Fhf2WT and Fhf2KO Nociceptor Models

Posted by Mitchell Goldfarb

Publication Reference: DOI: 10.1097/j.pain.0000000000002822

The Fhf2WT and Fhf2KO nociceptor models are described in Marra et al, Pain 2023, and explore the effects of FHF2-mediated altered sodium channel inactivation gating on heat-induced nociceptor excitation. The modeling is based upon recordings of sodium currents in the presence and absence of FHF2 protein, the observation that Fhf2-deficient nociceptors are insensitive to heat stimulation, and C-fiber conduction block in Fhf2-deficient saphenous nerves above 41oC. The wild-type (Fhf2WT) and mutant (Fhf2KO) models differ only in the inactivation gating parameters of tetrodotoxin-sensitive (nattxs) and tetrodotoxin-resistant (nav1p8) sodium conductances without altering the current densities of these conductances. I created these models by modification of the existing nociceptor model (ModelDB Accession #266850) as published in Barkai et al, J.Neurosci. 40:9346, 2020. The TTX-sensitive sodium conductance was converted to two Markov models (nattxs_withF and nattxs_noF), while the Nav1.8-like sodium conductance has inactivation (h) parameters modified in the Fhf2WT vs Fhf2KO models.

In addition to the differences between the Fhf2WT and Fhf2KO models, several additional modifications were made equivalently to both models:

1) In the original Barkai model 266850, the main peripheral axon (peri) with 0.8 micron diameter and the base of the terminal branch network (ter[0]) with 0.25 micron diameter are connected by a tapering element (cone) of 3 micron length. For retrograde conduction of actions potentials, this cone is a severe source/sink mismatch. While wild-type nociceptor generates enough sodium current during action potentials to conduct through the cone, action potentials in the Fhf2KO nociceptor experience conduction block at the cone. Since the cone element is a structurally unrealistic architectural element, I replaced the cone with a very gradual tapering of the peripheral axon to the terminal axon over a length of 5 millimeters.

2) In the original Barkai model, all gated channel models have a Q10 temperature scaling factor. Since I wished to examine the effects of heating in only the distal terminal branches of the nociceptors, the channel models required modification, since temperature (celsius) is a global variable. These new channel models apply a new term (localtemp) which enables localtemp_nav1p8, localtemp_km, etc. to be individually set in each cell compartment within the startup file Terminal Proj Main.hoc.

3) In the original Barkai model, the densities of all channel conductances are unvaried throughout the cell, with the exception of their absence from the terminal tip segments (Navless). In the Fhf2WT and Fhf2KO model cells, the conductance density (gbar) of nav1p8 is ~15-fold higher in terminal segments 3-26 than in the rest of the cell.

4) In the Fhf2WT and Fhf2KO models, I created acutely dissociated neuron variant models by disconnecting the soma from the stem and by setting gbar for nav1p8 to a level intermediate between its values in the soma and terminal branches of the intact models.

Instructions for running model simulations

This model deposit contains ten folders, consisting of Fhf2WT and Fhf2KO models each analyzed under five simulation conditions, as described below.

A) Fhf2WT and Fhf2KO neurons with heated distal terminals and current injection (“thermal nociception”):

1. Extract all files/folders from zip file Fhf2WTandKONociceptors.zip

2. Open folder WT or FHF2KO Model Terminal Stimulation and Heating

3. Build all MOD files using mknrndll.exe

4. Open Terminal Proj Main .hoc file

5. From NEURON Main Menu GUI, open File/load hoc/Capsaicin_stimulation.hoc

6. From NEURON Main Menu GUI, open File/load session/AllTerminalVoltages.ses

7. From Run Control GUI, click Init & Run.

The simulation sets localtemp for all channel models in most distal terminal branches to 45oC, and leaves localtemp through the rest of neuron compartments to 37oC. The embedded capsaicin stimulation hoc injects increasing current ramp into the gated channel-free (Navless) distal tip compartments starting at 0.5 sec and continuing for 1.7 sec, followed by down ramp. Results windows include voltage in terminal segments and at the central axon adjacent to the soma. A train of retrograde-propagating action potentials is induced by terminal stimulation of WT model, but not in the Fhf2KO model.

