Reversing GABA polarity corrects synaptic physiology and behavioural deficits in young adolescent Syngap1+/- mice

Abstract Haploinsufficiency in SYNGAP1 is implicated in Intellectual Disability (ID) and Autism Spectrum disorder (ASD) and affects the maturation of dendritic spines. The abnormal spine development has been suggested to cause disbalance of excitatory and inhibitory (E/I) neurotransmission at distinct developmental periods. In addition, E/I imbalances in Syngap1+/- mice might be due to abnormalities in K+-Cl- co-transporter function (NKCC1, KCC2), in a similar manner as in the murine models of Fragile-X and Rett syndromes. To study whether an altered intracellular chloride ion concentration represents an underlying mechanism of altered function of GABAergic synapses in Dentate Gyrus Granule Cells of Syngap1+/- recordings were performed at different developmental stages of the mice. We observed that neurons at P14-15 of Syngap1+/- mice had depolarised membrane potential and a decreased Cl- reversal potential. The KCC2 expression was decreased compared to Wild-type (WT) mice at P14-15. Furtherly, the small molecule GSK-3β inhibitor, 6-bromoindirubin-3`-oxime (6BIO), was tested in an attempt to restore the function of GABAergic synapses. We discovered that intraperitoneal administration of 6BIO during the critical period or young adolescents normalized an altered E/I balance, the deficits of synaptic transmission, and behavioral performance like social novelty, anxiety, and memory of the Syngap1+/- mice. In summary, altered functionality of GABAergic synapses in Syngap1+/- mice is based on a reduced KCC2 expression and a subsequent increase in the intracellular chloride concentration that can be counteracted by the small molecule 6BIO. The 6BIO sufficiently restored cognitive, emotional, and social symptoms by pharmacological intervention, particularly, in adulthood.

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GABAA-Dependent Chloride Influx Modulates Reversal Potential of GABAB-Mediated IPSPs in Hippocampal Pyramidal Cells

Journal of Neurophysiology ◽

10.1152/jn.2001.85.6.2381 ◽

2001 ◽

Vol 85 (6) ◽

pp. 2381-2387

Author(s):

Valeri Lopantsev ◽

Philip A. Schwartzkroin

Keyword(s):

Membrane Potential ◽

Mossy Fiber ◽

Chloride Concentration ◽

Reversal Potential ◽

Pyramidal Cells ◽

Intracellular Chloride ◽

The Impact ◽

Intracellular Chloride Concentration ◽

Chloride Influx ◽

Hippocampal Pyramidal Cells

Changes in intracellular chloride concentration, mediated by chloride influx through GABAA receptor–gated channels, may modulate GABAB receptor–mediated inhibitory postsynaptic potentials (GABAB IPSPs) via unknown mechanisms. Recording from CA3 pyramidal cells in hippocampal slices, we investigated the impact of chloride influx during GABAA receptor–mediated IPSPs (GABAA IPSPs) on the properties of GABAB IPSPs. At relatively positive membrane potentials (near −55 mV), mossy fiber–evoked GABAB IPSPs were reduced (compared with their magnitude at −60 mV) when preceded by GABAAreceptor–mediated chloride influx. This effect was not associated with a correlated reduction in membrane permeability during the GABAB IPSP. The mossy fiber–evoked GABAB IPSP showed a positive shift in reversal potential (from −99 to −93 mV) when it was preceded by a GABAA IPSP evoked at cell membrane potential of −55 mV as compared with −60 mV. Similarly, when intracellular chloride concentration was raised via chloride diffusion from an intracellular microelectrode, there was a reduction of the pharmacologically isolated monosynaptic GABABIPSP and a concurrent shift of GABAB IPSP reversal potential from −98 to −90 mV. We conclude that in hippocampal pyramidal cells, in which “resting” membrane potential is near action potential threshold, chloride influx via GABAA IPSPs shifts the reversal potential of subsequent GABAB receptor–mediated postsynaptic responses in a positive direction and reduces their magnitude.

