Computational protocol: Pacemaker Neuron and Network Oscillations Depend on a Neuromodulator-Regulated Linear Current

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[…] Experiments were carried on adult male crabs (Cancer borealis) purchased from local distributors (Newark, NJ, USA). The animals were maintained in artificial seawater tanks at 12–15°C until they were used, when they were first anesthetized by cooling on ice for 15–30 min prior to each dissection. The stomatogastric nervous system (including the stomatogastric ganglion, STG, the esophageal ganglion, OG, and the commissural ganglia, CoGs, Figure A) was removed using standard methods (Selverston et al., ; Harris-Warrick et al., ) and each preparation was pinned down in a Sylgard-coated Petri dish. The STG was desheathed to expose the cell bodies for microelectrode impalement, and to allow effective superfusion. Preparations were superfused using normal saline containing (in mM): 11 KCl, 440 NaCl, 13 CaCl2, 26 MgCl2, 11.2 Trizma base, 5.1 Maleic acid, pH 7.4–7.5, and kept at ∼12°C.Microelectrodes were pulled using a Flaming-Brown micropipette puller (P97, Sutter Instruments) and filled with 0.6 M K2SO4 + 0.02 M KCl (resistance of 15–25 MΩ). Extracellular recordings were made with two stainless steel pins, one placed in the bath and the other within a petroleum jelly well built around the nerve of interest. Neuronal identification was accomplished by matching the intracellular action potential recordings to their corresponding extracellular nerve recordings (Selverston et al., ; Harris-Warrick et al., ). Intracellular recordings were made from the soma of the neurons using Axoclamp 2B amplifiers (Molecular Devices) and extracellular recordings were amplified using a differential AC amplifier model 1700 (A–M Systems).We used the dynamic clamp technique (Sharp et al., ) to introduce ionic conductances into, or subtract them from, the biological neurons. A NI PCI-6070-E board (National Instruments, Austin, TX, USA) was used for current injection in dynamic clamp experiments. Data acquisition was performed using the Digidata 1332A data acquisition board and pClamp 9.2 software (Molecular Devices). The dynamic clamp software was developed in our laboratories (available for download at in the LabWindows/CVI software environment (National Instruments) on a Windows platform. In dynamic clamp, ionic currents are calculated using Hodgkin-Huxley-type equations as described below and continuously updated by recording the membrane potential (V) of the neurons in real time. IMI was described by:where EMI = −10 mV and τm = 4 ms. The values for g_MI varied depending on the experiment as described in the Results. The standard leak current is described by IL=gL(V−EL) where EL = −68 mV is the leak current reversal potential and gL is the leak conductance. When the value of leak conductance was set to be negative in the dynamic clamp software it resulted in a reduction of the total neuronal conductance. We describe this current asThe voltage-dependent current IMI was divided into regions approximating a linear I–V curve with positive linear conductance (MI–PL) or negative linear conductance (MI–NL). The linear conductance regions were restricted in their voltage range so that they represented the negative and positive-conductance regions of IMI:where τ = 4 ms. Although IMI is non-inactivating, to build IMI–NL, an inactivation variable hN with very fast kinetics is used in dynamic clamp as a method of restricting the voltage range of activation below the upper limit of −46 mV (see Figure A, red trace). A similar use of the activation variable mP was made to produce IMI–PL (Figure A, gray trace).It should be noted that dynamic clamp conductance manipulations introduce artificial ionic currents into the point of penetration, in this case the soma. The ionic currents in biological neurons are distributed over the entire structure of the cell and it is possible therefore that the size of the currents injected by dynamic clamp does not properly represent the equivalent biological ionic current. In particular, the currents injected in our dynamic clamp experiments may be much larger than what is actually needed for the pacemaker neurons to produce oscillations.We measured neuronal input conductance before and after injecting negative conductances with dynamic clamp to avoid reducing the total conductance of the neuron to less than zero. When simultaneous dynamic clamp current and constant current injections were required, a Brownlee Precision Amplifier (Brownlee Precision) was used to add the currents before input to the Axoclamp amplifier. The acquired data were saved as binary files and were analyzed with the software Readscope ( and Clampfit 9.2 (Molecular Devices).For experiments performed to compare the effect of neuromodulatory input, the STG was isolated from anterior neuromodulatory inputs (i.e. decentralized) by replacing the saline solution within a petroleum jelly well built around the single stomatogastric nerve (stn, Figure A) with 0.75-M sucrose and 1-μM TTX (Biotium) to block action potential transmission. In experiments to measure the input resistance of neurons 1-μM TTX was used to superfuse the ganglion.SAS (SAS Institute), SigmaStat 2.3 (Systat Software) and Origin 7 (OriginLab) software packages were used for statistical and graphical analysis and all final figures were made in CorelDraw 12 (Corel Corp.). Reported statistical significance indicated a significance level p ≤ 0.05. In all statistical tests the data have been checked for normality by the SAS software package. All error bars shown and error values reported are standard deviations of the mean (SD). […]

Pipeline specifications

Software tools pCLAMP, CorelDraw
Applications Miscellaneous, Patch-clamp
Diseases Myocardial Infarction