Walk-Through: Drugs, Optogenetics and Neuromodulation in the Network Module

If you have not already done the Network Walk-Through Warm-Up, I suggest you do that before proceeding, so that you are familiar with the general processes involved in building neural circuits.

• Start the Network module of Neurosim.
• In the Results view, check the Run on change and Auto clear boxes.
• Check the Monitor box at the bottom of the Drugs and/or Light list in the Experimental Control dialog (which is usually docked on the left of the Setup view) .
• This will draw a purple line in the Results view whenever an agent is applied. The line can be dragged vertically to a new location if desired. The colour can be changed by clicking the coloured button.

The default circuit shows 2 neurons, with N1 making a type a spiking chemical synapse onto N2. You can check the properties of the synapse through the Synapses: Spiking chemical menu command, but, in short, it is a nicotinic cholinergic type.

This circuit is used as the base for walk-throughs setting up simulations using drugs, optogenetics and neuromodulation.

Drugs

Available agents are listed in the Drugs and/or Light group of the Experimental Control dialog. Each agent has an apply checkbox to the left of its name.

Note: agents can be artificial pharceutical agents, natural neurotransmitters, light, or neuromodulators. However, for simplicity, the generic term "drug" is used in these walk-throughs except where the specific type is important.

Here is some general information about drugs in this module:

• Two drugs are system-defined and cannot be edited:
  • TTX (tetrodotoxin): This blocks all integrate-and-fire spikes in all neurons, and any voltage-dependent channels with a short name containing 'Na'.
  • Cd (cadmium): Cadmium is a potent calcium channel blocker, so this blocks all chemical synapses (by preventing pre-synaptic release), and any voltage-dependent channels with a short name containing 'Ca'.
  • Both these drugs have absolute blocking effects.
• Up to 10 user-defined drugs can be specified.
• Four such drugs are already defined in the default circuit, but these can be edited/removed if desired.
• User-defined drugs can have blocking, activating or neuromodulatory effects on synapses and/or voltage-dependent channels.
• In the latter case, an activator drug can simulate light in an optogenetic experiment (see below).
• Note that electrical synapses can be blocked by drugs, but not activated, although their conductance can be enhanced by neuromodulation.
• Each user-defined drugs can have specified variable effectiveness (equivalent to varying concentration).
• Drugs can be applied manually by clicking the appropriate checkbox, either before the start of an experiment, or while the experiment is actually running.
• Drugs can be automatically applied/removed at pre-set times during an experiment.

Note that the Experimental Control drug options can be hidden through the Options: Configure View menu command if they are not relevant for an activity and you want to simplify the configuration.

The drug properties (normally available through the Drugs menu) can be hidden through the Options: Set puzzle menu command. The drugs are still available for use, but their properties cannot be seen or changed.

Synapse Effects

• Select the Drugs/Light: Define menu command to open the Define Drugs and/or Light dialog.

There are 10 editable rows in the dialog, for the 10 possible user-defined agents.

The left column is headed Agent name, the remaining columns specify what the drug does. To be enabled for the user, the drug must have a name. If it has a name, but no other properties, it will appear in the Drugs/Light list, but it will do nothing.

Note that Drug 1 (αBT: alpha bungarotoxin) affects type a spiking and non-spiking chemical synapses, and its Action (right-hand column) is to Block. In real systems the snake venom αBT does indeed block nicotinic acetylcholine receptors, so this is appropriate. Drug 4 (ACh: acetyl choline) also affects type a spiking and non-spiking chemical synapses, but its action is to Activate. Since ACh is the natural transmitter for these synapses, this too is appropriate. (The third type of action, Modulate, is described later.)

• The Define Drugs dialog is modal, so click OK to dismiss it.
• Click Start to see the circuit activity without any drugs. Hopefully, the Results make sense.
• Check the αBT drug box.

The new N2 trace in the results is completely flat because we have completely blocked the synapse.

• Uncheck the αBT box and then check the ACh box.

N2 now spikes continuously because the ACh receptors are continuously activated.

Note: In Neurosim, receptors do not desensitize when an activator drug is applied.

• Uncheck the ACh box.