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Shift of Intracellular Chloride Concentration in Ganglion and Amacrine Cells of Developing Mouse Retina

Journal of Neurophysiology ◽

10.1152/jn.00578.2005 ◽

2006 ◽

Vol 95 (4) ◽

pp. 2404-2416 ◽

Cited By ~ 47

Author(s):

Ling-Li Zhang ◽

Hemal R. Pathak ◽

Douglas A. Coulter ◽

Michael A. Freed ◽

Noga Vardi

Keyword(s):

Gaba Receptors ◽

Chloride Concentration ◽

Reversal Potential ◽

Amacrine Cells ◽

Resting Potential ◽

Equilibrium Potential ◽

Mouse Retina ◽

Intracellular Chloride ◽

Excitatory Action ◽

Intracellular Chloride Concentration

GABA and glycine provide excitatory action during early development: they depolarize neurons and increase intracellular calcium concentration. As neurons mature, GABA and glycine become inhibitory. This switch from excitation to inhibition is thought to result from a shift of intracellular chloride concentration ([Cl−]i) from high to low, but in retina, measurements of [Cl−]i or chloride equilibrium potential ( ECl) during development have not been made. Using the developing mouse retina, we systematically measured [Cl−]i in parallel with GABA's actions on calcium and chloride. In ganglion and amacrine cells, fura-2 imaging showed that before postnatal day (P) 6, exogenous GABA, acting via ionotropic GABA receptors, evoked calcium rise, which persisted in HCO3−- free buffer but was blocked with 0 extracellular calcium. After P6, GABA switched to inhibiting spontaneous calcium transients. Concomitant with this switch we observed the following: 6-methoxy- N-ethylquinolinium iodide (MEQ) chloride imaging showed that GABA caused an efflux of chloride before P6 and an influx afterward; gramicidin-perforated-patch recordings showed that the reversal potential for GABA decreased from −45 mV, near threshold for voltage-activated calcium channel, to −60 mV, near resting potential; MEQ imaging showed that [Cl−]i shifted steeply around P6 from 29 to 14 mM, corresponding to a decline of ECl from −39 to −58 mV. We also show that GABAergic amacrine cells became stratified by P4, potentially allowing GABA's excitatory action to shape circuit connectivity. Our results support the hypothesis that a shift from high [Cl−]i to low causes GABA to switch from excitatory to inhibitory.

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Synergistic inhibition of histone modifiers produces therapeutic effects in adult Shank3-deficient mice

Translational Psychiatry ◽

10.1038/s41398-021-01233-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Freddy Zhang ◽

Benjamin Rein ◽

Ping Zhong ◽

Treefa Shwani ◽

Megan Conrow-Graham ◽

...

Keyword(s):

Pyramidal Neurons ◽

Social Preference ◽

Intervention Strategy ◽

Autism Spectrum ◽

Synaptic Function ◽

Pharmacological Intervention ◽

Therapeutic Effects ◽

Male Mice ◽

Behavioral Abnormalities ◽

Deficient Mice

AbstractAutism spectrum disorder (ASD) is a lifelong developmental disorder characterized by social deficits and other behavioral abnormalities. Dysregulation of epigenetic processes, such as histone modifications and chromatin remodeling, have been implicated in ASD pathology, and provides a promising therapeutic target for ASD. Haploinsufficiency of the SHANK3 gene is causally linked to ASD, so adult (3–5 months old) Shank3-deficient male mice were used in this drug discovery study. We found that combined administration of the class I histone deacetylase inhibitor Romidepsin and the histone demethylase LSD1 inhibitor GSK-LSD1 persistently ameliorated the autism-like social preference deficits, while each individual drug alone was largely ineffective. Another behavioral abnormality in adult Shank3-deficient male mice, heightened aggression, was also alleviated by administration of the dual drugs. Furthermore, Romidepsin/GSK-LSD1 treatment significantly increased transcriptional levels of NMDA receptor subunits in prefrontal cortex (PFC) of adult Shank3-deficient mice, resulting in elevated synaptic expression of NMDA receptors and the restoration of NMDAR synaptic function in PFC pyramidal neurons. These results have offered a novel pharmacological intervention strategy for ASD beyond early developmental periods.

