Elevated intracellular chloride level in albino visual cortex neurons is mediated by Na-K-Cl co-transporter
© Diykov et al; licensee BioMed Central Ltd. 2008
Received: 20 March 2008
Accepted: 30 June 2008
Published: 30 June 2008
During development the switch from a depolarizing to a hyperpolarizing action of GABA is a consequence of a decrease of the Na+-K+-2Cl- co-transporter (NKCC1, Cl--uptake) and increase of the K+-Cl- co-transporter (KCC2, Cl--extrusion) expression. However albino visual cortex neurons don't show a corresponding decrease in intracellular chloride concentration during development of the visual system as compared to pigmented animals.
Our study revealed that more cells express NKCC1 in albinos compared to pigmented rat visual cortex neurons whereas KCC2 is expressed in all cells in both strains. We determined a positive relationship between the presence of NKCC1 and an inhibitory deficit in single neurons of the albino visual cortex. After pharmacological blockade of NKCC1 function with its specific inhibitor, bumetanide, the reversal potential of electrically evoked GABAA receptor-mediated postsynaptic currents and, as a consequence, [Cl-]i in albino visual cortex neurons shifted to the pigmented rat brain value. In conclusion, our pharmacological experiments and subsequent single cell real time PCR analysis of the co-transporter mRNA demonstrated that the inhibitory deficit present in the albino visual cortical network is almost exclusively mediated by NKCC1.
Our findings suggest that blocking of NKCC1 in albino visual cortex neurons could improve processing in visual cortex and therefore might be beneficial for vision in albinos.
Albino mutations lead to a decrease in direction selectivity of neurons in the motion sensitive cortical and subcortical areas of albino mammals [1, 2]. This, in turn critically depends on GABAergic mechanisms [3–6]. While GABA is the main inhibitory transmitter in the adult brain, GABAergic transmission is excitatory during early postnatal development. This different action of GABA results from a reversed chloride concentration gradient with higher intracellular chloride concentration in immature neurons [7–10]. The Na+-K+-2Cl- co-transporter (NKCC1, Cl--uptake) and the K+-Cl- co-transporter (KCC2, Cl--extrusion) are the most important of the many known chloride regulators in neocortical neurons [11, 12]. The developmental switch to an inhibitory action of GABA is a consequence of a decrease of NKCC1 and increase of KCC2 expression after birth. Cl- uptake in immature neurons is mediated by Na+-K+-2Cl- cotransporter [8, 10, 13], as no GABAA mediated depolarization was found in NKCC1 knock out mice . Interestingly in cortical neurons, a shift in Cl- homeostasis toward a higher [Cl-]i is implicated in the determination of developmental stage: depolarizing GABAergic and glycinergic responses mediate various developmental processes, such as neuronal migration, differentiation, and synapse formation [7, 15–17]. In contrast to NKCC1, the KCC2 co-transporter's central role is promoting inhibition and preventing hyperexcitability [9, 18]. An inhibitory action of GABA is required to discriminate differences in input activities during processes accompanied by synaptic pruning , i.e. the presence of GABAA receptor-mediated inhibition is essential during the critical period of ocular-dominance plasticity . Zhu et al. showed in KCC2-/- mice  that cortical neurons lacking KCC2 not only fail to show a developmental decrease in [Cl-]i, but also are unable to regulate [Cl-]i on Cl- loading or maintain [Cl]i during membrane depolarization.
Results and discussion
NKCC1 positive and negative cells in albino and pigmented rat visual cortex
In order to evaluate the impact of elevated intracellular chloride levels in albino visual cortex neurons on cortical network properties we conducted gramicidin perforated patch clamp analysis of both albino and pigmented layer V visual cortex neurons without blocking of excitation during physiological recordings. We found that the reversal potential of postsynaptic currents (combined inhibitory and excitatory) significantly differ between albino and pigmented animals, being shifted into the depolarizing direction in albinos (-49.2 +/- 1.3 mV, n = 16, in albino vs. -58.2 +/- 2.3 mV, n = 10, in pigmented animals; Additional file 1, fig. 1). One should keep in mind, however, that the observed difference may result from variable inhibitory and/or excitatory inputs to characterized cells. Surprisingly, minimal interspike intervals were significantly longer in albinos than in pigmented (Additional file 1, fig. 2): 28.2 +/- 2.9 ms(n = 16) vs. 23.2 +/- 2.6 ms(n = 10). Oscillations within and across neuronal systems are believed to serve various complex functions, such as perception, cognition, movement initiation, plasticity and memory. GABAergic mechanisms play a major role in these oscillatory patterns. Therefore, the increase in minimal interspike intervals in albino neuron may indicate a reduced possibility for high frequency neuronal coding in albino visual cortex networks. Previous findings of Barmashenko et al. showed that albino rat visual cortex neurons also demonstrate a significantly lower rheobase (the current threshold to release a spike by long depolarizing pulses) and a smaller chronaxy (the minimal duration of a current pulse of twice the rheobase amplitude to release a spike) compared to pigmented animals. These data strengthen the hypothesis of a severe inhibitory deficit in albino visual cortex.
