both PV-INs and PCs. Focal GABA uncaging in whole-cell recordings reveal that KCC2 and NKCC1 are functional in both PV-INs and PCs but differentially contribute to transmembrane chloride gradients in their soma and dendrites. Blocking KCC2 function depolarizes the reversal potential of GABA A R-mediated currents in PV-INs and PCs, often beyond firing threshold, showing KCC2 is essential to maintain the inhibitory effect of GABA A Rs. Finally, we show that repetitive 10 Hz activation of GABA A Rs in both PV-INs and PCs leads to a progressive decline of the postsynaptic response independently of the ion flux direction or KCC2 function. This suggests intraneuronal chloride build-up may not predominantly contribute to activity-dependent plasticity of GABAergic synapses in this frequency range. Altogether our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal PV-INs and PCs and suggest KCC2 downregulation in the pathology may affect the valence of GABA signalling in both cell types., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

KCC2 regulates neuronal transmembrane chloride gradients and thereby controls GABA signaling in the brain. KCC2 downregulation is observed in numerous neurological and psychiatric disorders. Paradoxical, excitatory GABA signaling is usually assumed to contribute to abnormal network activity underlying the pathology. We tested this hypothesis and explored the functional impact of chronic KCC2 downregulation in the rat dentate gyrus. Although the reversal potential of GABAA receptor currents is depolarized in KCC2 knockdown neurons, this shift is compensated by depolarization of the resting membrane potential. This reflects downregulation of leak potassium currents. We show KCC2 interacts with Task-3 (KCNK9) channels and is required for their membrane expression. Increased neuronal excitability upon KCC2 suppression altered dentate gyrus rhythmogenesis, which could be normalized by chemogenetic hyperpolarization. Our data reveal KCC2 downregulation engages complex synaptic and cellular alterations beyond GABA signaling that perturb network activity thus offering additional targets for therapeutic intervention., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The main inhibitory neurotransmitter receptors in the adult central nervous system (CNS) are type A γ-aminobutyric acid receptors (GABA A Rs) and glycine receptors (GlyRs). Synaptic responses mediated by GlyR and GABA A R display a hyperpolarizing shift during development. This shift relies mainly on the developmental up-regulation of the K + -Cl - co-transporter KCC2 responsible for the extrusion of Cl - . In mature neurons, altered KCC2 function-mainly through increased endocytosis-leads to the re-emergence of depolarizing GABAergic and glycinergic signaling, which promotes hyperexcitability and pathological activities. Identifying signaling pathways and molecular partners that control KCC2 surface stability thus represents a key step in the development of novel therapeutic strategies. Here, we present our current knowledge on the cellular and molecular mechanisms governing the plasma membrane turnover rate of the transporter under resting conditions and in response to synaptic activity. We also discuss the notion that KCC2 lateral diffusion is one of the first parameters modulating the transporter membrane stability, allowing for rapid adaptation of Cl - transport to changes in neuronal activity.

The fundamental role of the brain-specific myelin transcription factor 1-like (MYT1L) gene in cases of intellectual disability and in the etiology of neurodevelopmental disorders is increasingly recognized. Yet, its function remains under-investigated. Here, we identify a network of helix-loop-helix (HLH) transcriptional regulators controlled by MYT1L, as indicated by our analyses in human neural stem cells and in the human brain. Using cell-based knockdown approaches and microarray analyses we found that (1) MYT1L is required for neuronal differentiation and identified ID1, a HLH inhibitor of premature neurogenesis, as a target. (2) Although MYT1L prevented expression of ID1, it induced expression of a large number of terminal differentiation genes. (3) Consistently, expression of MYT1L in the human brain coincided with neuronal maturation and inversely correlated with that of ID1 and ID3 throughout the lifespan. (4) Genetic polymorphisms that reduced expression of MYT1L in the hippocampus resulted in increased expression of ID1 and ID3, decreased levels of the proneural basic HLH (bHLH) transcriptional regulators TCF4 and NEUROD6 and decreased expression of genes involved in long-term potentiation and synaptic transmission, cancer and neurodegeneration. Furthermore, our neuroimaging analyses indicated that MYT1L expression associated with hippocampal volume and activation during episodic memory recall, as measured by blood-oxygen-level-dependent (BOLD) signals. Overall, our findings suggest that MYT1L influences memory-related processes by controlling a neuronal proliferation/differentiation switch of ID-bHLH factors.

The K + -Cl - co-transporter KCC2 (SLC12A5) tunes the efficacy of GABA A receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl - ] i . KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABA A receptor (GABA A R)-mediated transmission in mature hippocampal neurons. Enhancing GABA A R-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl - as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl - -sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl - ] i to GABA A R activity.

