Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation

A developmental “switch” in chloride transporters occurs in most neurons resulting in GABA_A mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABA_A receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH) neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABA_A receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABA_A receptor activation. Anion exchangers (AEs) are also potential modulators of responses to GABA_A activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s) underlying GABA_A mediated depolarization, GnRH neurons were analyzed for 1) expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2) responses to GABA_A receptor activation in NKCC1^-/- mice and 3) function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1^-/- mice retained relatively normal responses to the GABA_A agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO₃ ^- mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABA_A mediated transmission in GnRH neurons.     

Nonsynaptic glycine release is involved in the early KCC2 expression

The cation‐chloride co‐transporters are important regulators of the cellular Cl^‐ homeostasis. Among them the Na⁺‐K⁺?2Cl^? co‐transporter (NKCC1) is responsible for intracellular chloride accumulation in most immature brain structures, whereas the K⁺‐Cl^? co‐transporter (KCC2) extrudes chloride from mature neurons, ensuring chloride‐mediated inhibitory effects of GABA/glycine. We have shown that both KCC2 and NKCC1 are expressed at early embryonic stages (E11.5) in the ventral spinal cord (SC). The mechanisms by which KCC2 is prematurely expressed are unknown. In this study, we found that chronically blocking glycine receptors (GlyR) by strychnine led to a loss of KCC2 expression, without affecting NKCC1 level. This effect was not dependent on the firing of Na⁺ action potentials but was mimicked by a Ca²⁺‐dependent PKC blocker. Blocking the vesicular release of neurotransmitters did not impinge on strychnine effect whereas blocking volume‐sensitive outwardly rectifying (VSOR) chloride channels reproduced the GlyR blockade, suggesting that KCC2 is controlled by a glycine release from progenitor radial cells in immature ventral spinal networks. Finally, we showed that the strychnine treatment prevented the maturation of rhythmic spontaneous activity. Thereby, the GlyR‐activation is a necessary developmental process for the expression of functional spinal motor networks. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 764–779, 2016     

Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons

The equilibrium potential for GABA-A receptor mediated currents (E_GABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K⁺/Cl^- co-transporter (KCC2) but a relatively high expression of Na⁺-K⁺-Cl^- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in E_GABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on E_GABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus E_GABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in E_GABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in E_GABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in E_GABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.     

Topiramate attenuates cerebral ischemia/reperfusion injury in gerbils via activating GABAergic signaling and inhibiting astrogliosis

Impaired GABAergic inhibitory synaptic transmission plays an essential role in the pathogenesis of selective neuronal cell death following transient global ischemia. GABA_A receptor (GABA_AR), K⁺-Cl⁻ co-transporter 2 (KCC2), Na⁺-K⁺-Cl⁻ co-transporter 1 (NKCC1) and astrocytes are of particular importance to GABAergic transmission. The present study was designed to explore whether the neuroprotective effect of topiramate (TPM) was linked with the alterations of GABAergic signaling and astrocytes. The bilateral carotid arteries were occluded, and TPM (80 mg/kg/day (divided twice daily), i.p.) was injected into gerbils. At day 1, 3 and 7 post-ischemia, neurological deficit was scored and changes in hippocampal neuronal cell death were evaluated by Nissl staining. The apoptosis-related regulatory proteins (procaspase-3, caspase-3, Bax and Bcl-2) and GABAergic signal molecules (GABA_AR α1, GABA_AR γ2, KCC2 and NKCC1) were also detected using western blot assay. In addition, the fluorescent intensity and protein level of glial fibrillary acidic protein (GFAP), a major component of astrocyte, were examined by confocal and immunoblot analysis. Our results showed that TPM treatment significantly decreased neurological deficit scores, attenuated the ischemia-induced neuronal loss and remarkably decreased the expression levels of procaspase-3, caspase-3 as well as the ratio of Bax/Bcl-2. Besides, treatment with TPM also resulted in the increased protein expressions of GABA_AR α1, GABA_AR γ2 and KCC2 together with the decreased protein level of NKCC1 in gerbils hippocampus. Furthermore, fluorescent intensity and protein level of GFAP were evidently reduced in TPM-treated gerbils. These findings suggest that the therapeutic effect of TPM on global ischemia/reperfusion injury appears to be associated with the enhancement of GABAergic signaling and the inhibition of astrogliosis in gerbils.     

