INTRODUCTION This application implements the model described in Steephen, J. E., & Manchanda, R. (2009). Differences in biophysical properties of nucleus accumbens medium spiny neurons emerging from inactivation of inward rectifying potassium currents. J Comput Neurosci, doi:10.1007/s10827-009-0161-7 This help window provides instructions for replicating the results in the paper. The information in this window is available in the text file 'Help.txt' within the 'support_files' folder. If required, it could be printed for easy reference. Launching the program (main.hoc) invokes 4 windows: NEURON Main Menu, Command Window (nrniv), the Control Panel and a Graph Window. Note that the Control Panel is organized into 3 vertical panels. The instructions given below for each figure assume that the application is in its default settings. It is recommended that the application be restarted after each simulation as it would reset all parameters to their default values. Note that you may have to right click the graph panel and select View... -> View=plot to view some of the plots. Toggling ON the 'Beep' button in the middle panel under RUN CONTROL in the Control Panel will enable audible alert at the end of each simulation. For questions and queries, send e-mail to johneric@iitb.ac.in. FIGURE 1 Fig 1A: The KIR activation time constant curves (tau_m) shown in Fig 1A for the Existing and Tuned models may be generated as follows:Click the 'KIR tau_m...' button in the Control Panel (top of middle panel, under CELL PROPERTIES). In the new window, toggle the 'Tuned Model' button OFF to turn off the tuning. Now click the 'MSP Grapher' button (Right panel, under RUN) to launch the Grapher window. Click 'KIR tau_m' button and click 'Plot' to generate the tau_m curve for the Existing model. Toggle the 'Tuned Model' button ON and click 'Plot' in the Grapher window to generate the tau_m curve for the Tuned model. Fig 1B: The voltage responses may be generated by entering current values of -0.227, 0.248 and 0.271 in the 'amp(nA)' field editor and clicking the 'Execute' Button. FIGURE 2 Fig 2A: Turn on voltage clamp by toggling ON the 'Voltage Clamp' button under INPUT (left panel). Select VClamp in the 'I/V Clamp Electrode' window. Set Conditioning Level duration to 10 ms, and amplitude to -50 mV. Set the Testing Level duration to 200 ms and amplitude to -120 mV. Set the Return Level duration to 1000 ms and amplitude to -50 ms. Click the 'Execute' button in the Control Panel. Current response of non-inKir cell to a clamp voltage of -120 mV will be plotted. To plot the same for the inKir cell, select the 'inKIR' radio button under CELL VARIANT in the Control Panel and click the 'Execute' button. The clamp responses for other voltages can be generated by entering the appropriate voltages in the 'I/V Clamp Electrode' window and clicking the 'Execute' button. Fig 2C: Click 'MSP Grapher'. Click 'gKIR' in the Grapher window. Click 'Plot' to generate the gKIR curve for the non-inKIR cell. (You may have to right click the graph panel and select View...->View=plot to see the curve) Select 'inKIR' in the Control Panel window and click 'Plot' to generate the same for the inKIR cell. Fig 2D: Click 'MSP Grapher'. Click 'gK' in the Grapher window. Change 'Indep End' to -60. Click 'Plot' to generate the gK curve for the non-inKir cell. Select 'inKIR' in the Control Panel window and click 'Plot' to generate the same for the inKIR cell. FIGURE 3 Fig 3A: These plots can be generated by entering the appropriate current values (0.24 to 0.28, -0.2) in the 'amp(nA)' field editor in the Control Panel, selecting 'Non-inKIR' or 'inKIR' and clicking the 'Execute' button. Fig 3B: Open the 'Currents' window by clicking the 'Currents' button in the Control Panel. Scroll down to the KIR current graph panel. Enter appropriate injected current value (0.24 or 0.26) in the 'amp(nA)' field editor in the Control Panel. Select 'Non-inKIR' or 'inKIR' and click the 'Execute' button. Fig 3C: This figure is derived from Fig 3A. Fig 3D: To obtain data points for the strength duration curve, select 'Strength-Duration' under ACTION in the Control Panel window. Select 'Non-inKIR' 'under CELL VARIANT. Click 'Execute'. The NEURON command window will show the injected current values (strength) and the corresponding durations once the simulation (comprising 10 runs) completes. Select 'inKIR', change 'min' under RUN CONTROL to 0.23 and execute again. Fig 3E: To plot the I-V curve, input 750 in the field editor 'Tstop (ms)' under RUN CONTROL, select 'Non-inKIR' or 'inKIR' and click 'MSP Grapher' to open the Grapher window. Input -0.3, 0.2, 10 respectively in the field editors 'Indep Begin', 'Indep End' and 'Steps' respectively. Set 'Independent Var' to 'stim.amp' and 'Generator' to 'run()'. Click the 'v' button followed by 'Plot'. 