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GABA responses in rat dentate granule neurons are mediated by chloride

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y88-099 ◽

1988 ◽

Vol 66 (5) ◽

pp. 637-642 ◽

Cited By ~ 5

Author(s):

Timothy J. Blaxter ◽

Peter L. Carlen

Keyword(s):

Granule Cells ◽

Hippocampal Slices ◽

Reversal Potential ◽

Chloride Ion ◽

Chloride Ions ◽

Ion Concentration ◽

Aminobutyric Acid ◽

Granule Neurons ◽

Nernst Equation ◽

Central Neurons

The dendrites of granule cells in hippocampal slices responded to γ-aminobutyric acid (GABA) with a depolarization. The response was blocked by picrotoxin in a noncompetitive manner. Reductions in the extracellular chloride ion concentration changed the reversal potential of the response by an amount predicted from the Nernst equation for chloride ion. Chloride-dependent hyperpolarizing responses were sometimes also found in the cell body of the granule cells. Since the reversal potential followed that predicted from the Nernst equation for chloride, we conclude that the response was mediated by chloride ions alone with no contribution from other ions. This has not previously been shown for the depolarizing response to GABA in central neurons.

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The opposing Chloride Cotransporters KCC and NKCC control locomotor activity in constant light and during long days

10.1101/2021.09.14.460201 ◽

2021 ◽

Author(s):

Anna Katharina Eick ◽

Maite Ogueta ◽

Edgar Buhl ◽

James J. L. Hodge ◽

Ralf Stanewsky

Keyword(s):

Locomotor Activity ◽

Circadian Clock ◽

Opposite Effect ◽

Neuronal Response ◽

Reversal Potential ◽

Ion Concentration ◽

Day Length ◽

Constant Light ◽

Behavioral Activity ◽

Dependent Manner

AbstractCation Chloride Cotransporters (CCC’s) regulate intracellular chloride ion concentration ([Cl−]i) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA. Na+ K+ Cl− (NKCC) and K+ Cl− (KCC) cotransporters transport Cl− into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl−]i can lead to an excitatory and depolarizing response of the neuron upon GABAA receptor opening, while KCC dominance has the opposite effect. This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length, and its dysregulation is associated with neurodevelopmental disorders such as epilepsy. Constant light normally disrupts circadian clock function and leads to arrhythmic behavior. Here, we demonstrate a function for KCC in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of KCC expression in circadian clock neurons elicits rhythmic behavior in constant light. We observed the same effects after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl−]i-dependent manner. Patch-clamp recordings from clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC downregulation represses morning behavioral activity during long photoperiods, while downregulation of NKCC promotes morning activity. In summary, our results support a model in which the regulation of [Cl−]i by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions.

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Long-Term Imaging Reveals a Circadian Rhythm of Intracellular Chloride in Neurons of the Suprachiasmatic Nucleus

Journal of Biological Rhythms ◽

10.1177/07487304211059770 ◽

2022 ◽

pp. 074873042110597

Author(s):

Nathan J. Klett ◽

Olga Cravetchi ◽

Charles N. Allen

Keyword(s):

Suprachiasmatic Nucleus ◽

Chloride Concentration ◽

Intracellular Chloride ◽

Voltage Gated Calcium Channels ◽

Excitatory Gaba ◽

Ratiometric Probe ◽

Gaba Transmission ◽

Circadian Physiology ◽

Intracellular Chloride Concentration

Both inhibitory and excitatory GABA transmission exist in the mature suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. Whether GABA is inhibitory or excitatory depends on the intracellular chloride concentration ([Cl−]i). Here, using the genetically encoded ratiometric probe Cl-Sensor, we investigated [Cl−]i in AVP and VIP-expressing SCN neurons for several days in culture. The chloride ratio (RCl) demonstrated circadian rhythmicity in AVP + neurons and VIP + neurons, but was not detected in GFAP + astrocytes. RCl peaked between ZT 7 and ZT 8 in both AVP + and VIP + neurons. RCl rhythmicity was not dependent on the activity of several transmembrane chloride carriers, action potential generation, or the L-type voltage-gated calcium channels, but was sensitive to GABA antagonists. We conclude that [Cl−]i is under circadian regulation in both AVP + and VIP + neurons.