Decreased intracellular GABA levels contribute to spinal cord stimulation-induced analgesia in rats suffering from painful peripheral neuropathy: the role of KCC2 and GABA(A) receptor-mediated inhibition

Elevated spinal extracellular γ-aminobutyric acid (GABA) levels have been described during spinal cord stimulation (SCS)-induced analgesia in experimental chronic peripheral neuropathy. Interestingly, these increased GABA levels strongly exceeded the time frame of SCS-induced analgesia. In line with the former, pharmacologically-enhanced extracellular GABA levels by GABA_B receptor agonists in combination with SCS in non-responders to SCS solely could convert these non-responders into responders. However, similar treatment with GABA_A receptor agonists and SCS is known to be less efficient. Since K⁺ Cl⁻ cotransporter 2 (KCC2) functionality strongly determines proper GABA_A receptor-mediated inhibition, both decreased numbers of GABA_A receptors as well as reduced KCC2 protein expression might play a pivotal role in this loss of GABA_A receptor-mediated inhibition in non-responders. Here, we explored the mechanisms underlying both changes in extracellular GABA levels and impaired GABA_A receptor-mediated inhibition after 30 min of SCS in rats suffering from partial sciatic nerve ligation (PSNL). Immediately after cessation of SCS, a decreased spinal intracellular dorsal horn GABA-immunoreactivity was observed in responders when compared to non-responders or sham SCS rats. One hour later however, GABA-immunoreactivity was already increased to similar levels as those observed in non-responder or sham SCS rats. These changes did not coincide with alterations in the number of GABA-immunoreactive cells. C-Fos/GABA double-fluorescence clearly confirmed a SCS-induced activation of GABA-immunoreactive cells in responders immediately after SCS. Differences in spinal dorsal horn GABA_A receptor-immunoreactivity and KCC2 protein levels were absent between all SCS groups. However, KCC2 protein levels were significantly decreased compared to sham PSNL animals. In conclusion, reduced intracellular GABA levels are only present during the time frame of SCS in responders and strongly point to a SCS-mediated on/off GABAergic release mechanism. Furthermore, a KCC2-dependent impaired GABA_A receptor-mediated inhibition seems to be present both in responders and non-responders to SCS due to similar KCC2 and GABA_A receptor levels.     

High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters

Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl^- with K⁺ and/or Na⁺ in an electroneutral manner. They play important roles in myriad fundamental physiological processes––from cell volume regulation to transepithelial solute transport and intracellular ion homeostasis––and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacology of these transporters, there have been several breakthroughs in the structural determination of CCC transporters. The insights provided by these new structures for the Na⁺/K⁺/Cl^- cotransporter NKCC1 and the K⁺/Cl^- cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their molecular basis and transport function. This focused review discusses recent advances in the structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.     

Differential expression pattern of chloride transporters<Emphasis Type="Italic"> NCC,NKCC2, KCC1, KCC3, KCC4,</Emphasis> and<Emphasis Type="Italic"> AE3</Emphasis> in the developing rat auditory brainstem

During development of inhibitory synapses, the action of the two neurotransmitters GABA and glycine shifts from depolarizing to hyperpolarizing. The shift is due to an age-dependent regulation of the intracellular free chloride concentration ([Cl(-)](i)) in postsynaptic neurons. A model system to study this maturation process is a glycinergic projection in the mammalian auditory brainstem. It is formed in the superior olivary complex (SOC) by neurons of the medial nucleus of the trapezoid body, whose axons terminate in the lateral superior olive (LSO). LSO neurons of perinatal rats and mice are depolarized upon glycine application, whereas older cells (>postnatal day (P) 8) are hyperpolarized. Here we examined the expression of six secondary active chloride transporter genes ( NCC, NKCC2, KCC1, KCC3, KCC4, and AE3) in the rat SOC to unravel the molecular mechanisms underlying this change. RT-PCR analysis demonstrated brainstem expression of KCC1, KCC3, KCC4, and AE3, but not of NCC and NKCC2. RNA in situ hybridization showed that only AE3 is highly expressed both at P3 (high [Cl(-)](i)) and P12 (low [Cl(-)](i)) in LSO neurons. KCC1 and KCC4 are weakly expressed in LSO neurons at P3 and P12, respectively. This study completes the expression analysis of all known chloride transporters sensitive to loop diuretic drugs in the SOC and demonstrates differences in the maturation between hippocampal and brainstem inhibitory synapses.     