Timed Application

• Uncheck the Run on change box in the Results view.
• Select the Drugs/Light: Timed application, strength menu command to open the Timed Application and Strength dialog.
• This dialog is non-modal, so it can be left open while doing other things.

Each drug has an Auto checkbox which, if checked, will mean that the drug is applied at the specified On time and maintained for the specified Duration. time. If the drug is already on when the simulation reaches the specified On time, the drug remains on. A drug can be applied/removed repeatedly by checking the Repeat box, with a specified off-time Gap.

• In the ACh row (4) check the Auto box.

Note that an asterisk appears after the drug name in the Drugs/Light list of the Experimental Control dialog. This is a visual indication that the drug status may change during a simulation run without user intervention (if the Timed Application dialog were not showing, the user might not realize that Auto apply was active).

• Set the On time to 0.03 s and the Duration to 0.05 s, and then click Start.

A pulse of ACh is applied just before the start of the stimulus and consequently the neuron depolarizes and spikes. The timing of the pulse is shown by the magenta bar in the Results view, and while the ACh is applied the ACh box in the Setup view is checked. However, the pulse is very brief, and the post-synaptic neuron returns to its normal response when it terminates (and the box unchecks).

• Check the Repeat box for ACh, set the Gap to 0.03 s, and click Start.

Now pulses of ACh are applied repeatedly, with the specified gap between pulses.

• Set the Duration to 0.06 s to produce slightly longer pulses, and click Start.

Note that the simulation terminates with ACh still applied, and consequently the ACh box in the Drugs/Light frame remains checked.

• Click Start again.

Now ACh is applied right from the start of the simulation, and again it remains applied at the end of the run. To prevent the immediate application of ACh and return to just timed application, you must manually un-check the ACh box before clicking Start.

Hopefully, this gives you a good idea of how you can auto-apply drugs in the Network module.

Fraction Effect

In reality, drug effects often depend on the concentration of the drug when it is applied - low concentrations usually have weaker effects than high concentrations. This can be simulated in Neurosim as described next.

• Load and Start the file Fraction Effect.

The circuit is similar to the default, except that N2 has been set to be non-spikingThe neuron was made non-spiking by unchecking the 'Use integrate-and-fire spikes' box in the 'Neuron Properties' dialog, accessed by double clicking the neuron. so that its spikes do not interfere with the drug effects. The synapse type a is still a nicotinic cholinergic type, but it does not show facilitationThe 'Relative facilitation' has been set to 1 in the 'Spiking Synapse' properties dialog, accessed from the 'Synapses: Spiking chemical' menu command.. In the Results view, the bottom trace shows the chemical synaptic conductance in N2. The conductance changes underlying the EPSP are obvious.

• Open the Timed Application and Strength dialog as before.

The right hand column is labelled Strength. For drugs defined as blockers or activators, a value of 1 (the default) means that the effect is maximal (either total block, or total activation of all receptors), values less than 1 reduce the effect in proportion.

• Check the αBT box in the Experimental Control dialog and click Start.

A new sweep occurs, and the synaptic conductance is completely blocked.

• In the αBT row (1), set the Strength to 0.7, and click Start.

Another sweep occurs, but this time the block is partial. A Fraction effect of 0.7 means that 70% of the receptors are blocked, leaving 30% available to carry synaptic current. Hence the synaptic current is only about one third of that for the unblocked receptors, and the EPSPs are reduced in size but not abolished. The 30% can be regarded as the residual conductance fraction.

Multiple Blocking Drugs

You can specify more than one drug as a blocker of a particular synaptic type (although I would not normally recommend this when teaching).

• Select the Drugs: Define menu command to open the Define Drugs dialog.
• In the empty row 5, enter "ACh block 2" as the Agent name, and a as the Spiking synapse type.
• Click OK to close the dialog.

The new drug should appear both in the Drug/Light list in the Experimental Control dialog, and in row 5 in the Drug/Light Timed Application and Strength dialog (assuming that you kept that open - if not, re-open it now).

• Set the Strength of the new drug to 0.7.
• Apply the new drug by checking its box in the Experimental Control dialog.
• Click Start.

A fourth sweep occurs, and now the synaptic conductance is very small, as is the resulting EPSP.