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Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSC with a TSC2 mutation

Molecular Autism ◽

10.1186/s13229-020-00391-w ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Mouhamed Alsaqati ◽

Vivi M. Heine ◽

Adrian J. Harwood

Keyword(s):

Neuronal Network ◽

Electrode Array ◽

Network Connectivity ◽

Autism Spectrum ◽

Network Activity ◽

Inhibitory Synapses ◽

Pharmacological Intervention ◽

Spatial Connectivity ◽

Neuronal Network Activity

Abstract Background Tuberous sclerosis complex (TSC) is a rare genetic multisystemic disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes. It is characterised by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and has severe neurodevelopmental and neurological components including autism, intellectual disability and epilepsy. In human and rodent models, loss of the TSC proteins causes neuronal hyperexcitability and synaptic dysfunction, although the consequences of these changes for the developing central nervous system are currently unclear. Methods Here we apply multi-electrode array-based assays to study the effects of TSC2 loss on neuronal network activity using autism spectrum disorder (ASD) patient-derived iPSCs. We examine both temporal synchronisation of neuronal bursting and spatial connectivity between electrodes across the network. Results We find that ASD patient-derived neurons with a functional loss of TSC2, in addition to possessing neuronal hyperactivity, develop a dysfunctional neuronal network with reduced synchronisation of neuronal bursting and lower spatial connectivity. These deficits of network function are associated with elevated expression of genes for inhibitory GABA signalling and glutamate signalling, indicating a potential abnormality of synaptic inhibitory–excitatory signalling. mTORC1 activity functions within a homeostatic triad of protein kinases, mTOR, AMP-dependent protein Kinase 1 (AMPK) and Unc-51 like Autophagy Activating Kinase 1 (ULK1) that orchestrate the interplay of anabolic cell growth and catabolic autophagy while balancing energy and nutrient homeostasis. The mTOR inhibitor rapamycin suppresses neuronal hyperactivity, but does not increase synchronised network activity, whereas activation of AMPK restores some aspects of network activity. In contrast, the ULK1 activator, LYN-1604, increases the network behaviour, shortens the network burst lengths and reduces the number of uncorrelated spikes. Limitations Although a robust and consistent phenotype is observed across multiple independent iPSC cultures, the results are based on one patient. There may be more subtle differences between patients with different TSC2 mutations or differences of polygenic background within their genomes. This may affect the severity of the network deficit or the pharmacological response between TSC2 patients. Conclusions Our observations suggest that there is a reduction in the network connectivity of the in vitro neuronal network associated with ASD patients with TSC2 mutation, which may arise via an excitatory/inhibitory imbalance due to increased GABA-signalling at inhibitory synapses. This abnormality can be effectively suppressed via activation of ULK1.

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A Study into the Localized Corrosion of Magnesium Alloy Magnox Al-80

CORROSION ◽

10.5006/3574 ◽

2020 ◽

Author(s):

Ronald Clark ◽

James Humpage ◽

Robert Burrows ◽

Hugh Godfrey ◽

Mustufa Sagir ◽

...

Keyword(s):

