Veit Witzemann

Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels

Abstract1.Functional acetylcholine receptor (AChR) and sodium channels were expressed in the membrane ofXenopus laevis oocytes following injection with poly(A)+-mRNA extracted from denervated rat leg muscle. Wholecell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. Oocytes injected with specific mRNA, transcribed from cDNA templates and coding for the AChR ofTorpedo electric organ, expressed functional AChR channels at a much higher density.2.Single-channel currents were recorded from the oocyte plasma membrane following removal of the follicle cell layer and the vitelline membrane from the oocyte. The follicle cell layer was removed enzymatically with collagenase. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase.3.Stretch activated (s.a.) currents were observed in most recordings from cell-attached patches obtained with standard patch pipettes. S.a.-currents were evoked by negative or positive pressure (≥5 mbar) applied to the inside of the pipette, and were observed in both normal and mRNA injected oocytes indicating that they are endogenous to the oocyte membrane.4.The s.a.-channels are cation selective and their conductance is 28 pS in normal frog Ringers solution (20±1°C). Their gating is voltage dependent, and their open probability increases toward more positive membrane potentials.5.The density of s.a.-channels is estimated to be 0.5–2 channels per μm2 of oocyte plasma membrane. In cell-attached patches s.a.-currents are observed much less frequently when current measurement is restricted to smaller patches of 3–5 μm2 area using thick-walled pipettes with narrow tips. In outside-out patches s.a.-currents occur much less frequently than in cell-attached or inside-out patches.6.AChR-channel and sodium channel currents were observed only in a minority of patches from oocytes injected with poly(A)+-mRNA from rat muscle. AChR-channel currents were seen in all patches of oocytes injected with specific mRNA coding forTorpedo AChR. In normal frog Ringers solution (20±2°C) the conductance of implanted rat muscle AChR-channels was 38 pS and that of sodium channels 20 pS. The conductance of implantedTorpedo AChR channels was 40 pS. The conductance of implanted channels was similar in cell-attached and in cell-free patches.7.The conductances of rat muscle AChR and sodium channels implanted into the oocyte membrane were similar to those of channels in their native muscle membrane, suggesting that important functional properties of these channels are determined by their primary amino acid sequence.

Voluntary exercise induces anxiety-like behavior in adult C57BL/6J mice correlating with hippocampal neurogenesis.

Several studies investigated the effect of physical exercise on emotional behaviors in rodents; resulting findings however remain controversial. Despite the accepted notion that voluntary exercise alters behavior in the same manners as antidepressant drugs, several studies reported opposite or no effects at all. In an attempt to evaluate the effect of physical exercise on emotional behaviors and brain plasticity, we individually housed C57BL/6J male mice in cages equipped with a running wheel. Three weeks after continuous voluntary running we assessed their anxiety‐ and depression‐like behaviors. Tests included openfield, dark‐light‐box, elevated O‐maze, learned helplessness, and forced swim test. We measured corticosterone metabolite levels in feces collected over a 24‐h period and brain‐derived neurotrophic factor (BDNF) in several brain regions. Furthermore, cell proliferation and adult hippocampal neurogenesis were assessed using Ki67 and Doublecortin. Voluntary wheel running induced increased anxiety in the openfield, elevated O‐maze, and dark‐light‐box and higher levels of excreted corticosterone metabolites. We did not observe any antidepressant effect of running despite a significant increase of hippocampal neurogenesis and BDNF. These data are thus far the first to indicate that the effect of physical exercise in mice may be ambiguous. On one hand, the running‐induced increase of neurogenesis and BDNF seems to be irrelevant in tests for depression‐like behavior, at least in the present model where running activity exceeded previous reports. On the other hand, exercising mice display a more anxious phenotype and are exposed to higher levels of stress hormones such as corticosterone. Intriguingly, numbers of differentiating neurons correlate significantly with anxiety parameters in the openfield and dark‐light‐box. We therefore conclude that adult hippocampal neurogenesis is a crucial player in the genesis of anxiety.

Differential regulation of muscle acetylcholine receptor γ-and ϵ-subunit mRNAs

The contents of the mRNAs encoding the γ‐ and ϵ‐subunits of the nicotinic acetylcholine receptor as well as the single‐channel properties of the receptor have been assessed in innervated, denervated and reinnervated rat muscle. The changes in abundance of the γ‐ and ϵ‐subunit mRNAs correlate with the changes in relative density of two classes of acetylcholine receptor channels. The results support the view that a switch in the relative abundance of the γ‐ and ϵ‐subunit mRNAs is a major mechanism in regulating the properties of acetylcholine receptor channels in muscle.

Development of the neuromuscular junction

The differentiation of the neuromuscular junction is a multistep process requiring coordinated interactions between nerve terminals and muscle. Although innervation is not needed for muscle production, the formation of nerve-muscle contacts, intramuscular nerve branching, and neuronal survival require reciprocal signals from nerve and muscle to regulate the formation of synapses. Following the production of muscle fibers, clusters of acetylcholine receptors (AChRs) are concentrated in the central regions of the myofibers via a process termed “prepatterning”. The postsynaptic protein MuSK is essential for this process activating possibly its own expression, in addition to the expression of AChR. AChR complexes (aggregated and stabilized by rapsyn) are thus prepatterned independently of neuronal signals in developing myofibers. ACh released by branching motor nerves causes AChR-induced postsynaptic potentials and positively regulates the localization and stabilization of developing synaptic contacts. These “active” contact sites may prevent AChRs clustering in non-contacted regions and counteract the establishment of additional contacts. ACh-induced signals also cause the dispersion of non-synaptic AChR clusters and possibly the removal of excess AChR. A further neuronal factor, agrin, stabilizes the accumulation of AChR at synaptic sites. Agrin released from the branching motor nerve may form a structural link specifically to the ACh-activated endplates, thereby enhancing MuSK kinase activity and AChR accumulation and preventing dispersion of postsynaptic specializations. The successful stabilization of prepatterned AChR clusters by agrin and the generation of singly innervated myofibers appear to require AChR-mediated postsynaptic potentials indicating that the differentiation of the nerve terminal proceeds only after postsynaptic specializations have formed.

Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity

The levels of mRNAs coding for the myogenic factors MyoD and myogenin were measured during synapse formulation in developing muscle and in adult muscle, after denervation and reinnervation and after muscle paralysis induced by blocking of neuromuscular transmission by neurotoxins known to alter the density and localization of synaptic proteins such as the acetylcholine receptor (AChR). The mRNA levels of both factors depend on usage of the neuromuscular synapses, but they change to different extents. Myogenin mRNA levels decrease drastically with innervation and increase strongly following blocking of transmission whereas the level of MyoD mRNA showed only a small decrease in response to innervation, denervation of muscle paralysis by neurotoxins. Neither mRNA showed a synapse‐related cellular distribution. The results suggest that nerve‐induced electrical muscle activity determines the cellular ratio of MyoD and myogenin mRNAs in adult muscle.

Developmental regulation of five subunit specific mRNAs encoding acetylcholine receptor subtypes in rat muscle

The muscular content of the mRNAs encoding the five subunits of the nicotinic acetylcholine receptor was measured during postnatal development in the rat. Subunit specific mRNAs show differential regulation. The levels of the α‐, γ‐ and δ‐subunit specific mRNAs decrease steadily after birth, while the β and ε‐subunit mRNAs increase transiently and then decrease. The adult pattern of subunit specific mRNA levels is reached at 4–6 weeks postnatally. The content of γ‐ and ε‐subunit mRNA changes in a reciprocal fashion during the first 2 postnatal weeks, supporting the view that differential regulation of γ‐ and ε‐subunit mRNA during development is one mechanism mediating the appearance of the adult, ε‐subunit containing, subtype of end‐plate channel. Denervation of neonatal muscle increases the levels of all subunit‐specific mRNAs during further development. It prevents the postnatal decrease in γ‐subunit mRNA and enhances the initial increase in ε‐subunit mRNA. This makes it appear that the ε‐subunit gene is less sensitive to regulation by the nerve in the postnatal period than the γ‐subunit gene.

High-efficiency transfection of individual neurons using modified electrophysiology techniques.

Transfection of cells by electroporation is a widely used and efficient method. Recently, it has been shown that single neurons in brain slice cultures can be transfected using micropipettes loaded with plasmid DNA expression constructs. However, the transfection efficiencies were very low. Routine employment of single-cell electroporation (SCE) for transfection of neurons requires high and reliable efficiency together with good cell survival. Here, we describe the modification of electrophysiology techniques for SCE leading to very simple and efficient (up to 80%) transfection of neurons in organotypic rat hippocampus and mouse cortex slice cultures. Electroporation-mediated transfection was visualized in real-time by two-photon microscopy at the cellular level using fluorescently labeled oligonucleotides and plasmid DNA. Small oligonucleotides enter the cell immediately during pulse application while large plasmids remain localized for more than 10 min at the cell membrane before they enter the cell by an, as yet, unknown process. SCE does not affect the electrophysiology of transgene-expressing cells. Expression of several neuronal green fluorescent protein-tagged proteins demonstrates that the method can be employed to analyze subcellular trafficking and targeting in single living neurons.

The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non‐receptor proteins including the receptor‐associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one‐ and two‐dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane‐bound acetylcholine receptor and the cytoskeleton.

Differential Expression Patterns of Five Acetylcholine Receptor Subunit Genes in Rat Muscle During Development

The spatial and temporal expression patterns of five genes which encode the α‐, β‐, γ‐, δ‐ and ε‐subunits of the nicotinic acetylcholine receptor in skeletal muscle were followed during development in the rat by in situ hybridization analysis. Three major developmental phases, characterized by specific expression patterns, could be distinguished. (i) During myogenic differentiation α‐, β‐, γ‐ and δ‐subunit genes are activated and transcripts are expressed in muscle precursor cells at embryonic day 12 (E12) and during subsequent cell fusion. (ii) Following innervation of myotubes at ˜E15‐E17 the mRNA of the α‐, β‐, γ‐ and δ‐subunit genes accumulate in synaptic and decrease in extrasynaptic fibre regions during early synaptogenesis. The mRNA of the δ‐subunit gene becomes detectable first in subsynaptic nuclei 2–3 days after innervation has occurred. (iii) During postnatal development α‐, β‐ and δ‐ subunit transcript levels are reduced predominantly in extrasynaptic fibre segments and show significant differences in distribution depending on the muscle subtype whereas the γ‐subunit mRNA disappears completely within the first postnatal week in all muscles. In contrast, the γ‐subunit gene is transcribed only in subsynaptic myonuclei throughout development and in the adult muscle.

Cholinergic ligand-induced affinity changes in Torpedo californica acetylcholine receptor.

Abstract The binding of cholinergic ligands to Torpedo californica acetylcholine receptor has been studied in vitro by inhibition of the time course of 125 I-labeled α-bungarotoxin-receptor complex formation. The extent of inhibition was dependent on the duration of exposure to the ligand, the apparent affinity for ligand increasing with time, and was reversible upon removal of ligand. Ligand concentration, temperature, and Ca 2+ ions influenced this effect which is reminiscent of receptor desensitization in vivo . Such effects were observed both for a cholinergic agonist, carbamylcholine, and for an antagonist, bis(3-aminopyridinium)-1,10-decane diiodide. A minimal model is discussed which can account for these effects and for receptor ligand association leading to postsynaptic depolarization.