KCC2 Manipulation Alters Features of Migrating Interneurons in Ferret Neocortex.

KCC2 is a brain specific chloride-potassium cotransporter affecting neuronal development including migration and cellular maturation. It modulates chloride homeostasis influencing the switch of GABA from depolarizing to hyperpolarizing, which contributes to the cues that influence the termination of neuronal migration. The expression of KCC2 during migration of interneurons, therefore, correlates with the ability of these cells to respond to GABA as a stop signal. Manipulation of KCC2 in development can affect various aspects of migrating neurons, including the speed. We describe the effect of KCC2 downregulation and inhibition on features of migrating interneurons of normal ferret kits and those treated with methylazoxymethanol acetate, which increases KCC2. Treatment of organotypic cultures with Bisphenol A, an environmental toxin that alters gene expression, also downregulates KCC2 protein. In organotypic slices treated with the KCC2 antagonist VU0240551, chloride imaging shows inhibition of KCC2 via blockade of chloride flux. Time-lapse video imaging of organotypic cultures treated with either drug, shows a significant increase in the average speed, step size, and number of turns made by migrating neurons leaving the ganglionic eminence. Our findings demonstrate the harmful effect of environmental toxins on brain development and potential consequences in the pathogenesis of neurodevelopmental disorders.

Specific mesenchymal/epithelial induction of olfactory receptor, vomeronasal, and gonadotropin-releasing hormone (GnRH) neurons.

We asked whether specific mesenchymal/epithelial (M/E) induction generates olfactory receptor neurons (ORNs), vomeronasal neurons (VRNs), and gonadotropin-releasing hormone (GnRH) neurons, the major neuron classes associated with the olfactory epithelium (OE). To assess specificity of M/E-mediated neurogenesis, we compared the influence of frontonasal mesenchyme on frontonasal epithelium, which becomes the OE, with that of the forelimb bud. Despite differences in position, morphogenetic and cytogenic capacity, both mesenchymal tissues support neurogenesis, expression of several signaling molecules and neurogenic transcription factors in the frontonasal epithelium. Only frontonasal mesenchyme, however, supports OE-specific patterning and activity of a subset of signals and factors associated with OE differentiation. Moreover, only appropriate pairing of frontonasal epithelial and mesenchymal partners yields ORNs, VRNs, and GnRH neurons. Accordingly, the position and molecular identity of specialized frontonasal epithelia and mesenchyme early in gestation and subsequent inductive interactions specify the genesis and differentiation of peripheral chemosensory and neuroendocrine neurons.

Odorants suppress a voltage-activated K+ conductance in rat olfactory neurons.

Stimulation of olfactory receptor neurons (ORNs) with odors elicits an increase in the concentration of cAMP leading to opening of cyclic nucleotide-gated (CNG) channels and subsequent depolarization. Although opening of CNG channels is thought to be the main mechanism mediating signal transduction, modulation of other ion conductances by odorants has been postulated. To determine whether K+ conductances are modulated by odorants in mammalian ORNs, we examined the response of rat ORNs to odors by recording membrane current under perforated-patch conditions. We find that rat ORNs display two predominant types of responses. Thirty percent of the cells responded to odorants with activation of a CNG conductance. In contrast, in 55% of the ORNs, stimulation with odorants inhibited a voltage-activated K+ conductance (IKo). In terms of pharmacology, ion permeation, outward rectification, and time course for inactivation, IKo resembled a delayed rectifier K+ conductance. The effect of odorants on IKo was specific (only certain odorants inhibited IKo in each ORN) and concentration dependent, and there was a significant latency between arrival of odorants to the cell and the onset of suppression. These results indicate that indirect suppression of a K+ conductance (IKo) by odorants plays a role in signal transduction in mammalian ORNs.

Characterization of inositol-1,4,5-trisphosphate-gated channels in the plasma membrane of rat olfactory neurons.

It is generally accepted that inositol-1,4,5-trisphosphate (InsP3) plays a role in olfactory transduction. However, the precise mode of action of InsP3 remains controversial. We have characterized the conductances activated by the addition of 10 microM InsP3 to excised patches of soma plasma membrane from rat olfactory neurons. InsP3 induced current fluctuations in 25 of 121 inside-out patches. These conductances could be classified into two groups according to the polarity of the current at a holding potential of +40 to +60 mV (with Ringer's in the pipette and pseudointracellular solution in the bath). Conductances mediating outward currents could be further divided into large- (64 +/- 4 pS, n = 4) and small- (16 +/- 1.7 pS, n = 11) conductance channels. Both small- and large-conductance channels were nonspecific cation channels. The large-conductance channel displayed bursting behavior at +40 mV, with flickering increasing at negative holding potentials to the point where single-channel currents were no longer discernible. The small-conductance channel did not display flickering behavior. The conductance mediating inward currents at +40 to +60 mV reversed at +73 +/- 4 mV (n = 4). The current traces displayed considerable fluctuations, and single-channel currents could not be discerned. The current fluctuations returned to baseline after removal of InsP3. The power density spectrum for the excess noise generated by InsP3 followed a 1/f dependence consistent with conductance fluctuations in the channel mediating this current, although other mechanisms are not excluded. These experiments demonstrate the presence of plasma membrane InsP3-gated channels of different ionic specificity in olfactory receptor cells.