• If you are not sure about the superimposed sweeps in the Results view, use the Highlight sweep facility in the Results view to see the 4 sweeps highlighted in turn.

Activation and Occlusion

• Remove the blocking drugs by unchecking their apply boxes in the Experimental Control dialog.
• Click Clear, and then Start.
• Check the ACh box in the Experimental Control dialog, then click Start.

The N2 synaptic conductance immediately jumps to a high level, and N2 depolarizes.

Note: The post-synaptic conductance increase caused by maximum drug activation (Fraction effect = 1) is set heuristically at 2x the baselineThe baseline conductance and facilitation rate are set in the 'Synapse Properties' dialog, accessed through the appropriate Synapse sub-menu. synaptic conductance, with a further increase if the synapse facilitates. The increase is to allow for the possibility of temporal and spatial summation of the post-synaptic conductance.

When an activator drug is applied with a Fraction effect of 1, the conductance increase that would normally be caused by transmitter released from the pre-synaptic neuron is occluded - i.e. disappears. This is because all the post-synaptic receptors are assumed to be activated by the drug, so further release of transmitter from the pre-synaptic neuron has no effect.

• Set the Fraction effect of ACh to 0.7, and click Start.

Now the instant conductance increase caused by ACh application is smaller, and the pre-synaptic spikes cause a small further increase in conductance. This is because the receptors are not fully occluded with the weaker activator effect. This is meant to simulate a lower concentration of ACh.

• Check the αBT box to apply it. (Remember that earlier you set its Fraction effect to 0.7).
• Click Start.

Both the ACh and residual PSP conductance increases are reduced. This is because the blocking effect of αBT takes precedence over the activating effect of ACh.

Voltage-Dependent Channel Effects

Drugs that block voltage-dependent channels operate in a very similar way to those that block synaptic channels - they simply reduce the calculated channel conductance by the specified Fraction effect factor.

Activator agents act differently. The key difference is that they are obligate factors. A voltage-dependent channel can be specified as needing activation, and if this is done, that channel will have 0 conductance unless an activating agent is applied. This allows such an agent to act as light in a simulated optogenetic experiment, as described next. (However, note that agents specified as neuromodulators can enhance the normal conductance of a voltage-dependent channel, and this is described later.)

Optogenetics

An agent that activates an ion channel can simulate light in an optogenetic experiment (in which case, of course, the term "drug" is inappropriate).

• Load and Start the file Optogenetics start.
• Double-click the neuron in the Setup view to open the Neuron Properties dialog.

The neuron is an endogenous burster that contains 3 voltage-dependent channels. Na (V) and K (V) produce standard HH-type spikes (but with modified kinetics), while the third channel, Ca (inactivating), is a low-threshold, slowly inactivating calcium channel. It is this channel that drives the oscillations responsible for bursting (this is the same neuron used in the tutorial on endogenous bursting).

In the Results view, the top trace shows the membrane potential, and the bottom trace shows the conductance of the calcium channel.

Assume that a genetic engineer has induced the neuron to express two types of channelrhodopsinsChannelrhodopsins are light-activated ion channels that originally derived from unicellular green algae, but through genetic engineering are now available in a variety of forms. Their expression can be induced in particular cell lines in many model organisms used in neuroscience research, such as the fruit fly or mouse. in its membrane. The first is opened by blue light and is anion (i.e. chloride) selective, and therefore inhibits the neuron when activated. The second is a non-selective cation channel (like a nicotinic acetylcholine receptor) that is opened by red light and which excites the neuron when activated. The aim is to modify the existing model neuron to incorporate these new channels. This can be done by adding new "voltage-dependent" channels, but without making them voltage-dependent. If you want to jump ahead and see the finished model, it is available as the file Optogenetics finish.

• Click the Add button in the Voltage-dependent channels group in the Neuron Properties dialog.

This adds a new channel (4), but by default it is an HH sodium channel ('Another fast Na channel'), which is not what we want.