Passive Film ◽

Ph Dependence ◽

Chloride Concentration ◽

Ion Concentration ◽

Localized Corrosion ◽

High Concentration ◽

Transfer Resistance ◽

High Ph ◽

Intact Surface ◽

Chloride Concentrations

Magnesium (Mg) non-oxidizing alloy, known as Magnox, was historically used as a fuel cladding material for the first-generation of carbon dioxide (CO<sub>2</sub>) gas-cooled nuclear reactors in the UK. Waste Magnox is currently stored in cooling ponds, pending final disposal. The corrosion resistance of Mg and its alloys is relatively poor, compared to modern cladding materials such as zirconium (Zr) alloys, so it is important to have a knowledge of the chloride concentration/pH dependence on breakdown and localized corrosion characteristics prior to waste retrievals taking place. Our results show that Magnox exhibits passivity in high pH solutions, with charge transfer resistance and passive film thicknesses showing an increase with immersion time. When chloride is added to the system the higher pH maintains Magnox passivity, as shown through a combination of potentiodynamic and time-lapse/post corrosion imaging experiments. Potentiodynamic polarization of Magnox reveals a -229 mV<sup>-decade</sup> linear dependence of breakdown potential with chloride ion concentration. The use of the scanning vibrating electrode technique (SVET) enabled the localized corrosion characteristics to be followed. At high pH where Magnox is passive, at low chloride concentrations, the anodes which form predominantly couple to the visually intact surface in the vicinity of the anode. The high pH however means that visually intact Magnox in the vicinity of the anode is less prone to breakdown, restricting anode propagation such that they remain largely static. In high chloride concentrations the higher conductivity means that the anode and cathode can couple over greater distances and so propagation along the surface can occur at a much faster rate, with the visually intact surface acting as a distributed cathode. In addition, the chloride anion itself, when present at high concentration will play a role in rapid passive film dissolution, enabling rapid anode propagation.

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Nitric Oxide Transiently Converts Synaptic Inhibition to Excitation in Retinal Amacrine Cells

Journal of Neurophysiology ◽

10.1152/jn.01317.2005 ◽

2006 ◽

Vol 95 (5) ◽

pp. 2866-2877 ◽

Cited By ~ 27

Author(s):

Brian Hoffpauir ◽

Emily McMains ◽

Evanna Gleason

Keyword(s):

Nitric Oxide ◽

Hippocampal Neurons ◽

Reversal Potential ◽

Amacrine Cells ◽

Cell Types ◽

Synaptic Function ◽

Positive Shift ◽

Gabaergic Synapses ◽

Multiple Cell

Nitric oxide (NO) is generated by multiple cell types in the vertebrate retina, including amacrine cells. We investigate the role of NO in the modulation of synaptic function using a culture system containing identified retinal amacrine cells. We find that moderate concentrations of NO alter GABAA receptor function to produce an enhancement of the GABA-gated current. Higher concentrations of NO also enhance GABA-gated currents, but this enhancement is primarily due to a substantial positive shift in the reversal potential of the current. Several pieces of evidence, including a similar effect on glycine-gated currents, indicate that the positive shift is due to an increase in cytosolic Cl−. This change in the chloride distribution is especially significant because it can invert the sign of GABA- and glycine-gated voltage responses. Furthermore, current- and voltage-clamp recordings from synaptic pairs of GABAergic amacrine cells demonstrate that NO transiently converts signaling at GABAergic synapses from inhibition to excitation. Persistence of the NO-induced shift in ECl− in the absence of extracellular Cl− indicates that the increase in cytosolic Cl− is due to release of Cl− from an internal store. An NO-dependent release of Cl− from an internal store is also demonstrated for rat hippocampal neurons indicating that this mechanism is not restricted to the avian retina. Thus signaling in the CNS can be fundamentally altered by an NO-dependent mobilization of an internal Cl− store.

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GABA-mediated repulsive coupling between circadian clock neurons in the SCN encodes seasonal time

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421200112 ◽

2015 ◽

Vol 112 (29) ◽

pp. E3920-E3929 ◽

Cited By ~ 77

Author(s):

Jihwan Myung ◽

Sungho Hong ◽

Daniel DeWoskin ◽

Erik De Schutter ◽

Daniel B. Forger ◽

...

Keyword(s):

Circadian Clock ◽

Synaptic Input ◽

Chloride Concentration ◽

Phase Relationship ◽

Day Length ◽

Intracellular Chloride ◽

Circadian Oscillators ◽

Length Changes ◽

Excitatory Gaba ◽

Intracellular Chloride Concentration

The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock gene Bmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABAA-mediated synaptic input. We show that increasing day-length changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABAA signaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation–inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN.

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