Brain development involves extensive migration of neurons. Microtubules (MTs) are key cellular effectors of neuronal displacement that are assembled from α/β-tubulin heterodimers. Mutation of the α-tubulin isotype TUBA1A is associated with cortical malformations in humans. In this study, we provide detailed in vivo and in vitro analyses of Tuba1a mutants. In mice carrying a Tuba1a missense mutation (S140G), neurons accumulate, and glial cells are dispersed along the rostral migratory stream in postnatal and adult brains. Live imaging of Tuba1a-mutant neurons revealed slowed migration and increased neuronal branching, which correlated with directionality alterations and perturbed nucleus-centrosome (N-C) coupling. Tuba1a mutation led to increased straightness of newly polymerized MTs, and structural modeling data suggest a conformational change in the α/β-tubulin heterodimer. We show that Tuba8, another α-tubulin isotype previously associated with cortical malformations, has altered function compared with Tuba1a. Our work shows that Tuba1a plays an essential, noncompensated role in neuronal saltatory migration in vivo and highlights the importance of MT flexibility in N-C coupling and neuronal-branching regulation during neuronal migration., (© 2017 Belvindrah et al.)

The structure and function of spines and excitatory synapses are under the dynamic control of multiple signalling networks. Although tyrosine phosphorylation is involved, its regulation and importance are not well understood. Here we study the role of Pyk2, a non-receptor calcium-dependent protein-tyrosine kinase highly expressed in the hippocampus. Hippocampal-related learning and CA1 long-term potentiation are severely impaired in Pyk2-deficient mice and are associated with alterations in NMDA receptors, PSD-95 and dendritic spines. In cultured hippocampal neurons, Pyk2 has autophosphorylation-dependent and -independent roles in determining PSD-95 enrichment and spines density. Pyk2 levels are decreased in the hippocampus of individuals with Huntington and in the R6/1 mouse model of the disease. Normalizing Pyk2 levels in the hippocampus of R6/1 mice rescues memory deficits, spines pathology and PSD-95 localization. Our results reveal a role for Pyk2 in spine structure and synaptic function, and suggest that its deficit contributes to Huntington's disease cognitive impairments.

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

Expression of the neuronal K/Cl transporter KCC2 is tightly regulated throughout development and by both normal and pathological neuronal activity. Changes in KCC2 expression have often been associated with altered chloride homeostasis and GABA signaling. However, recent evidence supports a role of KCC2 in the development and function of glutamatergic synapses through mechanisms that remain poorly understood. Here we show that suppressing KCC2 expression in rat hippocampal neurons precludes long-term potentiation of glutamatergic synapses specifically by preventing activity-driven membrane delivery of AMPA receptors. This effect is independent of KCC2 transporter function and can be accounted for by increased Rac1/PAK- and LIMK-dependent cofilin phosphorylation and actin polymerization in dendritic spines. Our results demonstrate that KCC2 plays a critical role in the regulation of spine actin cytoskeleton and gates long-term plasticity at excitatory synapses in cortical neurons., (Copyright © 2015 the authors 0270-6474/15/3515772-15$15.00/0.)

Benzodiazepines (BZDs) are widely used in the treatment of a variety of neurological and psychiatric conditions including anxiety, insomnia and epilepsy. BZDs are thought to act predominantly by affecting the gating of GABAA receptor channels, resulting in enhanced GABA-mediated currents in neurons. However, mutations mimicking the effect of BZDs on GABAAR channel gating have been shown to also impact the membrane dynamics and synaptic anchoring of the receptors. Here, using single molecule tracking combined with electrophysiological recordings, we show that BZD ligands rapidly influence the dynamic behavior of GABAARs in hippocampal neurons. Application of the inverse BZD agonist DMCM rapidly increased the diffusion and reduced the clustering of GABAARs at synapses, resulting in reduced postsynaptic currents. Conversely, the BZD full agonist diazepam had little effect at rest but reduced lateral diffusion and increased synaptic stabilization and clustering of GABAARs upon sustained neuronal activity, resulting in enhanced potency of inhibitory synapses. These effects occurred in the absence of detectable changes in gephyrin clusters, suggesting they did not reflect a rapid dispersion of the synaptic scaffold. Thus, alterations of the diffusion and synaptic anchoring of GABAARs represent a novel, unsuspected mechanism through which BZDs rapidly modulate GABA signaling in central neurons., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: A long-term in vitro preparation of diseased brain tissue would facilitate work on human pathologies. Organotypic tissue cultures retain an appropriate neuronal form, spatial arrangement, connectivity and electrical activity over several weeks. However, they are typically prepared with tissue from immature animals. In work using tissue from adult animals or humans, survival times longer than a few days have not been reported and it is not clear that pathological neuronal activities are retained., New Method: We modified tissue preparation procedures and used a defined culture medium to make organotypic cultures of temporal lobe tissue obtained after operations on adult patients with pharmaco-resistant mesial temporal lobe epilepsies., Results: Organototypic culture preparation and maintenance techniques were judged on criteria of morphology and the generation of epileptiform activities. Short-duration (30-100 ms) interictal-like population activities were initiated spontaneously in either the subiculum, dentate gyrus or the CA2/CA3 region, but not the cortex, for up to 3-4 weeks in culture. Ictal-like discharges, of duration greater than 10s, were induced by convulsants. Epileptiform activities were modulated by both glutamatergic and GABAergic receptor antagonists., Comparison With Existing Methods: Our methods now permit the maintenance in organotypic culture of epileptic adult human tissue, generating appropriate epileptiform activity over 3-4 weeks., Conclusions: We have shown that characteristic morphology and pathological activities are maintained in organotypic cultures of adult human tissue. These cultures should permit studies on the effects of prolonged drug treatments and long-term procedures such as viral transduction., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The neuronal K/Cl transporter KCC2 exports chloride ions and thereby influences the efficacy and polarity of GABA signaling in the brain. KCC2 is also critical for dendritic spine morphogenesis and the maintenance of glutamatergic transmission in cortical neurons. Because KCC2 plays a pivotal role in the function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. Here, we studied the impact of membrane diffusion and clustering on KCC2 function. KCC2 forms clusters in the vicinity of both excitatory and inhibitory synapses. Using quantum-dot-based single-particle tracking on rat primary hippocampal neurons, we show that KCC2 is slowed down and confined at excitatory and inhibitory synapses compared with extrasynaptic regions. However, KCC2 escapes inhibitory synapses faster than excitatory synapses, reflecting stronger molecular constraints at the latter. Interfering with KCC2-actin interactions or inhibiting F-actin polymerization releases diffusion constraints on KCC2 at excitatory but not inhibitory synapses. Thus, F-actin constrains KCC2 diffusion at excitatory synapses, whereas KCC2 is confined at inhibitory synapses by a distinct mechanism. Finally, increased neuronal activity rapidly increases the diffusion coefficient and decreases the dwell time of KCC2 at excitatory synapses. This effect involves NMDAR activation, Ca(2+) influx, KCC2 S940 dephosphorylation and calpain protease cleavage of KCC2 and is accompanied by reduced KCC2 clustering and ion transport function. Thus, activity-dependent regulation of KCC2 lateral diffusion and clustering allows for a rapid regulation of chloride homeostasis in neurons.

