We thank Christian Erxleben for preliminary dynamic clamp experiments on HN(7) neurons that demonstrated their bursting capabilities. Angela Wenning aided the experiments with expert advice.&#160;We acknowledge the NIH for funding this work through Grant 1 R21 NS111355 to GSC and RLC.

NOTE: Invertebrate animal experimental subjects are not regulated by the NIH or Emory and Georgia State Universities. All measures were nevertheless taken to minimize the suffering of the leeches used in this work.
1. Prepare isolated ganglion 7 from the leech nerve cord
<ol>
	<li>Maintain leeches Hirudo verbana in artificial pond water (containing 0.05% w/v of sea salt) diluted in de-ionized water at 16 &#176;C on a 12:12 light-dark cycle.</li>
	<li>Prepare the leeches for dissection by cold-anesthetizing them in a bed of crushed ice for &#62;10 min until immobile.</li>
	<li>Fill a black, resin-lined dissecting dish to a depth of ~1 cm with chilled saline containing 115 mM NaCl, 4 mM KCl, 1.7 mM CaCl2, 10 mM D-glucose, and 10 mM HEPES in de-ionized water; pH adjusted to 7.4 with 1 M NaOH. Pin the leech dorsal side up in the black resin-lined chamber (at least 20 cm x 10 cm with a depth of at least 2 cm above the resin that is at least 2 cm thick).</li>
	<li>Under a steromicroscope at 20x magnification with oblique light guide illumination, make a longitudinal cut at least 3 cm long with 5 mm spring scissors through the body wall in the rostral 1/3rd portion of the body. Use pins to pull aside the body wall and expose the internal organs. 
	NOTE: Any stored blood meal can be removed by suction with a fire-polished Pasteur pipette.</li>
	<li>Isolate an individual mid-body ganglion 7 (seventh free segmental ganglion caudal to the brain).
	<ol>
		<li>Open the sinus in which the nerve cord resides using the 5 mm spring scissors. Be sure to split the sinus dorsally and ventrally leaving&#160;two strips of sinus. Use sharp #5 forceps to help guide the cutting and hold the sinus.</li>
		<li>Keep the sinus attached to each of the two bilateral nerve roots that emerge from the ganglion (it adheres tightly to each root) to use these strips of sinus for pinning out the ganglion.</li>
		<li>Remove the ganglion from the body by cutting the rostral and caudal connective nerve bundles that link the ganglia (as far from the 7th ganglion as possible) and the sinus strips, and then cut the roots lateral to where they emerge from the sinus.</li>
	</ol>
	</li>
	<li>Pin the ganglion (using old blunted #5 forceps) with shortened minuten insect pins, ventral side up, in clear, resin lined Petri dishes. Insert pins in the strips of sinus and loose tissue adhering to the roots and the rostral and caudal connectives, as far from the ganglion as possible. 
	NOTE: The resin must be no thicker than 3 mm if good illumination from below is to be achieved during recording. Make sure the ganglion is taut, both longitudinally and laterally</li>
	<li>Increase the magnification of the stereomicroscope to 40x or greater, and adjust the oblique illumination so the neuronal cell bodies can be easily seen on the ventral surface of the ganglion just below the perineurium.</li>
	<li>Remove the perineurium of the ganglion (desheath) with microscissors.
	<ol>
		<li>Start the desheathing by cutting the loose sheath between the roots on one side, and continue the cut laterally to the other side, making sure to keep the scissor blades superficial and not harm the neuronal cell bodies directly beneath the sheath.</li>
		<li>Make a similar superficial cut caudally from the lateral cut along the midline.</li>
		<li>Now grab the caudolateral flap of sheath on one side with the fine #5 forceps, pull it away from the ganglion, and cut it off with the microscissors.</li>
		<li>Repeat on the other side; this procedure exposes both HN(7) neurons for recording with microelectrodes.</li>
	</ol>
	</li>
	<li>Place the preparation dish in the recording setup, and superfuse with saline at a flow rate of 5&#160;mL/min at room temperature.</li>
</ol>
2. Identify and record leech heart interneurons with sharp microelectrodes
<ol>
	<li>For the duration of the recording of the HN(7) neuron (recordings last between 30 to 60 min), acquire and digitize the intracellular current and voltage traces from a neurophysiological electrometer sampling at rate of 5 kHz with a digital data acquisition (Analog to Digital, A to D) and stimulation (Digital to Analog, D to A) system, and display on a computer screen. 
