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

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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Contribution of changes in the chloride driving force to the fading of I GABA in frog melanotrophs

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.278.3.e430 ◽

2000 ◽

Vol 278 (3) ◽

pp. E430-E443 ◽

Cited By ~ 6

Author(s):

Frank le Foll ◽

Olivier Soriani ◽

Hubert Vaudry ◽

Lionel Cazin

Keyword(s):

Driving Force ◽

Chloride Concentration ◽

Reversal Potential ◽

Resting Potential ◽

Perforated Patch ◽

Induced Currents ◽

Patch Configuration ◽

Intracellular Chloride Concentration ◽

Current Reversal

Chloride redistribution during type A γ-aminobutyric acid (GABAA) currents ( I GABA) has been investigated in cultured frog pituitary melanotrophs with imposed intracellular chloride concentration ([Cl−]i) in the whole cell configuration or with unaltered [Cl−]i using the gramicidin-perforated patch approach. Prolonged GABA exposures elicited reproducible decaying currents. The decay of I GABAwas associated with both a transient fall of conductance ( g GABA) and shift of current reversal potential ( E GABA). The shift of E GABAappeared to be time and driving force dependent. In the gramicidin-perforated patch configuration, repeated GABA exposures induced currents that gradually vanished. The fading of I GABA was due to persistent shifts of E GABA as a result of g GABArecovering from one GABA application to another. In cells alternatively clamped at potentials closely flanking resting potential and submitted to a train of brief GABA pulses, a reversal of I GABA was observed after 150 s recording. It is demonstrated that, in intact frog melanotrophs, shifts of E GABA combine with genuine receptor desensitization to depress I GABA. These findings strongly suggest that shifts of E GABA may act as a negative feedback, reducing the bioelectrical and secretory responses induced by an intense release of GABA in vivo.

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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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Intracellular chloride concentration is higher in rod bipolar cells than in cone bipolar cells of the mouse retina

Neuroscience Letters ◽

10.1016/s0304-3940(01)02120-6 ◽

2001 ◽

Vol 310 (2-3) ◽

pp. 161-164 ◽

Cited By ~ 30

Author(s):

Hiromasa Satoh ◽

Makoto Kaneda ◽

Akimichi Kaneko

Keyword(s):

Chloride Concentration ◽

Bipolar Cells ◽

Mouse Retina ◽

Intracellular Chloride ◽

Intracellular Chloride Concentration ◽

Rod Bipolar Cells ◽

Cone Bipolar Cells

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NKCC1 Does Not Accumulate Chloride in Developing Retinal Neurons

Journal of Neurophysiology ◽

10.1152/jn.00288.2007 ◽

2007 ◽

Vol 98 (1) ◽

pp. 266-277 ◽

Cited By ~ 42

Author(s):

Ling-Li Zhang ◽

Eric Delpire ◽

Noga Vardi

Keyword(s):

Ganglion Cells ◽

Chloride Concentration ◽

Müller Cells ◽

Amacrine Cells ◽

Muller Cells ◽

Retinal Neurons ◽

High Concentration ◽

Intracellular Chloride ◽

Cellular Markers ◽

Intracellular Chloride Concentration

GABA excites immature neurons due to their relatively high intracellular chloride concentration. This initial high concentration is commonly attributed to the ubiquitous chloride cotransporter NKCC1, which uses a sodium gradient to accumulate chloride. Here we tested this hypothesis in immature retinal amacrine and ganglion cells. Western blotting detected NKCC1 at birth and its expression first increased, then decreased to the adult level. Immunocytochemistry confirmed this early expression of NKCC1 and localized it to all nuclear layers. In the ganglion cell layer, staining peaked at P4 and then decreased with age, becoming undetectable in adult. In comparison, KCC2, the chloride extruder, steadily increased with age localizing primarily to the synaptic layers. For functional tests, we used calcium imaging with fura-2 and chloride imaging with 6-methoxy- N-ethylquinolinium iodide. If NKCC1 accumulates chloride in ganglion and amacrine cells, deleting or blocking it should abolish the GABA-evoked calcium rise. However, at P0-5 GABA consistently evoked a calcium rise that was not abolished in the NKCC1-null retinas, nor by applying high concentrations of bumetanide (NKCC blocker) for long periods. Furthermore, intracellular chloride concentration in amacrine and ganglion cells of the NKCC1-null retinas was ∼30 mM, same as in wild type at this age. This concentration was not lowered by applying bumetanide or by decreasing extracellular sodium concentration. Costaining for NKCC1 and cellular markers suggested that at P3, NKCC1 is restricted to Müller cells. We conclude that NKCC1 does not serve to accumulate chloride in immature retinal neurons, but it may enable Müller cells to buffer extracellular chloride.