In sum, our findings suggest that blocking of NKCC1 in albino visual cortex neurons will bring their chloride homeostasis to the pigmented brain level. This could improve processing in visual cortex and therefore might be beneficial for vision in albinos. So far this speculation is without supporting data and the role of NKCC1 in neuronal excitability seems controversial  and blocking NKCC1 in neurons without blocking in glia is not practical. In addition caution is advisable because bumetanide therapy may be controversial for retinal processing where many cell types express functional NKCC1 and their EGABA is either known or predicted to be positive to resting membrane potential [5, 25] for normal function.
where: V Eq . is the equilibrium (measurable) reversible potential for a given ion, in our study it is Cl- ion; Ris the universal gas constant (8.314 J.K-1.mol-1); Tis the temperature in Kelvin (°K = °C + 273.15); zis the valence of the ionic species; Fis the Faraday's constant (96485 C.mol-1); [X] out is the concentration of the ionic species X in the extracellular fluid (Cl1-); [X] in is the concentration of the ionic species X in the intracellular fluid (Cl1-).
Gramicidin-perforated patch-clamp recordings
Techniques we used for perforated patch clamp recording have been reported in detail elsewhere . The recording chamber was perfused with oxygenated room temperature ACSF, 3 ml/min. Kynurenic acid (2 mM) was added directly to the ACSF to prevent excitatory activity in the neurons tested. Gramicidin perforated patch clamp recordings were performed under visual control. Borosilicate patch electrodes (5–9 MΩ) were filled with a solution containing 130 mM K-gluconate, 0.5 mM Na-gluconate, 20 mM HEPES, 4 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 0.5 mM EGTA (pH 7.2). Gramicidin (30 μg/ml, dissolved in DMSO, Sigma) was added to the solution as a membrane perforating agent. The measured membrane potentials were corrected for the junction potential of -10 mV . Inhibitory postsynaptic currents (IPSCs) were evoked through a concentric bipolar electrode placed approximately 50–100 μm lateral to the recorded neuron with stimuli (20–100 μA, 50 μs duration, 0.1 Hz) in voltage clamp mode by means of a PC-501A patch clamp amplifier (Warner Institute Corporation) connected via AD/DA-converters (CED 1401+, Cambridge Electronic Design, UK) to a personal computer. Holding potentials were raised from -100 to +30 mV in 10 mV steps in every recording. Recordings underwent low-pass filtering at 3 kHz and were sampled at 10 kHz. For recording and analyzing WinWCP software (John Dempster, University of Strathclyde, Glasgow, UK) was used.
Single cell real time PCR
cDNA synthesis and the first round of PCR was performed using OneStep RT-PCR kit (Qiagen, GmbH, Germany) as described in . Briefly, cytoplasm of a cell excluding the cell nucleus was aspirated by a single mild suction. This sample was then expelled into a reaction tube which contained 5 μl of RNAse-free water with 10 units of RNAse inhibitor (Qiagen, GmbH, Germany). Harvested cytoplasm was frozen and stored at -80°C for at most 8 hours. The master mix for the reverse transcription was prepared by mixing 10 μl of 5× Qiagen OneStep RT-PCR buffer, 2 μl of 10 mM of each dNTP mix, 1 μl of 10 μM β-actin outer primers, 3 μl of 10 μM KCC2 outer primers, 3 μl of 10 μl NKCC1 outer primers, 10 units of Qiagen OneStep RT-PCR enzyme mix, 10 μl of 5× Q-solution and RNAse-free water to obtain a total volume of 40 μl. Afterwards 40 μl of the master mix were combined with 10 μl sample and the reverse transcription performed for 30 min at 50°C in a thermal cycler. After the reverse transcription step first round PCR amplification was immediately started as follows: 15 min at 95°C, followed by 40 cycles (30 s at 94°C, 30 sec at 55°C, 1 min at 72°C) in a thermal cycler. Outer primers pair's sequences for NKCC1, KCC2 and β-actin were taken from . Subsequently we diluted first-round PCR products 500-fold and 1 μl of the diluted mixture was taken as a template for second round of PCR (40 cycles) in a real time PCR format using the Opticon2 detection system (Biorad, Hercules, USA) (see Additional file 1, fig. 3 for schematic of experimental procedures). For each template 10 μl SyberGreen, 0.25 μl of 10 μM inner primers and 9.75 μl of water were used (final reaction volume was 20 μl). The amplification involved a hot start activation of the polymerase at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 52°C for 30 s, extention at 72°C for 30 s in separate reactions using the inner primers pairs (β-actin, NKCC1, KCC2) for each template. These primers are specific to regions within the PCR products produced by the first round PCR amplification (nested PCR). The design of the inner primers was performed using Primer Express 2.0 software (Applied Biosystems, Foster city, USA). A possible cross-homology between NKCC1, KCC2 and β-actin sequences was excluded. The specificity of primers was further confirmed in agarose gel after amplification and by melting point analysis of the amplicons generated by Real Time PCR. Outer and inner primers sequences are presented in Additional file 1, Table 1.
The drugs applied were gramicidin D, bumetanide (Sigma, St. Louis, MO), kynurenic acid (KYN, an ionotropic glutamatergic receptor antagonist) and bicuculline (an ionotropic GABAA receptor antagonist) (Tocris Cookson, Bristol, UK). Substances were prepared as stock solutions and frozen, then added to the ACSF to reach the desired final concentration.
Mann-Whitney rank sum test, one way ANOVA and Chi-square tests (p < 0.001), SigmaStat software, were used to test the data for significant disparities. Numerical data are presented as mean ± S.D.
Authors want to thank Margareta Möllmann and Stephanie Kramer for excellent technical assistance. This work was supported by IGSN funds to Dmitry Diykov and Andrey Turchinovich and DFG grant SFB 509 A11 to K.-P. Hoffmann.
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