Neuron
The targeting of sensory afferent neurons in the leech CNS occurs in two discrete steps that are mediated via different carbohydrate recognitions, as shown by molecular perturbations of cultured embryos. A constitutive carbohydrate marker that is generic to all of these neurons mediates their initial defasciculation and arborization across the entire target region via mannose-specific recognition...
We are analyzing features of the K+ channel subunit proteins that are critical for function and regulation of these proteins. Our studies show biochemically that subunit proteins from the Shaker and Shaw subfamilies fail to assemble into a heteromultimer. The basis for this incompatibility is the sequences contained within the T1 assembly domain. For a subunit protein to heteromultimerize with a Shaker...
Dominant mutations of the Drosophila ninaE-encoded rhodopsin are described that reduce the expression of wild-type rhodopsin and cause a slow, age-dependent form of retinal degeneration. A posttranslational event subsequent to the requirement for the ninaA-encoded cyclophilin is disrupted by the dominant mutations. Most of these dominant mutations are missense mutations that affect the physical properties...
The continuous presence of nerve growth factor (NGF) is thought to be required for the elaboration of neuronal-like traits in PC12 cells. Surprisingly, we find that a 1 min exposure to NGF is sufficient to engage a longer-term genetic program leading to the acquisition of membrane excitability. Whereas continuous exposure to NGF causes the induction of a family of sodium channels, the effect of a...
Cerebral deposition of amyloid β protein (Aβ) is an early and critical feature of Alzheimer's disease. Here we analyze the substrate requirements of proteases (''β-secretases'') that cleave the R-amyloid precursor protein (βAPP) at the N-terminus of Aβ (Asp-597 of βAPP695)fis5) in intact human cells. The cleavage requires a membrane-bound substrate but tolerates shifts in the distance of the hydrolyzed...
Mammalian high conductance, calcium-activated potassium (maxi-K) channels are composed of two dissimilar subunits, α and β. We have examined the functional contribution of the β subunit to the properties of maxi-K channels expressed heterologously in Xenopus oocytes. Channels from oocytes injected with cRNAs encoding both a and β subunits were much more sensitive to activation by voltage and calcium...
Alternative splicing of the Shaker (Sh) locus of Drosophila generates several transcripts with divergent 5' and 3' domains that produce kinetically distinct K+ currents in Xenopus oocytes. Although suggestive that alternative splicing may be involved in generating K+ channel diversity, clear tissue-specific differences in the distribution of particular Sh gene products have not been demonstrated....
The α-Ca2+/calmodulin kinase II (αCaMKlI) is required for long-term potentiation in the CA1 region of the hippocampus. Here, we report that this kinase also has a crucial role in presynaptic plasticity. Paired-pulse facilitation is blunted in the CAI region of mice heterozygous for a targeted mutation of αCaMKlI, confirming that this kinase can promote neurotransmitter release. Unexpectedly, field...
We have identified, by affinity chromatography, a binding protein for the snake venom toxin taipoxin. The sequence of this 47 kDa protein is unique, is characteristic of a secreted protein, and has homology to the acute phase proteins serum amyloid P protein and C-reactive protein of the pentraxin family. We have named this protein neuronal pentraxin (NP), as Northern analysis and in situ hybridization...
The neurogenic gene Xotch acts to divert cellular determination during gastrulation in Xenopus embryos. We examined the role of Xotch in the developing retina, where cell signaling events are thought to affect differentiation. Xotch is expressed in undifferentiated precursor cells of the ciliary marginal zone and late embryonic central retina. It is not expressed in stem cells or in differentiated...
The flip and flop splice variants of AMPA receptors show strikingly different sensitivity to allosteric regulation by cyclothiazide; heteromers assembled from GluR-A and GIuR-B also exhibit splice variant-dependent differences in efficacy for activation by glutamate and kainate. The sensitivity for attenuation of desensitization by cyclothiazide for homomeric GIuR-A was solely dependent upon exchange...
Induction of prodynorphin gene expression by psychostimulant drugs may represent a compensatory adaptation to excessive dopamine stimulation and may contribute to the aversive aspects of withdrawal. We therefore investigated the molecular mechanisms by which dopamine psychostimulant drugs induce prodynorphin gene expression in vivo and in rat primary striatal cultures. We demonstrate that three recently...
We developed a rapid method that uses diphtheria toxin, the flp recognition target sequences, and the GAL4-UAS activation system, to ablate specific neurons in the Drosophila embryo and to examine the consequences in large numbers of embryos at many time points. We used this method to show that, in the absence of the aCC axon, which pioneers the intersegmental nerve in the PNS, the three U follower...
Sensory transduction in olfactory receptors and photoreceptors is mediated by cyclic nucleotide-activated ion channels. We have studied the gating mechanism in olfactory and rod channels expressed in Xenopus oocytes. We report that the differences in cyclic nucleotide affinity and efficacy between these channels resulted from sequence differences outside the cyclic nucleotide-binding domain, especially...
We describe the in situ modulation of potassium channels in a semi-intact preparation of the Drosophila retina. In whole-cell recordings of photoreceptors, rapidly inactivating Shaker channels are characterized by a conspicuously negative voltage operating range; together with a delayed rectifier, these channels are specifically modulated by the putative efferent neurotransmitter serotonin. Contrary...
Two rabbits immunized with a portion of glutamate receptor (GIuR) subunit GIuR' (amino acids 245-457) exhibited seizure-like behaviors, suggesting that antibodies to GIuR3 may modulate neuronal excitability. Using whole-cell recording, rabbit GIuR3 antisera were tested on cultured fetal mouse cortical neurons. In a subset of kainate-responsive neurons, miniperfusion of antisera and IgG evoked currents...
Using whole-cell recording from CA1 hippocampal pyramidal neurons and minimal stimulation of Schaffer collaterals, we have studied what seem to be single synapses. Although the transmission at a putative single synapse is quite unreliable, the synapse can be made to release transmitter reliably in response to the second stimulus in a pair of stimuli that are presented in rapid succession (e.g., 50...
The effect of various Ca2+-channel blockers on exocytosis has been studied at the level of single presynaptic terminals in rat hippocampal cell cultures. The fluorescence change of the styryl dye FM1-43 has been used as a measure of exocytosis during electrical stimulation. ω-Conotoxin GVIA (2-10 μM) completely inhibited exocytosis in ~45% of the boutons in the field of view, while in ~55% exocytosis...
Here we describe a technique that uses a recombinant vaccinia virus to transfect neurons in rat hippocampal slices. This technique allows the use of molecular biological manipulations on neuronal tissue while maintaining intact synaptic function. This method should be useful in testing specific hypotheses regarding the role of synaptic proteins.