Abstract
Whole cell recordings (WCRs) are frequently used to study neuronal properties, but may be problematic when studying neuromodulatory responses, due to dialysis of the cell's cytoplasm. Perforated patch recordings (PPR) avoid cellular dialysis and might reveal additional modulatory effects that are lost during WCR. We have previously used WCR to characterize the responses of the V2a class of Chx10-expressing neurons to serotonin (5-HT) in the neonatal mouse spinal cord (Zhong et al., 2010). Here we directly compare multiple aspects of the responses to 5-HT using WCR and PPR in Chx10-eCFP neurons in spinal cord slices from 2 to 4 day old mice. Cellular properties recorded in PPR and WCR were similar, but high-quality PP recordings could be maintained for significantly longer. Both WCR and PPR cells could respond to 5-HT, and although neurons recorded by PPR showed a significantly greater response to 5-HT in some parameters, the absolute differences between PPR and WCR were small. We conclude that WCR is an acceptable recording method for short-term recordings of neuromodulatory effects, but the less invasive PPR is preferable for detailed analyses and is necessary for stable recordings lasting an hour or more.
Highlights
Most studies investigating the effects of neuromodulators on vertebrate neurons have used traditional whole cell recordings (WCRs), which break the membrane under the recording pipette and within minutes replace the neuron’s cytoplasm with the pipette internal solution (Hamill et al, 1981)
Soon after the perforated patch technique was developed, it was shown that cultured mouse brown fat cells recorded with Perforated patch recordings (PPR) responded to norepinephrine with large changes in membrane conductance, whereas previous WCRs had shown no response to norepinephrine application (Lucero and Pappone, 1989, 1990)
Recordings were made in acute slices of 2–4 day old neonatal mouse spinal cord, in the presence of blockers of fast glutamatergic, GABAergic and glycinergic synaptic activity (Figure 1A)
Summary
Most studies investigating the effects of neuromodulators on vertebrate neurons have used traditional whole cell recordings (WCRs), which break the membrane under the recording pipette and within minutes replace the neuron’s cytoplasm with the pipette internal solution (Hamill et al, 1981). Many ionic currents and cellular processes are adversely affected by the washout of intracellular molecules (Becq, 1996; Sarantopoulos, 2007), including second messenger pathway enzymes (Armstrong and Eckert, 1987; Chad et al, 1987), protease inhibitors (Chad and Eckert, 1986; Belles et al, 1988a,b), and calcium buffers (Korn and Weight, 1987; Korn and Horn, 1989). Perforated patch recordings (PPR) mitigate intracellular dialysis by using ionophores to open small pores in the membrane, which allow small ions but not large signaling molecules to pass through the membrane (Lindau and Fernandez, 1986; Horn and Marty, 1988; Rae et al, 1991). Direct comparisons of the effect of recording type on the action of neuromodulators including serotonin, using large enough sample sizes to allow for thorough analysis, are still needed
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