Dentate gyrus granule cells (GCs) have been suggested to synthesize both GABA and glutamate immediately after birth and under pathological conditions in the adult. Expression of the GABA synthesizing enzyme GAD67 by GCs during the first few weeks of postnatal development may then allow for transient GABA synthesis and synaptic release from these cells. Here, using the GAD67-EGFP transgenic strain G42, we explored the phenotype of GAD67-expressing GCs in the mouse dentate gyrus. We report a transient, GAD67-driven EGFP expression in differentiating GCs throughout ontogenesis. EGFP expression correlates with the expression of GAD and molecular markers of GABA release and uptake in 2-4 weeks post-mitotic GCs. These rather immature cells are able to fire action potentials (APs) and are synaptically integrated in the hippocampal network. Yet they show physiological properties that differentiate them from mature GCs. Finally, GAD67-expressing GCs express a specific complement of GABAA receptor subunits as well as distinctive features of synaptic and tonic GABA signaling. Our results reveal that GAD67 expression in dentate gyrus GCs is a transient marker of late differentiation that persists throughout life and the G42 strain may be used to visualize newborn GCs at a specific, well-defined differentiation stage.

Hippocampal parvalbumin-expressing interneurons (PV INs) provide fast and reliable GABAergic signalling to principal cells and orchestrate hippocampal ensemble activities. Precise coordination of principal cell activity by PV INs relies in part on the efficacy of excitatory afferents that recruit them in the hippocampal network. Feed-forward (FF) inputs in particular from Schaffer collaterals influence spike timing precision in CA1 principal cells whereas local feedback (FB) inputs may contribute to pacemaker activities. Although PV INs have been shown to undergo activity-dependent long term plasticity, how both inputs are modulated during principal cell firing is unknown. Here we show that FF and FB synapses onto PV INs are endowed with distinct postsynaptic glutamate receptors which set opposing long-term plasticity rules. Inward-rectifying AMPA receptors (AMPARs) expressed at both FF and FB inputs mediate a form of anti-Hebbian long term potentiation (LTP), relying on coincident membrane hyperpolarization and synaptic activation. In contrast, FF inputs are largely devoid of NMDA receptors (NMDARs) which are more abundant at FB afferents and confer on them an additional form of LTP with Hebbian properties. Both forms of LTP are expressed with no apparent change in presynaptic function. The specific endowment of FF and FB inputs with distinct coincidence detectors allow them to be differentially tuned upon high frequency afferent activity. Thus, high frequency (>20 Hz) stimulation specifically potentiates FB, but not FF afferents. We propose that these differential, input-specific learning rules may allow PV INs to adapt to changes in hippocampal activity while preserving their precisely timed, clockwork operation.

Dentate gyrus granule cells have been suggested to corelease GABA and glutamate both in juvenile animals and under pathological conditions in adults. Although mossy fiber terminals (MFTs) are known to express glutamic acid decarboxylase (GAD) in early postnatal development, the functional role of GABA synthesis in MFTs remains controversial, and direct evidence for synaptic GABA release from MFTs is missing. Here, using GAD67-GFP transgenic mice, we show that GAD67 is expressed only in a population of immature granule cells in juvenile animals. We demonstrate that GABA can be released from these cells and modulate mossy fiber excitability through activation of GABAB autoreceptors. However, unitary postsynaptic currents generated by individual, GAD67-expressing granule cells are purely glutamatergic in all postsynaptic cell types tested. Thus GAD67 expression does not endow dentate gyrus granule cells with a full GABAergic phenotype and GABA primarily instructs the pre- rather than the postsynaptic element.