• Double-click the new channel to open the Voltage-Dependent Channel Properties dialog.
• Make the following changes:
  • Change the Description to 'Anion channelrhodopsin'.
  • Check the Requires Activation box (top-right in the dialog) so that its conductance can be light-dependent.
  • Note that the name in the channel list in the parent Neuron Properties dialog is updated to reflect the change, and that it has an asterisk after it because it requires activation.
  • Set the Equilibrium potential to -70 mV, which is appropriate for a chloride channel.
  • Set the Maximum conductance to 2.
  • Set the Short name to 'A-ChR'.
  • Select the second gate ('Na: h (inactivation)') in the Gates list.
  • Click the Del button to delete that gate.
  • With the first gate ('Na: m (activation') in the Gates list selected
    • Change the gate Description to just 'activation'.
    • Select User-defined ∞τ as the type in the Equations group.
    • The default values in the Gate kinetics equation edit boxes are OK. The value of 1 in the Inf var (time infinity open probability) variable box means that the channel will always be fully open, but only while it is activated by an external modulator (because Requires Activation was set earlier). The value in the Time constant edit box is irrelevant since the open probability has a fixed value (but the value 1 means that it would respond rapidly if the channel had any voltage-dependency).
    • Check its OK box, and uncheck the other OK boxes. This is just for consistency.
    • Change the Exp # to 1.

At this point we have an anion channelrhodopsin embedded in the membrane.

• Click Apply in the Neuron Properties dialog to lock in the changes.
• In the Neuron Properties dialog, select the entry '4: Anion channelrhodopsin *' in the channel list if it is not already selected.
• Click the Copy button.
• Click the Paste button.

This implants a new channel (5) which is identical to the one we just implanted.

• In the Voltage-Dependent Channel Properties dialog:
  • Set the ID to 5 (if it is not already set).
  • Change the Description to 'Cation channelrhodopsin'.
  • Change the Equilibrium potential to -10 mV (typical for a non-selective cation channel).
  • Change the Short name to 'C-ChR'.
  • Click the Trace colour button, and select blue.
• Click OK in the Neuron Properties dialog.

We have finished implanting the two channelrhodopsins into the neuron. Now we need a way to activate them.

• Select the Drugs/Light: Define menu command to open the Define Drugs Light etc. dialog.
• In the top row:
  • Enter 'blue light' in the left-hand Drug name column.
  • Enter 'A-ChR' in the Voltage-dependent types column
  • In the right-hand Action column, select Activate.
• In the second row:
  • Enter 'red light' and 'C-ChR' into their respective columns, and select Activate.
• Click OK to dismiss the Define Drugs dialog.

Now we have two agents (light) that can activate the channelrhodopsins. It would be nice to monitor the channelrhodopsin conductance directly.

• Clear the Results if data are showing.
• Click the Traces button in the Results view to open the Trace and Axis Setup dialog.
• In the Conductance group near the top-right of the dialog, add 4 and 5 to the Voltage-dependent channels box so that it reads '3 4 5'.
• Click OK to close the dialog.
• In the Results view select Scroll as the Trigger mode.
• In the main toolbar select a Slow down factor of 2 from the drop down list. (You may need to adjust this, depending on the speed of your computer.)

You are now ready to run an experiment. The idea is to manually "switch on" first the blue light, and then the red light, while observing what happens to the neuron's activity.

• Click Start.
• After a few seconds of activity, check the blue light box in the Drugs etc. group of the Experimental Control dialog.
• After a few more seconds, uncheck the blue light box.
• After activity has recovered, check the red light box.
• After a few more seconds, uncheck the red light box.
• After activity has recovered, click End in the Results view to terminate the experiment.
• Click the Show all button ()in the Results toolbar.

If you want to simulate the effect of light at reduced (sub-optimal) intensity:

• Select the Drugs: Timed application, strength menu command.
• Set a Fraction effect less than 1 for the appropriate light in the Timed Application and Strength dialog.

This has the same effect as would reducing the maximum conductance in the Voltage-Dependent Channel Properties dialog for the channelrhodopsin that the light activates.

You have now finished the walk through.

The final model is available as the file Optogenetics finish. The lights are switched on and off automaticallyAutomatic light switching is specified in the 'Timed Application and Strength' dialog, which is accessed through the 'Drugs: Timed application, strength' menu command., first the blue light, then the red light, so you can just Start the simulation and sit back and watch.