D1‐like dopamine receptors regulate GABAA receptor function to modulate hippocampal neural progenitor cell proliferation

The proliferation and differentiation of neural progenitor (NP) cells can be regulated by neurotransmitters including GABA and dopamine. The present study aimed to examine how these two neurotransmitter systems interact to affect post‐natal hippocampal NP cell proliferation in vitro. Mouse hippocampal NP cells express functional GABA_A receptors, which upon activation led to an increase in intracellular calcium levels via the opening of L‐type calcium channels. Activation of these GABA_A receptors also caused a significant decrease in proliferation; an effect that required the entry of calcium through L‐type calcium channels. Furthermore, while activation of D₁‐like dopamine receptors had no effect on proliferation, it abrogated the suppressive effects of GABA_A receptor activation on proliferation. The effects of D₁‐like dopamine receptors are associated with a decrease in the ability of GABA_A receptors to increase intracellular calcium levels, and a reduction in the surface expression of GABA_A receptors. In this way, D₁‐like dopamine receptor activation can increase the proliferation of NP cells by preventing GABA_A receptor‐mediated inhibition of proliferation. These results suggest that, in conditions where NP cell proliferation is under the tonic suppression of GABA, agonists which act through D₁‐like dopamine receptors may increase the proliferation of neural progenitors.     

Bumetanide Enhances Phenobarbital Efficacy in a Rat Model of Hypoxic Neonatal Seizures

Neonatal seizures can be refractory to conventional anticonvulsants, and this may in part be due to a developmental increase in expression of the neuronal Na⁺-K⁺-2 Cl⁻ cotransporter, NKCC1, and consequent paradoxical excitatory actions of GABA_A receptors in the perinatal period. The most common cause of neonatal seizures is hypoxic encephalopathy, and here we show in an established model of neonatal hypoxia-induced seizures that the NKCC1 inhibitor, bumetanide, in combination with phenobarbital is significantly more effective than phenobarbital alone. A sensitive mass spectrometry assay revealed that bumetanide concentrations in serum and brain were dose-dependent, and the expression of NKCC1 protein transiently increased in cortex and hippocampus after hypoxic seizures. Importantly, the low doses of phenobarbital and bumetanide used in the study did not increase constitutive apoptosis, alone or in combination. Perforated patch clamp recordings from ex vivo hippocampal slices removed following seizures revealed that phenobarbital and bumetanide largely reversed seizure-induced changes in E_GABA. Taken together, these data provide preclinical support for clinical trials of bumetanide in human neonates at risk for hypoxic encephalopathy and seizures.     

Involvement of the K+‐Cl− co‐transporter KCC2 in the sensitization to morphine‐induced hyperlocomotion under chronic treatment with zolpidem in the mesolimbic system

Benzodiazepines are commonly used as sedatives, sleeping aids, and anti‐anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K⁺‐Cl^? co‐transporter 2 (KCC2) in the sensitization to morphine‐induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of γ‐aminobutyric acid A‐type receptor (GABA_AR) α1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine‐induced hyperlocomotion, which is accompanied by the up‐regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down‐regulation of protein phosphatase‐1 (PP‐1) as well as the up‐regulation of phosphorylated protein kinase C γ (pPKCγ). Furthermore, PP‐1 directly associated with KCC2 and pPKCγ, whereas pPKCγ did not associate with KCC2. On the other hand, pre‐treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine‐induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKCγ‐PP‐1‐KCC2 pathway by chronic treatment with zolpidem.     

