The type 2 K/Cl cotransporter (KCC2) allows neurons to maintain low intracellular levels of Cl, a prerequisite for efficient synaptic inhibition. Reductions in KCC2 activity are evident in epilepsy; however, whether these deficits directly contribute to the underlying pathophysiology remains controversial. To address this issue, we created knock-in mice in which threonines 906 and 1007 within KCC2 have been mutated to alanines (KCC2-T906A/T1007A), which prevents its phospho-dependent inactivation. The respective mice appeared normal and did not show any overt phenotypes, and basal neuronal excitability was unaffected. KCC2-T906A/T1007A mice exhibited increased basal neuronal Cl extrusion, without altering total or plasma membrane accumulation of KCC2. Critically, activity-induced deficits in synaptic inhibition were reduced in the mutant mice. Consistent with this, enhanced KCC2 was sufficient to limit chemoconvulsant-induced epileptiform activity. Furthermore, this increase in KCC2 function mitigated induction of aberrant high-frequency activity during seizures, highlighting depolarizing GABA as a key contributor to the pathological neuronal synchronization seen in epilepsy. Thus, our results demonstrate that potentiating KCC2 represents a therapeutic strategy to alleviate seizures.

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The mechanisms by which intake of dietary protein is regulated are poorly understood despite their potential involvement in determining food choice and appetite. In particular, it is unclear whether protein deficiency results in a specific appetite for protein and whether influences on diet are immediate or develop over time. To determine the effects of protein restriction on consumption, preference, and palatability for protein we assessed patterns of intake for casein (protein) and maltodextrin (carbohydrate) solutions in adult rats. To induce a state of protein restriction, rats were maintained on a low protein diet (5% casein) and compared to control rats on non-restricted diet (20% casein). Under these dietary conditions, relative to control rats, protein-restricted rats exhibited hyperphagia without weight gain. After two weeks, on alternate conditioning days, rats were given access to either isocaloric casein or maltodextrin solutions that were saccharin-sweetened and distinctly flavored whilst consumption and licking patterns were recorded. This allowed rats to learn about the post-ingestive nutritional consequences of the two different solutions. Subsequently, during a preference test when rats had access to both solutions, we found that protein-restricted rats exhibited a preference for casein over carbohydrate whereas non-restricted rats did not. Analysis of lick microstructure revealed that this preference was associated with an increase in cluster size and number, reflective of an increase in palatability. In conclusion, protein-restriction induced a conditioned preference for protein, relative to carbohydrate, and this was associated with increased palatability.

Methylphenidate (MPD) modulates dopamine (DA) overflow in part by redistributing vesicle pools, a function shared by the presynaptic protein ␣-synuclein (␣-syn). We suggest that ␣-syn modifies the effect of MPD on DA neurotransmission. The effect was studied in the dorsal striatum in wild-type mice and two mouse lines lacking ␣-syn by using in vivo voltammetry and microdialysis. MPD (1 mg/kg) attenuated evoked DA overflow only in mice lacking ␣-syn but produced a similar increase in the extracellular DA levels in all three lines. A kinetic analysis showed that MPD decreased DA release per stimulus pulse in ␣-syn-deficient mice but increased in wild-type mice. MPD blocked DA reuptake and produced a similar increase in the apparent affinity (K m ) for DA reuptake in all three lines. Repeated-burst stimulation redistributes vesicular storage pools and facilitates DA overflow, and this form of facilitation is significantly enhanced in ␣-syn knockout mice. The DA reuptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) (10 mg/kg) completely blocked the facilitation of DA overflow in all three lines, whereas MPD (1 mg/kg) selectively decreased it only in mice lacking ␣-syn. MPD (5 mg/kg) and GBR12909 (10 mg/kg) produced equipotent inhibition of DA reuptake (in terms of K m ), indicating that reuptake inhibition does not explain the MPD selectivity. Our data indicate that MPD decreases DA release probability in the absence of ␣-syn and increases it in control animals, whereas the effect of MPD on DA reuptake is independent of ␣-syn. We suggest that this selectivity is based on ␣-syn-dependent compartmentalization of presynaptic DA.

The mechanisms by which intake of dietary protein is regulated are poorly understood despite their potential involvement in determining food choice and appetite. In particular, it is unclear whether protein deficiency results in a specific appetite for protein and whether influences on diet are immediate or develop over time. To determine the effects of protein restriction on consumption, preference, and palatability for protein we assessed patterns of intake for casein (protein) and maltodextrin (carbohydrate) solutions in adult rats. To induce a state of protein restriction, rats were maintained on a low protein diet (5% casein) and compared to control rats on non-restricted diet (20% casein). Under these dietary conditions, relative to control rats, protein-restricted rats exhibited hyperphagia without weight gain. After two weeks, on alternate conditioning days, rats were given access to either isocaloric casein or maltodextrin solutions that were saccharin-sweetened and distinctly flavoured whilst consumption and licking patterns were recorded. This allowed rats to learn about the post-ingestive nutritional consequences of the two different solutions. Subsequently, during a preference test when rats had access to both solutions, we found that protein-restricted rats exhibited a preference for casein over carbohydrate whereas non-restricted rats did not. Analysis of lick microstructure revealed that this preference was associated with an increase in cluster size and number, reflective of an increase in palatability. In conclusion, proteinrestriction induced a conditioned preference for protein, relative to carbohydrate, and this was associated with increased palatability.