Neuromodulation

This walk-through shows how to set up neuromodulation in a Network circuit. There is a tutorial showing neuromodulation in action here, and details of how neuromodulation is implemented in Neurosim are given here.

• Load and Start the file Neuromod start.

This is a slightly modified version of the default Network circuit.

• Right-click in the Setup view somewhere below the two existing neurons, and select Add neuron from the context menu.
• This adds a standard integrate-and-fire neuron to the circuit.
• Double-click on the new neuron to open its Neuron properties dialog.
  • Set the Trace colour to blue by clicking on the coloured button.
  • Change the description to "neuromodulator"
  • Click OK to dismiss the dialog.

In the Stimulus group of the Experimental Control panel:

• Make sure that 0 (new) is selected in the list.
• Enter 3 into the Target neuron(s) box.
• Set the stimulus Duration to 5 ms and the Delay to 300 ms.
• Select Gauss from the option list.
• This reveals options for repetitive stimulation.
• Set the Mean interval to 150 ms (leave the Interval S.D. at 0)
• Check the Burst box, and set the Duration to 250 ms.

The new neuron should now receive 2 supra-threshold stimulus pulses part way through the long stimulus applied to N1. It would be sensible to check that this is happening.

• Clear the Results view.
• Click the Traces button in the Results view.
  • Add 3 after the existing 1 in the axis 3 Stimulus list (leave a space between the numbers).
  • Check the Show box for axis 4.
  • Set the Axis label to N3, and enter 3 into the Neuron(s) edit box.
  • Click OK.
• Click Start.

You should see a new axis showing the activity of N3 as a blue trace. There should be 2 spikes in it.

We now need to make N3 release a neuromodulator. The first task is to define a neuromodulator. Since the main feature in the circuit is the nicotinic cholinergic synapse from N1 to N2, that is a good target for modulation in this walk-through.

• Select the Synapses: Neuromodulator men command to open the Neuromodulator dialog.
• This dialog is non-modal and can be left open so that you can easily adjust parameters. So if you have a big enough monitor, move the dialog so that it does not obscure the main window.
  • Enter ACh mod into the Name edit box.
  • Enter a into the Spiking synapse list. This means that the neuromodulator will affect all post-synaptic receptors of type a spiking synapses that exist in the circuit (although in this case there is only such synapse: the N1 to N2 synapse).
  • Change the ID colour to orange by clicking on the coloured button.
  • Check the Show graph box to expand the dialog to show the Release profile (concentration vs time) that would occur following a single spike in the releasing neurons. The vertical axis is the extracellular concentration (arbitrary units), and note that it has a peak value approaching 1. This is always the default peak concentration following a single spike.
  • Neuromodulators typically have quite slow release and quite slow effects, so select the 2 Exponential option in the Synapse release kinetics group, and change the Decay rate to 120 ms.
    • Change the right-hand x (time) scale in the graph to 1000 ms, to see the full profile.
  • Note that the Base multiplier (under the Name edit box) has a default value of 1.5 and a Maximum value of 5.
  • If the Base multiplier is greater than 1 the modulator is an up-regulator that causes an inrease in conductance/current above the normal value. If the Base multiplier is less than 1 it is a down-regulator that causes a decrease in conductance/current.
  • Select the Modulation multiplier vs concentration option above the graph.

The graph now shows the concentration-dependence of the multiplier that will be applied to the conductance of type a spiking synapses. When the concentration is 0 (left hand end) the multiplier is 1 (horizontal dashed line), meaning no modulation. When the concentration is 1 (vertical dashed line) the multiplier has its base value of 1.5. The multiplier then rises linearly with concentration until it reaches a value 1 below the maximum. After this, further increases in concentration cause the multiplier to rise asymptotically towards its maximum of 5.

• Change the Base multiplier to 0.5.
• Note that the Maximum is disabled, since it is not relevant for down-regulation.

The graph now shows the negative dependence of the multiplier on concentration. Again, when the concentration is 0 the multiplier is 1. When the concentratio is 1, the multiplier has its base value of 0.5. The multiplier has a power-dependence on the concentration, so the higher the concentration the closer the multiplier approaches to 0, meaning total block.

• Return the Base mutliplier to 1.5.
• Click Apply to lock in the changes (or OK to close the dialog, if you need the screens space).