Neuronal activity modulates the membrane diffusion of postsynaptic γ-aminobutyric acid (GABA)(A) receptors (GABA(A)Rs), thereby regulating the efficacy of GABAergic synapses. The K289M mutation in GABA(A)Rs subunit γ2 has been associated with the generalized epilepsy with febrile seizures plus (GEFS+) syndrome. This mutation accelerates receptor deactivation and therefore reduces inhibitory synaptic transmission. Yet, it is not clear why this mutation specifically promotes febrile seizures. We show that upon raising temperature both the number of GABA(A)Rs clusters and the frequency of miniature inhibitory postsynaptic currents decreased in neurons expressing the K289M mutant but not wild-type (WT) recombinant γ2. Single-particle tracking experiments revealed that raising temperature increases the membrane diffusion of synaptic GABA(A)Rs containing the K289M mutant but not WT recombinant γ2. This effect was mediated by enhanced neuronal activity as it was blocked by glutamate receptor antagonists and was mimicked by the convulsant 4-aminopyridine. Our data suggest the K289M mutation in γ2 confers GABA(A)Rs with enhanced sensitivity of their membrane diffusion to neuronal activity. Enhanced activity during hyperthermia may then trigger the escape of receptors from synapses and thereby further reduce the efficacy of GABAergic inhibition. Alteration of the membrane diffusion of neurotransmitter receptors therefore represents a new mechanism in human epilepsy.

Vezatin is an integral membrane protein associated with cell-cell adhesion complex and actin cytoskeleton. It is expressed in the developing and mature mammalian brain, but its neuronal function is unknown. Here, we show that Vezatin localizes in spines in mature mouse hippocampal neurons and codistributes with PSD95, a major scaffolding protein of the excitatory postsynaptic density. Forebrain-specific conditional ablation of Vezatin induced anxiety-like behavior and impaired cued fear-conditioning memory response. Vezatin knock-down in cultured hippocampal neurons and Vezatin conditional knock-out in mice led to a significantly increased proportion of stubby spines and a reduced proportion of mature dendritic spines. PSD95 remained tethered to presynaptic terminals in Vezatin-deficient hippocampal neurons, suggesting that the reduced expression of Vezatin does not compromise the maintenance of synaptic connections. Accordingly, neither the amplitude nor the frequency of miniature EPSCs was affected in Vezatin-deficient hippocampal neurons. However, the AMPA/NMDA ratio of evoked EPSCs was reduced, suggesting impaired functional maturation of excitatory synapses. These results suggest a role of Vezatin in dendritic spine morphogenesis and functional synaptic maturation.

The K-Cl co-transporter KCC2 plays multiple roles in the physiology of central neurons and alterations of its function and/or expression are associated with several neurological conditions. By regulating intraneuronal chloride homeostasis, KCC2 strongly influences the efficacy and polarity of the chloride-permeable γ-aminobutyric acid (GABA) type A and glycine receptor (GlyR) mediated synaptic transmission. This appears particularly critical for the development of neuronal circuits as well as for the dynamic control of GABA and glycine signaling in mature networks. The activity of the transporter is also associated with transmembrane water fluxes which compensate solute fluxes associated with synaptic activity. Finally, KCC2 interaction with the actin cytoskeleton appears critical both for dendritic spine morphogenesis and the maintenance of glutamatergic synapses. In light of the pivotal role of KCC2 in the maturation and function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. These include development and activity-dependent modifications both at the transcriptional and post-translational levels. We emphasize the importance of post-translational mechanisms such as phosphorylation and dephosphorylation, oligomerization, cell surface stability, clustering and membrane diffusion for the rapid and dynamic regulation of KCC2 function.

The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.

Expression of green fluorescent protein (GFP), its more fluorescent mutant forms (e.g., EGFP [enhanced GFP]), or their fusion protein derivatives, affords a number of informative possibilities in cellular neuroscience. EGFP is a soluble protein and appears to be homogeneously distributed within the cytosol of neurons when expressed. Thus, it reveals the structure of the neuron, including the cell body, and axonal and dendritic arbors. It is also sufficiently bright to reveal detailed structures such as axonal boutons and dendritic spines. When expressed as a fusion protein, EGFP can provide information about the distribution characteristics of the proteins within neurons. Furthermore, during single-cell electrophysiological studies, such expression can direct the investigator to record from a cell carrying a foreign gene. In this protocol, we describe the use of the Sindbis pseudovirus expression system to deliver GFP to neurons. Sindbis is a member of the alphaviruses, which are plus-stranded RNA viruses. This protocol uses the DH(26S) strain, which preferentially infects neurons over glia (50:1). Two infection methods are given: one for dissociated hippocampal cultured neurons and one for organotypic hippocampal slices.

