Some mutations of the LRRK2 gene underlie autosomal dominant form of Parkinson’s disease (PD). The G2019S is a common mutation that accounts for about 2% of PD cases. To understand the pathophysiology of this mutation and its possible developmental implications, we developed an in vitro assay to model PD with human induced pluripotent stem cells (hiPSCs) reprogrammed from skin fibroblasts of PD patients suffering from the LRKK2 G2019S mutation. We differentiated the hiPSCs into neural stem cells (NSCs) and further into dopaminergic neurons. Here we show that NSCs bearing the mutation tend to differentiate less efficiently into dopaminergic neurons and that the latter exhibit significant branching defects as compared to their controls.

Most serotonergic neurons display a prominent medium‐duration afterhyperpolarization (mAHP), which is mediated by small‐conductance Ca2+‐activated K+ (SK) channels. Recent ex vivo and in vivo experiments have suggested that SK channel blockade increases the firing rate and/or bursting in these neurons. The purpose of this study was therefore to characterize the source of Ca2+ which activates the mAHP channels in serotonergic neurons. In voltage‐clamp experiments, an outward current was recorded at −60 mV after a depolarizing pulse to +100 mV. A supramaximal concentration of the SK channel blockers apamin or (‐)‐bicuculline methiodide blocked this outward current. This current was also sensitive to the broad Ca2+ channel blocker Co2+ and was partially blocked by both ω‐conotoxin and mibefradil, which are blockers of N‐type and T‐type Ca2+ channels, respectively. Neither blockers of other voltage‐gated Ca2+ channels nor DBHQ, an inhibitor of Ca2+‐induced Ca2+ release, had any effect on the SK current. In current‐clamp experiments, mAHPs following action potentials were only blocked by ω‐conotoxin and were unaffected by mibefradil. This was observed in slices from both juvenile and adult rats. Finally, when these neurons were induced to fire in an in vivo‐like pacemaker rate, only ω‐conotoxin was able to increase their firing rate (by ~30%), an effect identical to the one previously reported for apamin. Our results demonstrate that N‐type Ca2+ channels are the only source of Ca2+ which activates the SK channels underlying the mAHP. T‐type Ca2+ channels may also activate SK channels under different circumstances.

The generation of neuronal cells from stem cells obtained from adult bone marrow is of significant clinical interest in order to design new cell therapy protocols for several neurological disorders. The recent identification in adult bone marrow of stem cells derived from the neural crest stem cells (NCSC) might explain the neuronal phenotypic plasticity shown by bone marrow cells. However, little information is available about the nature of these cells compared to mesenchymal stem cells (MSC), including their similarities and differences. In this paper, using transcriptomic as well as proteomic technologies, we compared NCSC to MSC and stromal nestin-positive cells, all of them isolated from adult bone marrow. We demonstrated that the nestin-positive cell population, which was the first to be described as able to differentiate into functional neurons, was a mixed population of NCSC and MSC. More interestingly, we demonstrated that MSC shared with NCSC the same ability to truly differentiate into Tuj1-positive cells when co-cultivated with paraformaldehyde-fixed cerebellar granule neurons. Altogether, those results suggest that both NCSC and MSC can be considered as important tools for cellular therapies in order to replace neurons in various neurological diseases.

Synaptic vesicle protein 2 (SV2) is a glycoprotein that exists in three isoforms, SV2A, SV2B, and SV2C. SV2A knockout (KO) mice and SV2A/SV2B double KO (DKO) mice, but not SV2B KO animals, start to experience severe seizures and weight loss 7 days after birth and die at about postnatal day (P)14–P23. Because excitatory and inhibitory inputs play a major role in controlling neuronal excitability in the hippocampus, we examined the effects of SV2A and/or SV2B deletions on glutamatergic and GABA A neurotransmission in hippocampal CA1 pyramidal neurons. Spontaneous and miniature excitatory and inhibitory postsynaptic currents (sEPSCs, mEPSCs, sIPSCs, and mIPSCs, respectively) were recorded using the whole‐cell patch‐clamp technique in slices from P6–P14 mice. The frequency of sEPSCs was increased in SV2A KO and SV2A/SV2B DKO mice, but their amplitude was unchanged. Such changes were not observed in SV2B KOs. On the contrary, the frequency and amplitude of sIPSCs were decreased in SV2A KO and SV2A/SV2B DKO mice but not in SV2B KO animals, as reported previously for the CA3 region. Kinetic parameters of sIPSCs and sEPSCs were unchanged. Importantly, no changes were observed in any genotype when examining mEPSCs and mIPSCs. We conclude that action potential‐ and Ca 2+ ‐dependent glutamatergic and GABAergic synaptic transmission are differentially altered in the hippocampus of SV2A‐deficient mice, whereas the mechanism of exocytosis itself is not changed. The altered balance between these major excitatory and inhibitory inputs is probably a contributing factor to seizures in SV2A KO and SV2A/SV2B DKO mice. © 2012 Wiley Periodicals, Inc.