Postnatal maturation of gamma-aminobutyric acidA and B-mediated inhibition in the CA3 hippocampal region of the rat

In the adult central nervous system, GABAergic synaptic inhibition is known to play a crucial role in preventing the spread of excitatory glutamatergic activity. This inhibition is achieved by a membrane hyperpolarization through the activation of postsynaptic γ-aminobutyric acid_A (GABA_A) and GABA_B receptors. In addition, GABA also depress transmitter release acting through presynaptic GABA_B receptors. Despite the wealth of data regarding the role of GABA in regulating the degree of synchronous activity in the adult, little is known about GABA transmission during early stages of development. In the following we report that GABA mediates most of the excitatory drive at early stages of development in the hippocampal CA3 region. Activation of GABA_A receptors induces a depolarization and excitation of immature CA3 pyramidal neurons and increases intracellular Ca²⁺ ([Ca²⁺]_i) during the first postnatal week of life. During the same developmental period, the postsynaptic GABA_B-mediated inhibition is poorly developed. In contrast, the presynaptic GABA_B-mediated inhibition is well developed at birth and plays a crucial role in modulating the postsynaptic activity by depressing transmitter release at early postnatal stages. We have also shown that GABA plays a trophic role in the neuritic outgrowth of cultured hippocampal neurons. © 1995 John Wiley & Sons, Inc.     

Pre‐synaptic GABAB receptors inhibit glutamate release through GIRK channels in rat cerebral cortex

Neuronal G protein‐gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post‐synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABA_B receptors. In this study, we show for the first time that GABA_B receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABA_B receptors reduces glutamate release and the Ca²⁺ influx mediated by N‐type Ca²⁺ channels in a mode insensitive to the GIRK channel blocker tertiapin‐Q and consistent with direct inhibition of this voltage‐gated Ca²⁺ channel. However, by means of weak stimulation protocols, we reveal that GABA_B receptors also reduce glutamate release mediated by P/Q‐type Ca²⁺ channels, and that these responses are reversed by the GIRK channel blocker tertiapin‐Q. Consistent with the functional interaction between GABA_B receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre‐synaptic boutons of asymmetric synapses co‐express GABA_B receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post‐synaptic level, also occurs at glutamatergic nerve terminals.     

K+-Cl− co-transporter 2 (KCC2) – a membrane trafficking perspective

K⁺-Cl^? co-transporter 2 (KCC2/SLC12A5) is a neuronal specific cation chloride co-transporter which is active under isotonic conditions, and thus a key regulator of intracellular Cl^? levels. It also has an ion transporter-independent structural role in modulating the maturation and regulation of excitatory glutamatergic synapses. KCC2 levels are developmentally regulated, and a postnatal upregulation of KCC2 generates a low intracellular chloride concentration that allows the neurotransmitters γ-aminobutyric acid (GABA) and glycine to exert inhibitory neurotransmission through its Cl^? permeating channel. Functional expression of KCC2 at the neuronal cell surface is necessary for its activity, and impairment in KCC2 cell surface transport and/or internalization may underlie a range of neuropathological conditions. Although recent advances have shed light on a range of cellular mechanisms regulating KCC2 activity, little is known about its membrane trafficking itinerary and regulatory proteins. In this review, known membrane trafficking signals, pathways and mechanisms pertaining to KCC2’s functional surface expression are discussed.     

Opposite effect of membrane raft perturbation on transport activity of KCC2 and NKCC1

In the majority of neurons, the intracellular Cl⁻ concentration is set by the activity of the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC1) and the K⁺-Cl⁻ cotransporter (KCC2). Here, we investigated the cotransporters' functional dependence on membrane rafts. In the mature rat brain, NKCC1 was mainly insoluble in Brij 58 and co-distributed with the membrane raft marker flotillin-1 in sucrose density flotation experiments. In contrast, KCC2 was found in the insoluble fraction as well as in the soluble fraction, where it co-distributed with the non-raft marker transferrin receptor. Both KCC2 populations displayed a mature glycosylation pattern. Disrupting membrane rafts with methyl-β-cyclodextrin (MβCD) increased the solubility of KCC2, yet had no effect on NKCC1. In human embryonic kidney-293 cells, KCC2 was strongly activated by a combined treatment with MβCD and sphingomyelinase, while NKCC1 was inhibited. These data indicate that membrane rafts render KCC2 inactive and NKCC1 active. In agreement with this, inactive KCC2 of the perinatal rat brainstem largely partitioned into membrane rafts. In addition, the exposure of the transporters to MβCD and sphingomyelinase showed that the two transporters differentially interact with the membrane rafts. Taken together, membrane raft association appears to represent a mechanism for co-ordinated regulation of chloride transporter function.     

