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DRG nociceptors from wild-type and Fhf2-KO mice. Fhf2 gene knockout blocks heat nociception. (Marra et al., 2023)
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<p class=MsoNormal><b><u>Fhf2WT and Fhf2KO Nociceptor Models</u></b></p>
<p class=MsoNormal>Posted by Mitchell Goldfarb</p>
<p class=MsoNormal>Publication Reference: DOI:<span style='font-size:10.5pt;
line-height:107%;font-family:"Arial",sans-serif;color:#3B3030;background:white'>
10.1097/j.pain.0000000000002822</span></p>
<p class=MsoNormal>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.</p>
<p class=MsoNormal>In addition to the differences between the Fhf2WT and Fhf2KO
models, several additional modifications were made equivalently to both models:</p>
<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in'>1)<span
style='font:7.0pt "Times New Roman"'> </span>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.</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>2)<span
style='font:7.0pt "Times New Roman"'> </span>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.</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>3)<span
style='font:7.0pt "Times New Roman"'> </span>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.</p>
<p class=MsoListParagraphCxSpLast style='text-indent:-.25in'>4)<span
style='font:7.0pt "Times New Roman"'> </span>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.</p>
<p class=MsoNormal><b>Instructions for running model simulations</b></p>
<p class=MsoNormal>This model deposit contains ten folders, consisting of
Fhf2WT and Fhf2KO models each analyzed under five simulation conditions, as
described below.</p>
<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in'>A)<span
style='font:7.0pt "Times New Roman"'> </span><u>Fhf2WT and
Fhf2KO neurons with heated distal terminals and current injection (“thermal
nociception”):</u></p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>1.<span
style='font:7.0pt "Times New Roman"'> </span>Extract
all files/folders from zip file Fhf2WTandKONociceptors.zip</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>2.<span
style='font:7.0pt "Times New Roman"'> </span>Open
folder WT or FHF2KO Model Terminal Stimulation and Heating</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>3.<span
style='font:7.0pt "Times New Roman"'> </span>Build all
MOD files using mknrndll.exe</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>4.<span
style='font:7.0pt "Times New Roman"'> </span>Open
Terminal Proj Main .hoc file</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>5.<span
style='font:7.0pt "Times New Roman"'> </span>From
NEURON Main Menu GUI, open File/load hoc/Capsaicin_stimulation.hoc</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>6.<span
style='font:7.0pt "Times New Roman"'> </span>From
NEURON Main Menu GUI, open File/load session/AllTerminalVoltages.ses</p>
<p class=MsoListParagraphCxSpLast style='text-indent:-.25in'>7.<span
style='font:7.0pt "Times New Roman"'> </span>From Run
Control GUI, click Init & Run.</p>
<p class=MsoNormal style='margin-left:.25in'>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.</p>
<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in'>B)<span
style='font:7.0pt "Times New Roman"'> </span><u>Fhf2WT
and Fhf2KO neurons without heating and distal current injection
(“mechanonociception”):</u></p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>1.<span
style='font:7.0pt "Times New Roman"'> </span>Extract
all files/folders from zip file Fhf2WTandKONociceptors</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>2.<span
style='font:7.0pt "Times New Roman"'> </span>Open
folder WT or FHF2KO Model Terminal Stimulation Without Heating</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>3.<span
style='font:7.0pt "Times New Roman"'> </span>Build all
MOD files using mknrndll.exe</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>4.<span
style='font:7.0pt "Times New Roman"'> </span>Open
Terminal Proj Main .hoc file</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>5.<span
style='font:7.0pt "Times New Roman"'> </span>From
NEURON Main Menu GUI, open File/load hoc/Capsaicin_stimulation.hoc</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>6.<span
style='font:7.0pt "Times New Roman"'> </span>From
NEURON Main Menu GUI, open File/load session/AllTerminalVoltages.ses</p>