We studied gamma-aminobutyric acid (GABA)-mediated currents in short-term cultured dorsal unpaired median (DUM) neurons of cockroach Periplaneta americana using the whole cell patch-clamp technique in symmetrical chloride solutions. All DUM neurons voltage-clamped at -50 mV displayed inward currents (I(GABA)) when 10(-4) M of GABA was applied by pneumatic pressure-ejection pulses. The semi-logarithmic curve of I(GABA) amplitude versus the ejection time yielded a Hill coefficient of 4.0. I(GABA) was chloride (Cl-) because the reversal potential given by the current-voltage (I-V) curve varied according to the value predicted by the Nernst equation for Cl- dependence. In addition, I(GABA) was almost completely blocked by bath application of the chloride channel blockers picrotoxin (PTX) or 3,3-bis(trifluoromethyl)bicyclo-[2,2,1]heptane-2,2-diacarbonitrile (BIDN). The I-V curve for I(GABA) displayed a unexpected biphasic aspect and was best fitted by two linear regressions giving two slope conductances of 35.6 +/- 2.1 and 80.9 +/- 4.1 nS for potentials ranging from 0 to -30 and -30 to -70 mV, respectively. At -50 mV, the current amplitude was decreased by cadmium chloride (CdCl2, 10(-3) M) and calcium-free solution. The semi-logarithmic curve for CdCl2-resistant I(GABA) gave a Hill coefficient of 2.4. Hyperpolarizing voltage step from -50 to -80 mV was known to increase calcium influx through calcium-resting channels. According to this protocol, a significant increase of I(GABA) amplitude was observed. However, this effect was never obtained when the same protocol was applied on cell body pretreated with CdCl2. When the calmodulin blocker N-(6-aminohexyl)-5-chloro-1-naphtalene-sulfonamide or the calcium-calmodulin-dependent protein kinase blocker 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) was added in the pipette solution, I(GABA) amplitude was decreased. Pressure ejection application of the cis-4-aminocrotonic acid (CACA) on DUM neuron cell body held at -50 mV, evoked a Cl- inward current which was insensitive to CdCl2. The Hill plot yielded a Hill coefficient of 2.3, and the I-V curve was always linear in the negative potential range with a slope conductance of 32.4 +/- 1.1 nS. These results, similar to those obtained with GABA in the presence of CdCl2 and KN-62, indicated that CACA activated one subtype of GABA receptor. Our study demonstrated that at least two distinct subtypes of Cl--dependent GABA receptors were expressed in DUM neurons, one of which is regulated by an intracellular Ca2+-dependent mechanism via a calcium-dependent protein kinase. The consequences of the modulatory action of Ca2+ in GABA receptors function and their sensitivity to insecticide are discussed.

In the central nervous system of both vertebrates and invertebrates inhibitory neurotransmission is mainly achieved through activation of gamma-aminobutyric acid (GABA) receptors. Extensive studies have established the structural and pharmacological properties of vertebrate GABA receptors. Although the vast majority of insect GABA-sensitive responses share some properties with vertebrate GABAA receptors, peculiar pharmacological properties of these receptors led us to think that several GABA-gated chloride channels are present in insects. We describe here the pharmacological properties of two GABA receptor subtypes coupled to a chloride channel on dorsal unpaired median (DUM) neurones of the adult male cockroach. Long applications of GABA induce a large biphasic hyperpolarization, consisting of an initial transient hyperpolarization followed by a slow phase of hyperpolarization that is not quickly desensitized. With GABA, the transient hyperpolarization is sensitive to picrotoxinin, fipronil and dieldrin whereas the slow response is insensitive to these insecticides.When GABA is replaced by muscimol and cis-4-aminocrotonic acid (CACA) a biphasic hyperpolarization consisting of an initial transient hyperpolarization followed by a sustained phase is evoked which is blocked by picrotoxinin and fipronil. Exposure to dieldrin decreases only the early phase of the muscimol and CACA-induced biphasic response, suggesting that two GABA-gated chloride channel receptor subtypes are present in DUM neurones. This study describes, for the first time, a dieldrin resistant component different to the dieldrin- and picrotoxinin-resistant receptor found in several insect species.

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to microM agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.