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<h2 align=center style='margin-top:0in;margin-right:0in;margin-bottom:5.75pt;
margin-left:0in;text-align:center'>Usage Instructions</h2>

<p class=Textbody>&nbsp;</p>

<p class=Textbody style='margin-left:.5in;text-indent:-.5in'>How to use the
source code from <br>
Popovic, MA, Carnevale, N, Rozsa, B and Zecevic, D. <br>
Electrical behaviour of dendritic spines as revealed by voltage imaging. <br>
Nature Communications 6:8436, 2015. DOI 10.1038/ncomms9436. PMID 26436431.</p>

<p class=Textbody>Compile the mod files by executing</p>

<p class=Textbody>            <span style='font-family:"Liberation Mono"'>nrnivmodl</span></p>

<p class=Textbody>Then execute</p>

<p class=Textbody><span style='font-family:"Liberation Mono"'>            nrngui
init.hoc</span></p>

<p class=Textbody>This will start NEURON, which will create the model cell and
present this user interface: </p>

<p class=Textbody align=center style='text-align:center'><img width=498
height=424 src="readme.fld/image001.jpg"></p>

<p class=Textbody>&nbsp;</p>

<p class=Textbody style='page-break-after:avoid'>The items in the interface are
(from left to right, top to bottom):</p>

<p class=Textbody>1. NEURON Main Menu panel</p>

<p class=Textbody>2. Graph that shows time course of membrane potential at
three locations: middle of spine head, proximal end of spine neck (== potential
at the point in the parent neurite to which the spine neck is attached), and
root node of the soma (&quot;most proximal point in the soma&quot;).</p>

<p class=Textbody>3. &quot;Controls&quot; panel for specifying model properties
and performing various analyses.</p>

<p class=Textbody>4. CellBuilder that contains the detailed anatomical
specification of the model cell. The name of the file that contains this data
(070502-exp2-zB_noax.ses) is reported at the top of the Controls panel.</p>

<p class=Textbody>5. RunControl panel for launching individual simulation runs.</p>

<p class=Textbody>6. Graph that shows time course of course of current
delivered by the synapse attached to the spine head (syn.i).</p>

<p class=Textbody>7. PointProcessManagers (PPMs) for: specifying properties of
the NetStim that activates the synapse; showing the actual location of the
synapse, and controlling the reversal potential and dynamics of the model
synapse (an Exp2Syn); Loc[0], which you can use to control where the spine is
attached.</p>

<p class=Textbody>Notice that each Graph and PPM has a white canvas with a
little square in its top left corner. The square is called a &quot;menu
box&quot;.</p>

<h3>To reproduce Fig. 8a</h3>

<p class=Textbody>First, make sure that the cell, synapse, and spine parameters
in the Controls panel have these values:</p>

<p class=Textbody style='margin-bottom:2.9pt'><b>Cell parameters:</b></p>

<p class=Textbody style='margin-bottom:2.9pt'>            Ra 100 ohm cm</p>

<p class=Textbody style='margin-bottom:2.9pt'>            cm 1 uf/cm2</p>

<p class=Textbody style='margin-bottom:2.9pt'>            g_pas 5e-5 S/cm2</p>

<p class=Textbody>            spine area factor 0</p>

<p class=Textbody>Peak gsyn 0.000291 uS</p>

<p class=Textbody style='margin-bottom:2.9pt'><b>Spine parameters:</b></p>

<p class=Textbody style='margin-bottom:2.9pt'>            Ra 100 ohm cm</p>

<p class=Textbody style='margin-bottom:2.9pt'>            Neck L 1 um</p>

<p class=Textbody>            Neck diam 0.18 um</p>

<p class=Textbody>Use the GUI to change any values that are not correct. When
the spine parameters are correct, Rspine will be 40.5 megohms.</p>

<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=665
 style='border-collapse:collapse'>
 <tr>
  <td width=510 valign=top style='width:382.6pt;padding:0in .5pt 0in .5pt'>
  <p class=Textbody>Next, make sure the spine is attached to dend[3](0.642857).</p>
  <p class=Textbody>The PointProcessManager (PPM) for Loc[0] should look like
  this:</p>
  <p class=Textbody align=center style='text-align:center'><img width=108
  height=165 id="Picture 1" src="readme.fld/image002.jpg"
  alt="A screenshot of a computer&#10;&#10;Description automatically generated"></p>
  <p class=Textbody>Note the position of the blue dot in the basilar field of
  the cell, and the message just above the PPM's canvas which says</p>
  <p class=Textbody style='margin-left:.5in'>Loc[0]</p>
  <p class=Textbody style='margin-left:.5in'>at: dend[3](0.642857)</p>
  <p class=Textbody>If you see something different, click on the cell image at
  the correct location and verify that the blue dot is now where it should be.
  It will be easier to do this if you first make the cell image larger by
  clicking and dragging the right lower corner of Loc[0]'s PPM down and to the
  right.</p>
  </td>
  <td width=155 valign=top style='width:116.0pt;padding:0in .5pt 0in .5pt'>
  <p class=Textbody align=right style='text-align:right'>&nbsp;</p>
  </td>
 </tr>
</table>

<p class=Textbody style='page-break-after:avoid'>When the dot is where you want
it, click on the</p>