Computational Modeling Reveals Dendritic Origins of GABAA-Mediated Excitation in CA1 Pyramidal Neurons

GABA is the key inhibitory neurotransmitter in the adult central nervous system, but in some circumstances can lead to a paradoxical excitation that has been causally implicated in diverse pathologies from endocrine stress responses to diseases of excitability including neuropathic pain and temporal lobe epilepsy. We undertook a computational modeling approach to determine plausible ionic mechanisms of GABA_A-dependent excitation in isolated post-synaptic CA1 hippocampal neurons because it may constitute a trigger for pathological synchronous epileptiform discharge. In particular, the interplay intracellular chloride accumulation via the GABA_A receptor and extracellular potassium accumulation via the K/Cl co-transporter KCC2 in promoting GABA_A-mediated excitation is complex. Experimentally it is difficult to determine the ionic mechanisms of depolarizing current since potassium transients are challenging to isolate pharmacologically and much GABA signaling occurs in small, difficult to measure, dendritic compartments. To address this problem and determine plausible ionic mechanisms of GABA_A-mediated excitation, we built a detailed biophysically realistic model of the CA1 pyramidal neuron that includes processes critical for ion homeostasis. Our results suggest that in dendritic compartments, but not in the somatic compartments, chloride buildup is sufficient to cause dramatic depolarization of the GABA_A reversal potential and dominating bicarbonate currents that provide a substantial current source to drive whole-cell depolarization. The model simulations predict that extracellular K⁺ transients can augment GABA_A-mediated excitation, but not cause it. Our model also suggests the potential for GABA_A-mediated excitation to promote network synchrony depending on interneuron synapse location - excitatory positive-feedback can occur when interneurons synapse onto distal dendritic compartments, while interneurons projecting to the perisomatic region will cause inhibition.     

The KCl‐cotransporter KCC2 linked to epilepsy

In the mature brain, the neurotransmitter GABA can cause a postsynaptic hyperpolarization via activation of chloride permeant GABA_A receptor channels. This hyperpolarizing response critically depends on chloride extrusion via the KCl‐cotransporter KCC2 1 . Its knockdown in mice impairs synaptic inhibition by changing the electrochemical potential for chloride and thus increases neuronal excitability 2 3 . Two independent groups provide first evidence now, published in EMBO reports, that rare variants of KCC2 confer an increased risk of epilepsy in men 4 5 .     

Two Metabotropic γ-Aminobutyric Acid Receptors Differentially Modulate Calcium Currents in Retinal Ganglion Cells

Metabotropic γ-aminobutyric acid (GABA) receptors were studied in amphibian retinal ganglion cells using whole cell current and voltage clamp techniques. The aim was to identify the types of receptor present and their mechanisms of action and modulation. Previous results indicated that ganglion cells possess two ionotropic GABA receptors: GABA_AR and GABA_CR. This study demonstrates that they also possess two types of metabotropic GABA_B receptor: one sensitive to baclofen and another to cis-aminocrotonic acid (CACA). The effects of these selective agonists were blocked by GDP-β-S. Baclofen suppressed an ω-conotoxin–GVIA-sensitive barium current, and this action was reversed by prepulse facilitation, indicative of a direct G-protein pathway. The effect of baclofen was also partially occluded by agents that influence the protein kinase A (PKA) pathway. But the effect of PKA activation was unaffected by prepulse facilitation, indicating PKA acted through a parallel pathway. Calmodulin antagonists reduced the action of baclofen, whereas inhibitors of calmodulin phosphatase enhanced it. Antagonists of internal calcium release, such as heparin and ruthenium red, did not affect the baclofen response. Thus, the baclofen-sensitive receptor may respond to influx of calcium. The CACA-sensitive GABA receptor reduced current through dihydropyridine-sensitive channels. Sodium nitroprusside and 8-bromo-cGMP enhanced the action of CACA, indicating that a nitric oxide system can up-regulate this receptor pathway. CACA-sensitive and baclofen-sensitive GABA_B receptors reduced spike activity in ganglion cells. Overall, retinal ganglion cells possess four types of GABA receptor, two ionotropic and two metabotropic. Each has a unique electrogenic profile, providing a wide range of neural integration at the final stage of retinal information processing.     

