Intracellular Cl- plays a key role in cellular volume regulation, cell cycle control and shaping the polarity of inhibitory postsynaptic responses mediated by anion-permeable GABA and glycine receptors. In this study, we have investigated the expression patterns of members of the cation-chloride cotransporters (CCCs), including the K-Cl cotransporters KCC1-4 and the Na-K-2 Cl cotranporter NKCC1 during rodent embryonic brain development. At the time of neurogenesis (embryonic days; E12.5-14.5), KCC1 was only detectable in the developing choroid plexus. KCC2 mRNA was detectable as early as E12.5 in the ventral part of the (cervical) spinal cord, and by E14.5, the expression had spread to TUJ1-positive differentiating regions of the rhombencephalon, diencephalon and olfactory bulb, in parallel with neuronal maturation. KCC3 mRNA was scarce in the cortical plate at E14.5, and slightly up-regulated at birth. In contrast, KCC4 mRNA was abundantly expressed in the ventricular zone and was down-regulated perinatally. At E14.5, NKCC1 was highly expressed in the vimentin-positive radial glia of the proliferative zone of the subcortical region. At later embryonic stages, during gliogenesis (E17-P0), there was a shift in NKCC1 expression to the neuron specific Class III beta-tubulin (betaIII) positive region of the cortical plate. These unique spatiotemporal expression patterns of distinct CCCs during embryonic development suggests that Cl- regulatory mechanisms are critically involved in the control of neuronal development.

Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.heparan sulfotransferase | Kallmann syndrome T he coordinated assembly of the nervous system in metazoans requires the migration of the large majority of neurons from their place of origin to their final destination in the brain (1). These processes require the complex interplay of many factors, including secreted and transmembrane proteins that mediate communication between cells. The activity of such factors is greatly influenced by the extracellular environment (2). For example, heparan sulfates (HSs), a class of molecularly diverse extracellular glycosaminoglycans, have been shown to be crucial for neural development in mice (3). From work in model organisms, it has become clear that much of the function of HS during neural development is embedded within complex modification patterns of the HS sugar residues (reviewed in refs. 4 and 5). HS modification patterns serve specific and instructive functions during neural development and are believed to regulate ligand-receptor interactions (6-8). These patterns arise as the consequence of nonuniform modifications of the sugar moieties, including sulfations, deacetylations, and epimerizations that are introduced by specific HS-modifying enzymes (9) (Fig. 1A). It is unknown whether the function of HS modifications impinges on normal human development and disease susceptibility.Idiopathic hypogonadotropic hypogonadism (IHH) is a clinically and genetically heterogeneous condition that is characterized by lack of sexual maturation and infertility in the absence of other organic etiologies (10). Patients with IHH either have a normal sense of smell [normosmic IHH (nIHH)] or have an impaired sense of smell (anosmia

Hyperpolarizing fast inhibitory neurotransmission by gamma-aminobutyric acid and glycine requires an efficient chloride extrusion mechanism in postsynaptic neurons. A major effector of this task in adult animals is the potassium-chloride co-transporter KCC2 that is selectively and abundantly expressed postsynaptically in most CNS neurons. Yet, the role of KCC2 in adult brain at the systems level is poorly known. Here, we characterize the behaviour of mice doubly heterozygous for KCC2 null and hypomorphic alleles that retain 15-20% of normal KCC2 protein levels in the brain. These hypomorphic KCC2-deficient mice were viable and fertile but weighed 15-20% less than wild-type littermates at 2 weeks old and thereafter. The mice displayed increased anxiety-like behaviour in several tests including elevated plus-maze and were more susceptible to pentylenetetrazole-induced seizures. Moreover, the mice were impaired in water maze learning and showed reduced sensitivity to tactile and noxious thermal stimuli in von Frey hairs, hot plate and tail flick tests. In contrast, the mice showed normal spontaneous locomotor activity in open field and Y-maze tests, and intact motor coordination in rotarod and beam tests. The results suggest that requirements for KCC2-dependent fast hyperpolarizing inhibition may differ among various functional systems of the CNS. As shunting inhibition is expected to be intact in KCC2-deficient neurons, these mice may provide a useful tool to study the specific functions and relative importance of hyperpolarizing fast synaptic inhibition in adult CNS that may have implications for human neuropsychiatric disorders, such as epilepsy, pain and anxiety.

GABA A receptors mediate both fast phasic inhibitory postsynaptic potentials and slower tonic extrasynaptic inhibition. Hyperpolarizing phasic GABAergic inhibition requires the activity of neuron-specific chloride-extruding potassium-chloride cotransporter KCC2 in adult CNS. However, the possible role of KCC2 in tonic GABAergic inhibition and the associated behaviors is unknown. Here, we have examined the role of KCC2 in phasic vs tonic GABA inhibition by measuring the behavioral effects of pharmacological agents that presumably enhance phasic vs tonic inhibition in mice that retain 15-20% of normal KCC2 protein levels. These KCC2-deficient mice show decreased sensitivity to diazepam-induced sedation and motor impairment consistent with the reported role for KCC2 in fast hyperpolarizing inhibition. In contrast, the mice exhibit normal responses to low-dose alcohol-induced motor impairment, gaboxadolinduced sedation, and neurosteroid-induced hypnosis; behaviors thought to be associated with tonic GABAergic inhibition. Electrophysiological recordings show that the tonic conductance is not affected. The results suggest that KCC2 activity is more critical for behaviors dependent on phasic than tonic GABAergic inhibition. Neuropsychopharmacology (2007) 32, 911-918.