Heterozygous mutations in the CLCN2 gene encoding the voltage-gated chloride channel CLC2 have been identified in patients with idiopathic generalized epilepsy (IGE). Yet the involvement of CLCN2 in epilepsy remains controversial. To investigate the involvement of CLCN2 in another independent sample, we screened 52 unrelated patients from IGE families and 23 patients with Doose syndrome for mutations in CLCN2. No mutations were found in patients with Doose syndrome. In three unrelated IGE families, we identified two novel missense mutations, p.Arg235Gln and p.Arg577Gln, which were absent in large ethnically-matched control populations, and one novel p.Arg644Cys variant, which was also found in five Indian controls. Functional characterization of mutant channels using heterologous expression in mammalian cells and whole-cell patch-clamp recordings revealed faster deactivation kinetics as the major phenotype of both missense mutations. This finding predicts a loss of function that may contribute to intracellular chloride accumulation or neuronal hyperexcitability. However, the incomplete segregation of the mutations among affected members and the transmission by unaffected parents suggests that these CLCN2 mutations alone are not sufficient to induce epilepsy. They may instead represent susceptibility factors among other so far undetected genetic alterations in the respective families., (2009 Wiley-Liss, Inc.)

Background: Epidemiological studies have linked maternal infection during pregnancy to later development of neuropsychiatric disorders in the offspring. In mice, experimental inflammation during embryonic development impairs behavioral and cognitive performances in adulthood. Synaptic dysfunctions may be at the origin of cognitive impairments, however the link between prenatal inflammation and synaptic defects remains to be established., Methodology/principal Findings: In this study, we show that prenatal alteration of microglial function, including inflammation, induces delayed synaptic dysfunction in the adult. DAP12 is a microglial signaling protein expressed around birth, mutations of which in the human induces the Nasu-Hakola disease, characterized by early dementia. We presently report that synaptic excitatory currents in mice bearing a loss-of-function mutation in the DAP12 gene (DAP12(KI) mice) display enhanced relative contribution of AMPA. Furthermore, neurons from DAP12(KI) P0 pups cultured without microglia develop similar synaptic alterations, suggesting that a prenatal dysfunction of microglia may impact synaptic function in the adult. As we observed that DAP12(KI) microglia overexpress genes for IL1beta, IL6 and NOS2, which are inflammatory proteins, we analyzed the impact of a pharmacologically-induced prenatal inflammation on synaptic function. Maternal injection of lipopolysaccharides induced activation of microglia at birth and alteration of glutamatergic synapses in the adult offspring. Finally, neurons cultured from neonates born to inflamed mothers and cultured without microglia also displayed altered neuronal activity., Conclusion/significance: Our results demonstrate that prenatal inflammation is sufficient to induce synaptic alterations with delay. We propose that these alterations triggered by prenatal activation of microglia provide a cellular basis for the neuropsychiatric defects induced by prenatal inflammation.

GABA acts on GABA(A) receptors to evoke both phasic inhibitory synaptic events and persistent, tonic currents. The gamma2 subunit of the GABA(A) receptor is involved in both phasic and tonic signaling in the hippocampus. Several mutations of this subunit are linked to human epileptic syndromes with febrile seizures, yet it is not clear how they perturb neuronal activity. Here, we examined the expression and functional impact of recombinant gamma2 in hippocampal neurons. We show that the K289M mutation has no effect on membrane trafficking and synaptic aggregation of recombinant gamma2, but accelerates the decay of synaptic currents. In contrast, the R43Q mutation primarily reduces surface expression of recombinant gamma2. However, it has no dominant effect on synaptic currents but instead reduces tonic GABA currents, at least in part by reducing surface expression of the alpha5 subunit. Our data suggests that the phenotypic specificity of mutations affecting the GABA(A) receptor gamma2 gene may result from different actions specific to distinct modes of GABAergic signaling.

Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals.

Several proteins are expressed in both immune and nervous systems. However, their putative nonimmune functions in the brain remain poorly understood. KARAP/DAP12 is a transmembrane polypeptide associated with cell-surface receptors in hematopoeitic cells. Its mutation in humans induces Nasu-Hakola disease, characterized by presenile dementia and demyelinization. However, alteration of white matter occurs months after the onset of neuropsychiatric symptoms, suggesting that other neuronal alterations occur in the early phases of the disease. We hypothesized that KARAP/DAP12 may impact synaptic function. In mice deficient for KARAP/DAP12 function, long-term potentiation was enhanced and was partly NMDA receptor (NMDAR) independent. This effect was accompanied by changes in synaptic glutamate receptor content, as detected by the increased rectification of AMPA receptor EPSCs and increased sensitivity of NMDAR EPSCs to ifenprodil. Biochemical analysis of synaptic proteins confirmed these electrophysiological data. In mutants, the AMPA receptor GluR2 subunit expression was decreased only in the postsynaptic densities but not in the whole membrane fraction, demonstrating specific impairment of synaptic receptor accumulation. Alteration of the BNDF-tyrosine kinase receptor B (TrkB) signaling in the mutant was demonstrated by the dramatic decrease of synaptic TrkB with no change in other regulatory or scaffolding proteins. Finally, KARAP/DAP12 was detected only in microglia but not in neurons, astrocytes, or oligodendrocytes. KARAP/DAP12 may thus alter microglial physiology and subsequently synaptic function and plasticity through a novel microglia-neuron interaction.