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Relative contributions of passive equilibrium and active transport to the distribution of chloride in mammalian cortical neurons

Journal of Neurophysiology ◽

10.1152/jn.1988.60.1.105 ◽

1988 ◽

Vol 60 (1) ◽

pp. 105-124 ◽

Cited By ~ 106

Author(s):

S. M. Thompson ◽

R. A. Deisz ◽

D. A. Prince

Keyword(s):

Time Constant ◽

Cortical Neurons ◽

Time Course ◽

Chloride Concentration ◽

Reversal Potential ◽

Resting Potential ◽

Equilibrium Potential ◽

Gamma Aminobutyric Acid ◽

The Mean ◽

Mean Time

1. Active and passive factors affecting the chloride gradient of cortical neurons were assessed using intracellular recordings from neurons in slices of cingulate cortex maintained in vitro. The chloride equilibrium potential (ECl-) was estimated indirectly from the reversal potentials of responses to perisomatic gamma-aminobutyric acid (GABA) application and the Cl(-)-dependent inhibitory postsynaptic potential (IPSP). Under control conditions the mean resting potential (Vm; -69.7 mV) was not significantly different than the mean IPSP reversal potential (EIPSP; -70.1 mV). 2. Increasing the external potassium concentration ([K+]o) from 1 to 10 mM shifted the mean EIPSP from -80.4 to -61.8 mV. The mean EIPSP was approximately equal to the mean Vm at all [K+]oS. The conditions of Donnan equilibrium are not met in [K+]o less than 10 mM. 3. Polarization of Vm up to 20 mV away from EIPSP for 4 min with maintained current injection had no significant effect on EIPSP. 4. The GABA reversal potential was maintained 37-52 mV less negative than Vm after equilibration in saline in which the external chloride concentration had been reduced from 133 to 5 mM by substitution with isethionate. Vm and input resistance were not significantly different from control values in cells recorded under these conditions. 5. We conclude that Cl- is not passively distributed in cortical neurons, perhaps due to a low resting Cl- permeability. 6. Impalement with electrodes containing 2 M KCl resulted in a rapid 10 mV depolarizing shift in EIPSP that then remained relatively constant. Intracellular iontophoresis of Cl- resulted in a further depolarizing shift of EIPSP of 5-10 mV that returned to control in less than 1 min. The time course of recovery of IPSP amplitude could be fit with a single exponential having a mean time constant of 6.9 +/- 1.5 s and was independent of the amount of Cl- injected or stimulation frequency. 7. Reductions in temperature from 37 to 32 degrees C significantly increased the mean time constant of IPSP recovery from Cl- injection to 11.1 +/- 3.3 s, corresponding to Q10 = 2.6.(ABSTRACT TRUNCATED AT 400 WORDS)

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

10.21203/rs.3.rs-682039/v1 ◽

2021 ◽

Author(s):

Vijaya Verma ◽

MJ Vijay Kumar ◽

Kavita Sharma ◽

Sridhar Rajaram ◽

Ravi Muddashetty ◽

...

Keyword(s):

Small Molecule ◽

Intraperitoneal Administration ◽

Chloride Concentration ◽

Reversal Potential ◽

Autism Spectrum ◽

Ion Concentration ◽

Pharmacological Intervention ◽

Subsequent Increase ◽

Intracellular Chloride ◽

Gabaergic Synapses

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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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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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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Control of intracellular calcium by membrane potential in human melanoma cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.6.c1501 ◽

1993 ◽

Vol 265 (6) ◽

pp. C1501-C1510 ◽

Cited By ~ 48

Author(s):

B. Nilius ◽

G. Schwarz ◽

G. Droogmans

Keyword(s):

Cell Proliferation ◽

Membrane Potential ◽

Intracellular Calcium ◽

Reversal Potential ◽

Melanoma Cells ◽

Human Melanoma ◽

Resting Potential ◽

Equilibrium Potential ◽

Patch Clamp Technique ◽

Human Melanoma Cells

The modulation of intracellular calcium ([Ca2+]i) by the membrane potential was investigated in human melanoma cells by combining the nystatin-perforated patch-clamp technique with Ca2+ measurements. Voltage steps to -100 mV induced a rise in [Ca2+]i and a creeping inward current. These effects were absent in Ca(2+)-free solution and could be blocked by Ni2+ or La3+. Voltage ramps revealed a close correlation between [Ca2+]i and voltage, with the strongest voltage dependence around the resting potential. Long-lasting tail currents, closely correlated with the rise in [Ca2+]i and a reversal potential close to the K+ equilibrium potential, occurred if the membrane potential was clamped back to 0 mV. They were absent if intracellular K+ was replaced by Cs+ and blocked by extracellular tetraethylammonium (5 mM), Ba2+ (1 mM), or a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue. These observations are discussed in relation to cell proliferation. The enhanced expression of K+ channels during cell proliferation provides a positive-feedback mechanism resulting in long-term changes in [Ca2+]i required for the G1-S transition in the cell cycle.

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