GABAB receptors suppress burst-firing in reticular thalamic neurons

Burst-firing in thalamic neurons is known to play a key role in mediating thalamocortical (TC) oscillations that are associated with non-REM sleep and some types of epileptic seizure. Within the TC system the primary output of GABAergic neurons in the reticular thalamic nucleus (RTN) is thought to induce the de-inactivation of T-type calcium channels in thalamic relay (TR) neurons, promoting burst-firing drive to the cortex and the propagation of TC network activity. However, RTN neurons also project back onto other neurons within the RTN. The role of this putative negative feedback upon the RTN itself is less well understood, although is hypothesized to induce de-synchronization of RTN neuron firing leading to the suppression of TC oscillations. Here we tested two hypotheses concerning possible mechanisms underlying TC oscillation modulation. Firstly, we assessed the burst-firing behavior of RTN neurons in response to GABA_B receptor activation using acute brain slices. The selective GABA_B receptor agonist baclofen was found to induce suppression of burst-firing concurrent with effects on membrane input resistance. Secondly, RTN neurons express Ca_V3.2 and Ca_V3.3 T-type calcium channel isoforms known to contribute toward TC burst-firing and we examined the modulation of these channels by GABA_B receptor activation. Utilizing exogenously expressed T-type channels we assessed whether GABA_B receptor activation could directly alter T-type calcium channel properties. Overall, GABA_B receptor activation had only modest effects on Ca_V3.2 and Ca_V3.3 isoforms. The only effect that could be predicted to suppress burst-firing was a hyperpolarized shift in the voltage-dependence of inactivation, potentially causing lower channel availability at membrane potentials critical for burst-firing. Conversely, other effects observed such as a hyperpolarized shift in the voltage-dependence of activation of both Ca_V3.2 and Ca_V3.3 as well as increased time constant of activation of the Ca_V3.3 isoform would be expected to enhance burst-firing. Together, we hypothesize that GABA_B receptor activation mediates multiple downstream effectors that combined act to suppress burst-firing within the RTN. It appears unlikely that direct GABA_B receptor-mediated modulation of T-type calcium channels is the major mechanistic contributor to this suppression.     

Taurine release modified by GABAergic agents in hippocampal slices from adult and developing mice

Summary. In order to characterize the possible regulation of taurine release by GABAergic terminals, the effects of several agonists and antagonists of GABA receptors on the basal and K⁺-stimulated release of [³H]taurine were investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice using a superfusion system. Taurine release was concentration-dependently potentiated by GABA, which effect was reduced by phaclofen, saclofen and (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) at both ages, suggesting regulation by both GABA_B and GABA_C receptors. The involvement of GABA_A receptors could not be excluded since the antagonist bicuculline was able to affect both basal and K⁺-evoked taurine release. Furthermore, several GABA_B receptor effectors were able to inhibit K⁺-stimulated taurine release in the adults, while the GABA_C receptor agonists trans-4-aminocrotonic acid (TACA) and cis-4-aminocrotonic acid (CACA) potentiated this release. The potentiation of taurine release by agents acting on the three types of GABA receptors in both adult and developing hippocampus further indicates the involvement of transporters operating in an outward direction. This inference is corroborated by the moderate but significant inhibition of taurine uptake by the same compounds. Received June 28, 1999, Accepted August 31, 1999     

Recent structural and functional insights into the family of sodium calcium exchangers

Maintenance of calcium homeostasis is necessary for the development and survival of all animals. Calcium ions modulate excitability and bind effectors capable of initiating many processes such as muscular contraction and neurotransmission. However, excessive amounts of calcium in the cytosol or within intracellular calcium stores can trigger apoptotic pathways in cells that have been implicated in cardiac and neuronal pathologies. Accordingly, it is critical for cells to rapidly and effectively regulate calcium levels. The Na⁺/Ca²⁺ exchangers (NCX), Na⁺/Ca²⁺/K⁺ exchangers (NCKX), and Ca²⁺/Cation exchangers (CCX) are the three classes of sodium calcium antiporters found in animals. These exchanger proteins utilize an electrochemical gradient to extrude calcium. Although they have been studied for decades, much is still unknown about these proteins. In this review, we examine current knowledge about the structure, function, and physiology and also discuss their implication in various developmental disorders. Finally, we highlight recent data characterizing the family of sodium calcium exchangers in the model system, Caenorhabditis elegans, and propose that C. elegans may be an ideal model to complement other systems and help fill gaps in our knowledge of sodium calcium exchange biology. genesis 52:93–109. © 2013 Wiley Periodicals, Inc.