Presynaptic calcium influx at most excitatory central synapses is carried by both Cav2.1 and Cav2.2 channels. The kinetics and modulation of Cav2.1 and Cav2.2 channels differ and may affect presynaptic calcium influx. We compared release dynamics at CA3/CA1 synapses in rat hippocampus after selective blockade of either channel subtype and subsequent quantal content restoration. Selective blockade of Cav2.1 channels enhanced paired-pulse facilitation, whereas blockade of Cav2.2 channels decreased it. This effect was observed at short (50 msec) but not longer (500 msec) intervals and was maintained during prolonged bursts of presynaptic activity. It did not reflect differences in the distance of the channels from the calcium sensor. The suppression of this effect by preincubation with the G(o/i)-protein inhibitor pertussis toxin suggests instead that high-frequency stimulation relieves inhibition of Cav2.2 by G(o/i), thereby increasing the number of available channels.

Long-term, activity-driven synaptic plasticity allows neuronal networks to constantly and durably adjust synaptic gains between synaptic partners. These processes have been proposed to serve as a substrate for learning and memory. Long-term synaptic potentiation (LTP) has been observed at many central excitatory synapses and perhaps most extensively studied at Schaffer collaterals synapses onto hippocampal CA1 neurons. Multiple contradictory models were proposed to account for this form of LTP. However, recent evidence suggests that some synapses are initially devoid of functional AMPA receptors which can be incorporated during LTP. This new model appears to account for most, but not all, properties of this form of plasticity. Indeed, several mechanisms seem to act in parallel to specifically enhance AMPA-receptor mediated synaptic transmission.

Some forms of activity-dependent synaptic potentiation require the activation of postsynaptic Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Activation of CaMKII has been shown to phosphorylate the glutamate receptor 1 subunit of the AMPA receptor (AMPAR), thereby affecting some of the properties of the receptor. Here, a recombinant, constitutively active form of alphaCaMKII tagged with the fluorescent marker green fluorescent protein (GFP) [alphaCaMKII(1-290)-enhanced GFP (EGFP)] was expressed in CA1 pyramidal neurons from hippocampal slices. The changes in glutamatergic transmission onto these cells were analyzed. AMPA but not NMDA receptor-mediated EPSCs were specifically potentiated in infected compared with nearby noninfected neurons. This potentiation was associated with a reduction in the proportion of synapses devoid of AMPARs. In addition, expression of alphaCaMKII(1-290)-EGFP increased the quantal size of AMPAR-mediated responses. This effect reflected, at least in part, an increased unitary conductance of the channels underlying the EPSCs. These results reveal that several key features of long-term potentiation of hippocampal glutamatergic synapses are reproduced by the sole activity of alphaCaMKII.

Synaptic transmission relies on both the gain and the dynamics of synapses. Activity-dependent changes in synaptic gain are well-documented at excitatory synapses and may represent a substrate for information storage in the brain. Here we examine the mechanisms of changes in transmission dynamics at excitatory synapses. We show that paired-pulse ratios (PPRs) of AMPAR and NMDAR EPSCs onto dentate gyrus granule cells are often different; this difference is reduced during LTP, reflecting PPR changes of AMPAR but not NMDAR EPSCs. Presynaptic manipulations, however, produce parallel changes in AMPAR and NMDAR EPSCs. LTP at these synapses reflects a reduction in the proportion of silent synapses lacking functional AMPARs. Changes in PPR during LTP therefore reflect the initial difference between PPRs of silent and functional synapses. Functional conversion of silent synapses permits postsynaptic sampling from additional release sites and thereby affects the dynamics and gain of signals conveyed between neurons.

1. Paired recordings from monosynaptically connected CA3 interneurons and pyramidal cells of rat hippocampal slice cultures were used to compare the modulation of GABA release at synapses from distinct interneurons. 2. The group II metabotropic glutamate receptor (mGluR) agonist (2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl) glycine (DCG-IV, 5 muM) reduced the amplitude of IPSPs originating from stratum radiatum but not stratum oriens interneurons. In contrast, the GABAB receptor agonist (-)baclofen (10 muM) reduced the amplitude of unitary IPSPs elicited by all interneurons. 3. IPSPs mediated by stratum oriens interneurons were unaffected by the N-type calcium channel blocker omega-conotoxin MVIIA (1 muM) but were suppressed by the P/Q-type blocker omega-agatoxin IVA (200 nM). In contrast, IPSPs mediated by stratum radiatum interneurons were abolished by omega-conotoxin MVIIA. 4. Transmission dynamics were different at synapses from the two groups of interneurons. IPSPs mediated by stratum oriens interneurons showed marked paired-pulse depression (PPD) at intervals of 50 400 ms. IPSPs mediated by stratum radiatum interneurons showed paired-pulse facilitation (PPF) at 50 ms and PPD at longer intervals. 5. The amplitude of unitary IPSPs from all interneurons was unaffected by the GABAB receptor antagonist CGP52432 (2 muM) as was PPD at both 50 and 400 ms intervals. However, CGP52432 did reduce PPD of extracellularly evoked IPSPs. 6. Our results show that two groups of inhibitory synapses impinging onto CA3 pyramidal cells can be distinguished according to their dynamic and modulatory properties.