<p class=MsoListParagraphCxSpLast style='text-indent:-.25in'>7.<span
style='font:7.0pt "Times New Roman"'> </span>From Run
Control GUI, click Init & Run.</p>
<p class=MsoNormal>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.</p>
<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in'>C)<span
style='font:7.0pt "Times New Roman"'> </span><u>Fhf2WT
and Fhf2KO axon stimulation and conduction at 37oC</u></p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>1.<span
style='font:7.0pt "Times New Roman"'> </span>Extract
all files/folders from zip file Fhf2WTandKO Nociceptors</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>2.<span
style='font:7.0pt "Times New Roman"'> </span>Open
folder WT or FHF2KO Model Axon Stimulation 37oC</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>3.<span
style='font:7.0pt "Times New Roman"'> </span>Build all
MOD files using mknrndll.exe</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>4.<span
style='font:7.0pt "Times New Roman"'> </span>Open
Terminal Proj Main .hoc file</p>
<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in'>5.<span
style='font:7.0pt "Times New Roman"'> </span>From
NEURON Main Menu GUI, open File/load session/AxonStimulation.ses</p>
<p class=MsoListParagraphCxSpLast style='text-indent:-.25in'>6.<span
style='font:7.0pt "Times New Roman"'> </span>From Run
Control GUI, click Init & Run.</p>
<p class=MsoNormal style='margin-left:.25in'>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</p>
<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in'>D)<span
style='font:7.0pt "Times New Roman"'> </span><u>Fhf2WT and
Fhf2KO axon stimulation and conduction at 44oC</u></p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>1.<span
style='font:7.0pt "Times New Roman"'> </span>Extract
all files/folders from zip file Fhf2WTandKO Nociceptors</p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>2.<span
style='font:7.0pt "Times New Roman"'> </span>Open
folder WT or FHF2KO Model Axon Stimulation 44oC</p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>3.<span
style='font:7.0pt "Times New Roman"'> </span>Build all
MOD files using mknrndll.exe</p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>4.<span
style='font:7.0pt "Times New Roman"'> </span>Open
Terminal Proj Main .hoc file</p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>5.<span
style='font:7.0pt "Times New Roman"'> </span>From
NEURON Main Menu GUI, open File/load session/AxonStimulation.ses</p>
<p class=MsoListParagraphCxSpLast style='margin-left:.75in;text-indent:-.25in'>6.<span
style='font:7.0pt "Times New Roman"'> </span>From Run
Control GUI, click Init & Run.</p>
<p class=MsoNormal style='margin-left:.25in'>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.</p>
<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in'>E)<span
style='font:7.0pt "Times New Roman"'> </span><u>Fhf2WT
and Fhf2KO acutely dissociated neurons and heat stimulation at 45oC</u></p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>1.<span
style='font:7.0pt "Times New Roman"'> </span>Extract
all files/folders from zip file Fhf2WTandKO Nociceptors</p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>2.<span
style='font:7.0pt "Times New Roman"'> </span>Open
folder WT or FHF2KO Model Dissociated Neuron with Heating</p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>3.<span
style='font:7.0pt "Times New Roman"'> </span>Build all
MOD files using mknrndll.exe</p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>4.<span
style='font:7.0pt "Times New Roman"'> </span>Open
Terminal Proj Main .hoc file</p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>5.<span
style='font:7.0pt "Times New Roman"'> </span>From
NEURON Main Menu GUI, open File/load hoc/SOMA_Capsaicin_stimulation.hoc</p>
<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;text-indent:-.25in'>6.<span
style='font:7.0pt "Times New Roman"'> </span>From
NEURON Main Menu GUI, open File/load session/SomaStimulation.ses</p>
<p class=MsoListParagraphCxSpLast style='margin-left:.75in;text-indent:-.25in'>7.<span
style='font:7.0pt "Times New Roman"'> </span>From Run
Control GUI, click Init & Run</p>
<p class=MsoNormal style='margin-left:.5in'>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.</p>
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DRG nociceptors from wild-type and Fhf2-KO mice. Fhf2 gene knockout blocks heat nociception. (Marra et al., 2023)