	NOTE: Any commercial or custom-built software and A to D/D to A board can be used for data acquisition (A to D). D to A and custom-built software are required for dynamic clamp.</li>
	<li>Under a steromicroscope at 50-100x with dark field illumination from below, tentatively identify an HN(7) neuron of the bilateral pair by its canonical location at the posteriolateral position in midbody ganglion seven.</li>
	<li>Now aim to penetrate the putative HN(7) neuron with a sharp microelectrode filled with 2 M potassium acetate and 20 mM KCl using a micromanipulator.
	<ol>
		<li>Place the microelectrode very near the target cell body.</li>
		<li>Continuously observe the recorded potential with the electrometer, and set this potential to zero mV before penetrating the neuron.</li>
		<li>Penetrate the neuron with the microelectrode, by slowly&#160;driving&#160;the electrode along its long axis with the manipulator. Using the electrometer buzz function, set to 100 ms buzz duration, until a negative shift in membrane potential and vigorous spiking activity is observed.</li>
	</ol>
	</li>
	<li>Set the electrometer in discontinuous current-clamp mode (DCC) &#8805; 3 kHz to simultaneously record membrane potential&#160;and pass current with the single microelectrode (capacity compensation set to just below ringing and then dialed back 10%).
	<ol>
		<li>Monitor the settling of the electrode during DCC on an oscilloscope.</li>
		<li>Inject a steady current of -0.1 nA with the electrometer steady current injector for a minute or two to stabilize the recording.</li>
	</ol>
	</li>
	<li>Definitively identify the HN(7) neuron by its characteristic spike shape and weak bursting activity (Figure 1Ci).</li>
	<li>Perform any data analysis offline after the experiment is completed, and save all data on a disk.</li>
</ol>
3. Build a real-time HN or another model neuron
<ol>
	<li>Build custom software using a digital signal processing board (DSB; D to A and A to D) in a desk-top computer to implement in real time the model currents described in2,4 or different model currents for other neurons or experiments.
	<ol>
		<li>Use Hodgkin-Huxley style equations as they are the generally preferred method for representing model currents.</li>
		<li>See7 for a detailed description of the implementation of the real-time HN model and dynamic clamp prior to the addition of the pump current. Refer to the introduction section for the description of the currents, intracellular Na+ concentration, and conductances of the&#160;living HN(7) neuron in the HN model.</li>
	</ol>
	</li>
</ol>
4. Implement and vary dynamic clamp conductances/currents
<ol>
	<li>Use the custom-built dynamic clamp software for the DSB to implement and change in real time dynamic clamp any of the graphical user interface (Figure 3) (GUI)-accessible, programmed conductances and currents of the HN real-time model of the HN(7) neuron. 
	NOTE: As a reminder, <img alt="Equation 5" src="/files/ftp_upload/61473/61473eqgp.jpg" /> and <img alt="Equation 6" src="/files/ftp_upload/61473/61473eqimp.jpg" />&#160;are the maximal conductance of the persistent Na+ current (IP) and the maximal pump current (Ipump), respectively.</li>
	<li>Use GUI entry boxes in the software to make changes, as the model is running, in the <img alt="Equation 7" src="/files/ftp_upload/61473/61473eqimp.jpg" /> (PumpMaxL box) and <img alt="Equation 8" src="/files/ftp_upload/61473/61473eqgp.jpg" /> &#8203;(GpinHNLive box) (Figure 3). 
	NOTE: The GUI input boxes accept typed values, and steps of 0.1 nA are recommended for <img alt="Equation 9" src="/files/ftp_upload/61473/61473eqimp.jpg" /> and steps of 1 nS are recommended for <img alt="Equation 12" src="/files/ftp_upload/61473/61473eqgp.jpg" />.
	<ol>
		<li>Add small amounts of <img alt="Equation 10" src="/files/ftp_upload/61473/61473eqimp.jpg" /> and <img alt="Equation 11" src="/files/ftp_upload/61473/61473eqgp.jpg" /> with dynamic clamp to stabilize bursting of the HN(7) neuron,&#160;which is weakened by a microelectrode-induced leak, as shown in Figure 1Cii. 