To elucidate mechanisms that control and execute activity-dependent synaptic plasticity, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal neurons. Long-term potentiation (LTP) or increased activity of the calcium/calmodulin-dependent protein kinase II (CaMKII) induced delivery of tagged AMPA-Rs into synapses. This effect was not diminished by mutating the CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but was blocked by mutating a predicted PDZ domain interaction site. These results show that LTP and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the association between GluR1 and a PDZ domain protein.

Thin slices (200-300 microm) of adrenal glands were prepared from Wistar rats. Patch-clamp recordings were made from visually identified chromaffin cells using the whole-cell and amphotericin B perforated-patch techniques. Electrophysiological properties of chromaffin cells in slices were similar to those in cultured cells. Catecholamine release from single chromaffin cells or cell clusters in slices was also measured by amperometry. Immunostaining of slices with an antineurofilament antibody revealed the presence of neuronal fibers. Acetylcholine release was stimulated either by raising external [K+] or by focally applying voltage pulses. Nicotinic excitatory postsynaptic currents (EPSCs) were detected, ranging from 20 pA to several hundreds of pA. Amplitude distributions of spontaneous EPSCs revealed clear equidistant peaks, supporting a quantal model for acetylcholine release onto chromaffin cells. The adrenal slice preparation therefore appears to be an excellent model for studying both the cholinergic innervation of chromaffin cells as well as catecholamine release from these cells.

Miniature, gamma-aminobutyric acid A receptor mediated inhibitory postsynaptic currents (mIPSCs) were recorded from CA3 pyramidal cells in hippocampal slice cultures using whole-cell techniques in the presence of tetrodotoxin. The kinetics and amplitudes of the mIPSCs were analyzed with the aim of determining whether subclasses of events arising from distinct populations of presynaptic interneurons could be distinguished. Histograms of mIPSC amplitude, rise time constant, and decay time constant were all positively skewed, but discrete subsets of events could not be distinguished. The positive skew did not appear to result from electrotonic filtering of distal synaptic currents because there was no correlation among mIPSC amplitudes and the kinetic parameters. Analysis of the intervals between mIPSCs indicated that each event occurred independently. The analysis of spontaneous mIPSCs does not provide evidence of the innervation of pyramidal cells by heterogeneous interneurons.

Transmitter release at most central synapses depends on multiple types of calcium channels. Identification of the channels mediating GABA release in hippocampus is complicated by the heterogeneity of interneurons. Unitary IPSPs were recorded from pairs of inhibitory and pyramidal cells in hippocampal slice cultures. The N-type channel antagonist omega-conotoxin MVIIA abolished IPSPs generated by interneurons in st. radiatum, whereas the P/Q-type antagonist omega-agatoxin IVA had no effect. In contrast, omega-agatoxin IVA abolished IPSPs generated by st. lucidum and st. oriens interneurons, but omega-conotoxin MVIIA had no effect. After unitary IPSPs were blocked by toxin, transmission could not be restored by increasing presynaptic calcium entry. The axons of the two types of interneurons terminated within distinct strata of area CA3. Thus, GABA release onto pyramidal cells, unlike glutamate release, is mediated entirely by either N- or P-type calcium channels, depending on the presynaptic cell and the postsynaptic location of the synapse.

Paired recording is a powerful and versatile tool to examine communication between and within neurones. This technique has provided new insights in studies of synaptic function and plasticity, of neuronal integration, and of the decoding of neuronal circuits. Recent studies using dual recordings in combination with morphology have successfully determined the number of transmitter release sites between synaptically connected neurones. Important progress in understanding the dynamics of signal transmission within individual cells has been made possible using infra-red microscopy, which permits dual recordings from visualized somatic and dendritic sites on a single neurone.

The effects of the benzodiazepine agonist midazolam on GABAA receptor-mediated inhibition were investigated in area CA3 of hippocampal slice cultures. Midazolam (100 nM) increased the decay time constant (tau OFF) of miniature inhibitory postsynaptic currents (mIPSCs) recorded from pyramidal cells by approximately 40%, but did not significantly affect their activation rate or amplitude, consistent with saturation of postsynaptic GABAA receptors by a quantum of GABA. Non-stationary variance analysis of mIPSCs revealed that the unitary conductance of synaptic GABAA channels (approximately 31 pS) was unaffected by midazolam. Midazolam increased not only the tau OFF (51%), but also the amplitude (23%) of unitary IPSPs, recorded from pairs of monosynaptically connected inhibitory and pyramidal cells. Simulation of unitary IPSPs indicated that the increased amplitude was primarily due to the slow time constant of pyramidal cells. Finally, the mean amplitude, tau OFF, and single-channel conductance of mIPSCs recorded in cultures chronically exposed to midazolam (0.1-10 microM) for 2 weeks were not different from control mIPSCs, nor was their response to midazolam. We conclude that benzodiazepines increase synaptic GABAA channel open time, as described previously, and that this results in an increase in both the amplitude and duration of IPSPs in pyramidal cells.