10 runs will be performed. Right click the graph panel and select View...->View=plot to view the plotted curve. FIGURE 4 Fig 4A: Select one of the cell variants in the Control Panel. Select 'Min KIR gmax for specified Spike no.' under ACTION. Enter the number of spikes in 'No. of spikes' field editor. Click 'Execute'. The NEURON command window will have the minimum gmax value that is needed to generate the specified number of spikes for the specified input current. Fig 4B: Select 'inKIR'. Set inKIR gmax under CELL PROPERTIES to 0.0014, 0.0012 or 0.0016. Enter values between 0 (No inactivation) and 1 (Full inactivation) in 'Inactivation level' field editor. Execute and observe spike frequency. Fig 4C: Select one of the cell variants. Input 0.25 in 'amp(nA)' field editor. Click 'Execute'. FIGURE 5 Fig 5A: Select a cell variant. Select the ACTION 'Earliest Spike Onset'. Input a value in the 'No. of spikes' field editor. Click 'Execute'. NEURON command window will display the current corresponding to the earliest spike onset. Fig 5B & 5C: Derived from Fig 5A. FIGURE 6 Fig 6A: Select 'Non-inKIR' or 'inKIR'. Input appropriate injected current duration in 'dur (ms)' field editor. Select 'Earliest Spike Onset' under ACTION. Click 'Execute'. See the NEURON command window for the output. Fig 6B & 6C: Derived from Fig 6A Fig 6D: Injected current inputs of 0.24741 nA and 0.237 nA to the non-inKIR and inKIR cells respectively can generate this figure. FIGURE 7 Fig 7A-D: These plots can be obtained by using the procedures described earlier (For Fig 5 & 6). The current plots will appear in the 'Currents' window which should be invoked by clicking the 'Currents' button prior to execution. Fig 7E: These can be obtained by executing the program after entering the specified values of current and duration. FIGURE 8 Fig 8A: Select 'Non-inKIR' or 'inKIR'. Activate synaptic input by toggling the 'Synaptic input' button ON under INPUT. Specify a seed value (eg. 0, 1, 2 etc.) in the 'Seed' field editor under RUN CONTROL. Execute. Fig 8B,C: These are derived from multiple runs using different seed values. Fig 8D: Select 'Non-inKIR' or 'inKIR'. Toggle the 'Synaptic input' button ON. Enter a value for input up-state frequency in the 'f(Hz)' field editor under INPUT. Specify a seed value. Execute. Note down the firing frequency for each up-state. Repeat for different seeds and input up-state frequencies. Calculate the mean firing frequency for each input up-state synaptic frequency. Fig 8E: To obtain the strength duration curve, toggle 'Synaptic Input' ON and select 'Strength-Duration' under ACTION in the Control Panel window. Select 'Non-inKIR' or 'inKIR' under CELL VARIANT. Under RUN CONTROL, input the minimum and maximum values of the range of input synaptic up-state frequencies for which the strength-duration curve is desired in the 'min' and 'max' field editors respectively (eg: 5.5 & 10) and set a seed value in the 'Seed' field editor. Input the desired number of values within the frequency range for which the duration needs to be calculated in the 'iterations' field editor (eg: 10). Click Execute. The NEURON command window will show the input synaptic frequency values (strength) and the corresponding durations once the simulation completes for the selected seed value. Repeat for different seeds and find the mean duration for each input frequency and plot. Fig 8F: Select 'Frequency Vs Voltage' under ACTION. Execute. Once the simulation completes, select 'inKIR' and execute again. Once simulation completes, both the plots will be shown in a graph window. FIGURE 9 Fig 9A: Select 'Advanced Run' under ACTION. Execute. Repeat after selecting 'inKIR'. Fig 9B: Select 'Advanced Run' under ACTION. Set 'dur(ms)' to 200, 'f(Hz)' to 7, 'Tstop(ms)' to 1200. Repeat for 'inKIR'.