Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.
Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.
We have been using zebrafish paired recordings (Wen and Brehm, 2005) principally to study the processes of vesicle release and recycling during high frequency stimulation. This process is disrupted in motility mutants wherein normal synaptic transmission is compromised due to point mutations in key synaptic components. For example, mutations that disrupt postsynaptic receptor aggregates (Ono et al., 2001, 2002, 2004) and synthesis of presynaptic transmitter (Wang et al., 2008) result in un-coordinated swimming. Paired recordings provide the information that can pinpoint the source of the functional defect, thus linking behavior, genetics and physiology. It should now be possible to further dissect the processes underlying synaptic transmission by means of transient expression in the CaP motor-neurons using judicious promoters. In this way dominant negative or mutated genes can be specifically expressed in these neurons and both behavioral and electrophysiological consequences determined. The additional advantage offered by the whole cell configuration allows dialysis of the CaP soma with agents that can potentially alter vesicle recycling, such as clostridial toxins and calcium buffers. Due to the short distance between the soma and muscle, diffusion should occur well within the time frame of recordings. The potential of this preparation has only begun to be tapped. The transparency of the fish at this age and superficial location of synapses also facilitates use of optical tools (Fetcho and O’Malley, 1997; Fetcho and Higashijima, 2004). We have been very successful in measuring of endo and exocytosis in living fish using FM dyes and genetic indicators such as Synaptophluorin (Li et al., 2003). Additionally, fluorescent conjugated toxins can be used to label synaptic proteins for imaging of live fish (Ibanez-Tallon et al., 2004). Given the simplicity of this preparation it holds great promise for future study.
The authors have nothing to disclose.
Funded by the NIH (NS-18205).
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Pneumatic transducer tester | Fluke Biomedical Instruments | DPM1B | ||
0.002 x 3 inch tungsten rod | A-M Systems Inc | 715000 | ||
Sylgard 184 Elastomer | Dow Corning Corp. | The thickness of application will affect the DIC optics |