<p class=Textbody>            Put spine at the mark</p>

<p class=Textbody>button at the bottom of the Controls panel. Three things will
happen:</p>

<p class=Textbody>1. NEURON will print this message to the terminal (which
might be behind some of NEURON's windows)</p>

<p class=Textbody>            oc&gt;moving spine to dend[3] 0.64285714</p>

<p class=Textbody>            dend[3]  nseg = 7  spine at 0.64285714</p>

<p class=Textbody>2. The blue dot in Exp2Syn[0]'s PPM will move to the location
shown in Fig. 8a, confirming that the entire spine is now where it should be.
If a new Shape Plot appears on top of the user interface, you can dismiss it by
clicking on its Close button.</p>

<p class=Textbody>3. A new graph (called a &quot;shape plot&quot;) will appear
on the screen that shows the model cell with the spine's parent dendrite
highlighted in red. To discard this shape plot, click on its Close button.</p>

<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=665
 style='border-collapse:collapse'>
 <tr>
  <td width=402 valign=top style='width:301.5pt;padding:0in .5pt 0in .5pt'>
  <p class=Textbody>Now you're ready to run a simulation.</p>
  <p class=Textbody>In the RunControl panel, click on Init &amp; Run and look
  at the graph that shows plots of head.v(0.5), neck.v(0), and soma.v(0) vs.
  time.</p>
  <p class=Textbody>To rescale this graph so it looks more like the graph in
  Fig. 8a, click on the little white menu box in the left upper corner of its
  canvas, drag the mouse pointer up slightly to reveal the graph's secondary
  menu, and then drag the pointer over to &quot;View = plot&quot;. Release the
  mouse button and the graph should look like the inset in Fig. 8a.</p>
  <p class=Textbody>&nbsp;</p>
  <p class=Textbody align=center style='text-align:center'>&nbsp;</p>
  </td>
  <td width=263 valign=top style='width:197.1pt;padding:0in .5pt 0in .5pt'>
  <p class=TableContents align=right style='text-align:right'><img width=270
  height=261 src="readme.fld/image003.jpg" align=right hspace=12></p>
  </td>
 </tr>
</table>

<h3>To reproduce Fig. 8b</h3>

<p class=Textbody style='page-break-after:avoid'>Click on the</p>

<p class=Textbody style='page-break-after:avoid'>            Run &amp; Analyze</p>

<p class=Textbody>button near the top of the Controls panel. This will march
the spine over all of the segments in the model cell (you'll see the blue dot
in the Exp2Syn PPM move), running a simulation at each point, for a total of
760 simulations over the course of about 20 seconds or less depending on the
speed of your computer.</p>

<p class=Textbody>When this completes, many new graphs will appear, including
XY plots of</p>

<p class=Textbody style='margin-left:.5in'>voltage attenuation from spine head
to dendrite (actually &quot;site of spine attachment&quot;) vs. distance from
soma to spine location</p>

<p class=Textbody style='margin-left:.5in'>maximum depolarization in mV at
spine head (red) and point of spine attachment (black) vs. distance in um from
soma to spine. The difference between these is the voltage drop across the
spine neck.</p>

<p class=Textbody style='margin-left:.5in'>normalized maximum depolarization
vs. distance from soma to spine. The Y coordinates of this graph are the Y
values of the black points in the previous plot divided by the very largest
depolarization observed in the cell</p>

<p class=Textbody style='page-break-after:avoid'>There will also be three color
coded plots that show how spine location affects</p>

<p class=Textbody>            maximum depolarization at the site of spine
attachment (max depol @ spine loc)</p>

<p class=Textbody>            maximum depolarization in the spine head (max
depol @ spine head)</p>

<p class=Textbody>            voltage attenuation from the spine head to the
site of spine attachment (head-&gt;dend V atten)</p>

<p class=Textbody>These three graphs correspond to the EPSP<span
style='position:relative;top:1.0pt'>dend</span>, EPSP<span style='position:
relative;top:1.0pt'>spine</span>, and EPSP<span style='position:relative;
top:1.0pt'>spine</span>/EPSP<span style='position:relative;top:1.0pt'>dend</span> components
of Fig.&nbsp;8b.</p>

<p class=Textbody>&nbsp;</p>

<p class=Textbody align=center style='text-align:center'><img width=331
height=132 id="Picture 3" src="readme.fld/image004.jpg"
alt="A screenshot of a computer&#10;&#10;Description automatically generated"></p>

<p class=Textbody align=center style='text-align:center'>&nbsp;</p>

<p class=Textbody>To generate a color coded plot that shows how input impedance
Zin of the cell varies with location, in the Controls panel enter the frequency
of interest (70 Hz for Fig. 8b) into the numeric field next to the Frequency
button, then click on the button labeled</p>

<p class=Textbody>            Click to show on cell</p>

<p class=Textbody>This will produce shape plots that show how Zin and
normalized Zin vary over the cell, and how spine location affects normalized
EPSP amplitude at the site of spine attachment. The one labeled &quot;Zin
70.0&nbsp;Hz&quot; corresponds to the &quot;Z<span style='position:relative;
top:1.0pt'>dendrite</span> (70Hz)&quot; component of Fig. 8b.</p>

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