B) Fhf2WT and Fhf2KO neurons without heating and distal current injection (“mechanonociception”):

1. Extract all files/folders from zip file Fhf2WTandKONociceptors

2. Open folder WT or FHF2KO Model Terminal Stimulation Without Heating

3. Build all MOD files using mknrndll.exe

4. Open Terminal Proj Main .hoc file

5. From NEURON Main Menu GUI, open File/load hoc/Capsaicin_stimulation.hoc

6. From NEURON Main Menu GUI, open File/load session/AllTerminalVoltages.ses

7. From Run Control GUI, click Init & Run.

The simulation sets localtemp for all channel models to 37oC in all compartments. The embedded capsaicin stimulation hoc injects increasing current ramp into the gated channel-free (Navless) distal tip compartments starting at 0.5 sec and continuing for 1.7 sec, followed by down ramp. Results windows include voltage in terminal segments and at the central axon adjacent to the soma. A train of retrograde-propagating action potentials is induced by terminal stimulation in both models.

C) Fhf2WT and Fhf2KO axon stimulation and conduction at 37oC

1. Extract all files/folders from zip file Fhf2WTandKO Nociceptors

2. Open folder WT or FHF2KO Model Axon Stimulation 37oC

3. Build all MOD files using mknrndll.exe

4. Open Terminal Proj Main .hoc file

5. From NEURON Main Menu GUI, open File/load session/AxonStimulation.ses

6. From Run Control GUI, click Init & Run.

The simulation sets localtemp for all channel models to 37oC in all compartments. The axon stimulation session positions an electrode at the midpoint of axon compartment peri[0] and applies a sharp current pulse at 100 msec. The graph reports voltage at the stimulation site and distally in 1 mm increments. Both Fhf2WT and Fhf2KO axon can conduct an action potential at 37oC

D) Fhf2WT and Fhf2KO axon stimulation and conduction at 44oC

1. Extract all files/folders from zip file Fhf2WTandKO Nociceptors

2. Open folder WT or FHF2KO Model Axon Stimulation 44oC

3. Build all MOD files using mknrndll.exe

4. Open Terminal Proj Main .hoc file

5. From NEURON Main Menu GUI, open File/load session/AxonStimulation.ses

6. From Run Control GUI, click Init & Run.

The simulation sets localtemp for all channel models to 44oC in all compartments. The axon stimulation session positions an electrode at the midpoint of axon compartment peri[0] and applies a sharp current pulse at 100 msec. The graph reports voltage at the stimulation site and distally in 1 mm increments. While Fhf2WT axon conducts the action potential at 44oC faster than at 37oC, the Fhf2KO axon experiences conduction block.

E) Fhf2WT and Fhf2KO acutely dissociated neurons and heat stimulation at 45oC

1. Extract all files/folders from zip file Fhf2WTandKO Nociceptors

2. Open folder WT or FHF2KO Model Dissociated Neuron with Heating

3. Build all MOD files using mknrndll.exe

4. Open Terminal Proj Main .hoc file

5. From NEURON Main Menu GUI, open File/load hoc/SOMA_Capsaicin_stimulation.hoc

6. From NEURON Main Menu GUI, open File/load session/SomaStimulation.ses

7. From Run Control GUI, click Init & Run

The simulation isolates the soma from axonal processes, sets soma localtemp for all channel models to 45oC, and injects increasing current ramp into the soma starting at 0.5 sec and continuing for 1.7 sec, followed by down ramp. Somatic voltage is plotted. Both Fhf2WT and Fhf2KO soma generate spike trains at 45oC in response to somatic current injection.