1. The effects of metabotropic glutamate receptor (mGluR) activation on synaptic inhibition were examined using whole-cell recordings of spontaneous and miniature inhibitory synaptic currents from CA3 pyramidal cells in rat hippocampal slices. 2. The mGluR agonist (1S,3R)trans-1-aminocyclopentane-1,3-dicarboxylic acid (tACPD) increased spontaneous IPSC (spIPSC) frequency by up to 5-fold. At doses above 5 microM the increase was transient (15-45 s) and was followed by a decline to control frequency. In these conditions, elevating external K+ from 2 to 8 mM could still increase spIPSC frequency. 3. Miniature IPSCs (mIPSCs) were recorded in the presence of 1 microM TTX, 5 mM Mg2+ and nominally zero Ca2+. At concentrations above 50 microM, tACPD induced a sustained, reversible reduction in mIPSC frequency by up to 43%. 4. Quisqualate, at doses as low as 50 nM, increased spIPSC frequency, but did not affect mIPSC frequency at concentrations up to 10 microM. 5. The specific mGluR2 and 3 agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV, 3 microM) reduced mIPSC frequency by 40 +/- 4% but did not increase spIPSC frequency. 6. The putative mGluR antagonist L-2-amino-3-phosphonopropionate (L-AP3, 1 mM) blocked the effect of tACPD on mIPSC but not spIPSC frequency. The broad-spectrum antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM) blocked both responses. 7. mGluR activation also had dual effects on IPSCs evoked by focal extracellular stimulation. Application of 5 microM tACPD increased the mean amplitude of evoked IPSCs by 112 +/- 9%, largely by reducing the proportion of response failures. In contrast, IPSC amplitude was reduced to 44 +/- 1% of control values by 3 microM DCG-IV. 8. These results suggest hippocampal inhibitory cells express two distinct mGluR subtypes. One receptor (possibly mGluR1 or 5) is located on somato-dendritic membrane and enhances cell excitability. Another (mGluR2 or 3) is present at inhibitory terminals and reduces the probability of GABA release.

Pyramidal cells form excitatory synaptic connections with local inhibitory neurons in the hippocampus. This recurrent synapse plays a crucial stabilizing role in the control of hippocampal activity, since it transforms pyramidal cell activity into inhibition of the same pyramidal cell population. Using a combination of dual recording from presynaptic and postsynaptic cells and anatomical techniques, we show that these synaptic connections often comprise a single site for liberation of excitatory transmitter. The resulting excitatory postsynaptic potentials (EPSCs) have a fast time course and a similar amplitude to miniature EPSCs recorded in tetrodotoxin and cobalt. In contrast, activation of metabotropic glutamate receptors (mGluRs) by transmitter liberated during repetitive activation of these synapses produces an excitation with a much slower time course. In addition to somatodendritic mGluRs, which excite inhibitory cells, a different species of mGluR is present on inhibitory cell terminals. This mGluR is activated by higher concentrations of the agonist t-1-amino-cyclopentyl-1,3-decarboxylate and acts to reduce gamma-aminobutyric acid release. mGluRs, thus, have a dual action to enhance and to depress synaptic inhibition in the hippocampus.

1. Inhibitory cell activity and inhibitory postsynaptic potentials impinging spontaneously on pyramidal cells were recorded in the CA3 region of hippocampal slices from guinea-pig. We compared the effects on synaptic inhibition, of tetanic stimuli in the presence of antagonists of ionotropic excitatory amino acid receptors, and of application of agonists of metabotropic glutamate receptors. 2. Tetanic stimulation of afferent fibres caused an increase, of duration 0.5-2.5 min, in the frequency of spontaneous Cl(-)-mediated IPSPs. Inhibitory cell firing increased due to a depolarization and a reduction of after-hyperpolarizing potentials. 3. Tetanic stimulation induced, in some experiments, rhythmic bursts of IPSPs and transformed the firing pattern of some inhibitory cells from a discharge of single action potentials to rhythmic bursts of three to five action potentials. 4. Application of the metabotropic glutamate receptor agonist, trans-1-amino-cyclopentane-1,3-dicarboxylic acid (tACPD), at concentrations from 3-10 microM increased the frequency of spontaneous IPSPs. In some slices tACPD caused IPSPs to occur rhythmically. IPSP frequency did not continue to increase with concentrations of tACPD above 20 microM. 5. tACPD depolarized inhibitory cells and reduced after-hyperpolarizing potentials. High concentrations (50-100 microM) of tACPD excited inhibitory cells to potentials at which they no longer discharged fast action potentials. 6. Both tetanic stimulation and tACPD led to the appearance in pyramidal cell pairs of simultaneous IPSPs which were not previously observed, suggesting that the same group of inhibitory cells was excited in both cases. 7. Low concentrations of tACPD (3-10 microM) enhanced IPSP responses to tetanic stimuli, while the effects of tetanic stimuli were occluded in the presence of high concentrations (20-30 microM) of tACPD. 8. We suggest that activation of metabotropic glutamate receptors during tetanic stimulation leads to a post-tetanic excitation of inhibitory cells that mediate Cl(-)-dependent IPSPs.