InsP3 causes an increase in apical [Ca2+]i by activating two distinct current components in vertebrate olfactory receptor cells.

1. Effects of inositol-1,4,5-trisphosphate (InsP3) applied through a patch pipette to Xenopus laevis olfactory receptor cells (ORCs) were studied using the patch-clamp technique in conjunction with calcium imaging with fura-2. 2. InsP3 activated, first, a novel voltage-independent Ca2+ current (ICa) and, second, a nonselective cation current (Icat). 3. The activation of these currents occurred at different intracellular calcium concentrations, and the activation of either current led to a marked increase of [Ca2+]i in the dendritic knob. 4. The results suggest that InsP3 might act as a second messenger in vertebrate olfactory receptor cells by activating, through different mechanisms, a plasma membrane Ca2+ conductance (ICa) and a nonselective cation conductance (Icat).

Sodium/calcium exchanger in olfactory receptor neurones of Xenopus laevis.

Ca2+ ions enter neurones through various types of calcium and cation channels. The mechanisms by which Ca2+ ions are spatially buffered and expelled from neurones have been studied considerable less. Using calcium imaging in conjunction with the patch clamp technique, we investigated the Na/Ca exchanger in olfactory neurones and found evidence for its localization on the dendrite. It is suggested that this tends to decouple increases in [Ca2+]i occurring in the transduction compartments of the cell from processes in the soma.

Amiloride-insensitive cation conductance in Xenopus laevis olfactory neurons: a combined patch clamp and calcium imaging analysis.

We used digital calcium imaging with Fura-2 in conjunction with the tight-seal whole-cell patch clamp technique to describe a novel cation conductance in olfactory neurons of the clawed toad Xenopus laevis. Substitution of extracellular Ca2+ and Na+ was used as a tool to change [Ca2+]i. When [Ca2+]i was increased to about 450 nM, a conductance gcat activated that was permeable for cations. Upon gcat activation, an increase in [Ca2+]i occurred in the dendritic knob. Once activated, gcat showed no further dependence upon [Ca2+]i. Icat is shown to be different from the current activated by a mixture of the odorants citralva and amyl acetate. We conclude that there are two different cation conductances in the peripheral compartments of olfactory neurons in X. laevis.

Standing calcium gradients in olfactory receptor neurons can be abolished by amiloride or ruthenium red.

Digital imaging and the patch clamp technique were used to investigate the intracellular calcium concentration in olfactory receptor neurons using the Ca2+ indicator dyes fura-2 and fura-2/AM. The spatial distribution of Cai2+ as well as its modification by the drugs Amiloride and Ruthenium Red were studied. Resting calcium concentrations in cells loaded with fura-2/AM were between 10 and 200 nM. In cells that were loaded with the pentapotassium salt of fura-2 through the patch pipette, calcium concentrations were in the same range if ATP was added to the pipette solution. Otherwise, Ca2+ reached concentrations of approximately 500 nM. Most of the observed cells showed a standing gradient of calcium, the calcium concentrations in the distal dendritic end of the cell being higher than in the soma. In some cells, the gradient was markedly reduced or abolished by adding either Amiloride or Ruthenium Red to the bath solution. In a few cells, neither drug had any effect upon the gradient. It is suggested that the inhomogenous spatial distribution of intracellular calcium in olfactory cells of Xenopus laevis is brought about by an influx of calcium ions through two different calcium permeable conductances in the peripheral compartments of the cells. The fact that only either Ruthenium Red or Amiloride abolished the standing calcium gradient further suggested that the two conductances blocked were presumably not coexpressed in the same cells.

Effects of nitric oxide upon olfactory receptor neurones in Xenopus laevis.

We have studied olfactory receptor neurones under voltage-clamp conditions using the patch clamp technique. Application of sodium nitroprusside (SNP) activated a cation conductance if the GTP concentration in the pipette was in the millimolar range. With 10 microM GTP, SNP had no effect on the holding current but it abolished the normally occurring wash-out of responses to odorants. This effect could also be obtained by adding 150 nM cGMP to the pipette solution. We conclude that there is a NO/cGMP system in Xenopus' olfactory neurones and that cGMP plays an important role in the transduction process in that it guarantees ongoing responsiveness to odorants.