Now we need to make N3 release the neuromodulator.

• Right-click N3.
• Select Add neuromodulator output to neuron from the context menu.
• This option is also available through the Neuron and Connections menus.

The neuromodulator release site is shown in the Setup view as an orange circle attached to N3.

It would be good to monitor the extracellular concentration and multiplier value of the neuromodulator.

• Click the Traces button in the Results view.
• Note that the Neuromodulator options check box at the bottom of the dialog is pre-selected, because a neuron releasing a neuromodulator exists in the circuit.
• At the bottom of the dialog, check the Concentration and Multiplier boxes in the Neuromodulation group.
• Enter a into each of the adjacent Types edit boxes.
• Click OK to dismiss the dialog.

Two new axes now show in the Results view.

• Click Start.

The bottom 2 traces show the extracellular neuromodulator concentration (upper) and the conductance multiplier (lower).

Note that before N3 spikes the neuromodulator concentration is 0 and the multiplier is 1, but that following each spike in N3 there is an increase in the neuromodulator concentration, an increase in the value of the multiplierThis, of course, is theoretical value resulting from the mechanism chosen to implement modulation. It could not be measured directly in a real experiment, although its equivalent could be calculated by measuring synaptic current in a voltage-clamp protocol., and a consequent increase in the amplitude of the EPSP in N2. The neuromodulator is acting as an up-regulator of the synaptic conductance.

• Change the Mean interval for stimulus 2 (applied to N3, the neuromodulator neuron) to 70 ms, and click Start.

N3 now spikes 4 times, at higher frequency. The neuromodulator concentration summates (as does the multiplier), and the EPSPs in N2 are now large enough to generate spikes.

• If it is not still open, re-open the Neuromodulator dialog through the Synapses: Neuromodulator menu command).
• Change the Base multiplier to 0.5.
• Click Start.

The increase in neuromodulator concentration is the same, but now the multiplier decreases below the initial value of 1, and the EPSPs in N2 decreases in amplitude. The neuromodulator is now acting as a down-regulator of the synaptic conductance.

• Return the modulator Base multiplier to 1.5.
• Close the Neuromodulator dialog.
• Clear the Results view.

Exogenous Neuromodulator Application

A neuromodulator can be specified as a drug that can be applied manually or automatically during an experiment.

• Select the Drugs/Light: Define menu command to open the Define Drugs and/or Light dialog.
• In row 5, select the Modulate option in the Action set towards the right.

The drug name is automatically set to the name of the neuromodulator specified in the Type column at the right, which defaults to type a, which we have set up earlier as ACh mod. (If you change to Type b, the name is blank because we have not defined a type b neuromodulator.)

• Click OK to dismiss the dialog.

Note that ACh mod now appears in the Drugs/Light list at the bottom of the Experimental Control panel.

• Check the Monitor box under the list.
• Un-check the Auto clear box in the Results view.
• Click Start.
• This just generates a comparison trace.
• Check the ACh mod box in the Drugs/Light list.
• Click Start.

The neuromodulator is now applied at a concentration of 1 throughout the experiment, and so the N2 EPSPs are enhanced throughout. However, when N3 spikes, additional neuromodulator is released, whose effect sums with the exogenously-applied neuromodulator.

• If desired, use the Highlight sweep facility in the Results to view each sweep in turn.

Timed Application and Exogenous Concentration

As described above for Drugs, neuromodulators can have timed exogenous application and can be applied at different concentrations, using options in the Timed Application and Strength dialog (accessed through the Drugs/Light: Timed Application and Strength menu command). Note that for neuromodulators, the Strength parameter in the dialog refers directly to the concentration at which the neuromodulator is applied (rather than to the Fraction Effect parameter used for activators and blockers).

Other Modulation Targets

This walk-through describes how to apply neuromodulation to the conductance of spiking chemical synapses, but neuromodulators can be defined to affect non-spiking chemical synapses, electrical synapses or voltage-dependent channels by editing the appropriate Target row in the Neuromodulator dialog. Neuromodulators can also affect the current generated by the Na/K exchange pump by entering "Pmp" as a target in the voltage-dependent channel row.

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