		NOTE: Sharp microelectrode penetration causes membrane damage that is expressed as increased leak conductance or decreased input resistance.</li>
		<li>Start by adding a value of <img alt="Equation 13" src="/files/ftp_upload/61473/61473eqimp.jpg" /> of 0.1-0.2 nA, which makes up for the microelectrode-induced leak, but depresses excitability, and then gradually increase <img alt="Equation 14" src="/files/ftp_upload/61473/61473eqgp.jpg" />, which increases excitability, until regular bursting ensues, usually at <img alt="Equation 15" src="/files/ftp_upload/61473/61473eqgp.jpg" /> of ~1-4 nS (Figure 4A).</li>
	</ol>
	</li>
	<li>Systematically co-vary these currents (increments of 0.1 nA for <img alt="Equation 16" src="/files/ftp_upload/61473/61473eqimp.jpg" /> and 1 nS for <img alt="Equation 17" src="/files/ftp_upload/61473/61473eqgp.jpg" />) to the recorded HN(7) neuron with dynamic clamp (Figure 3), and assess their effects on burst characteristics: spike frequency (f: the reciprocal of the average of the interspike interval during a burst), interburst interval (IBI: the time between the last spike in one burst to the first spike in the next burst), burst duration (BD: the time between the first spike in a burst and the last spike in a burst), and burst period (T: the time between the first spike in a burst and the first spike in the subsequent burst).
	<ol>
		<li>Change the values of <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimp.jpg" /> and <img alt="Equation 19" src="/files/ftp_upload/61473/61473eqgp.jpg" />, as in the video demonstration, to become familiar with the technique and then venture out.
		<ol>
			<li>Hold <img alt="Equation 20" src="/files/ftp_upload/61473/61473eqimp.jpg" /> at a specific fixed value and sweep in 1 nS increments over a range of <img alt="Equation 21" src="/files/ftp_upload/61473/61473eqgp.jpg" /> supporting regular bursting activity.</li>
			<li>Now increase the fixed value of <img alt="Equation 22" src="/files/ftp_upload/61473/61473eqimp.jpg" /> by 0.1 nA and again sweep over a range of <img alt="Equation 23" src="/files/ftp_upload/61473/61473eqgp.jpg" /> &#8203;supporting regular bursting activity.</li>
			<li>For each implemented parameter pair, collect data containing at least 8 bursts so that reliable average measures of f, IBI, BD, and T can be made.</li>
			<li>Continue with sweeps for as long as the neuron remains viable, as assessed by strong spiking and a stable baseline potential of oscillation.</li>
			<li>Collect data from several neurons (from different animals) to generate a composite graph (Figure 5).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cymbalyuk, G. S., Gaudry, Q., Masino, M. A., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bursting+in+leech+heart+interneurons:+cell-autonomous+and+network-based+mechanisms.">Bursting in leech heart interneurons: cell-autonomous and network-based mechanisms.</a> Journal of Neuroscience. 22, 10580-10592 (2002).</li><li>Hill, A. A., Lu, J., Masino, M. A., Olsen, O. H., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+a+segmental+oscillator+in+the+leech+heartbeat+neuronal+network.">A model of a segmental oscillator in the leech heartbeat neuronal network.</a> Journal of Computational Neuroscience. 10, 281-302 (2001).</li><li>Tobin, A. E., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myomodulin+increases+Ih+and+inhibits+the+NA/K+pump+to+modulate+bursting+in+leech+heart+interneurons.">Myomodulin increases Ih and inhibits the NA/K pump to modulate bursting in leech heart interneurons.</a> Journal of Neurophysiology. 94, 3938-3950 (2005).</li><li>Kueh, D., Barnett, W. H., Cymbalyuk, G. S., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na(+)/K(+)+pump+interacts+with+the+h-current+to+control+bursting+activity+in+central+pattern+generator+neurons+of+leeches.">Na(+)/K(+) pump interacts with the h-current to control bursting activity in central pattern generator neurons of leeches.</a> eLife. 5, 19322 (2016).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clamp:+computer-generated+conductances+in+real+neurons.">Dynamic clamp: computer-generated conductances in real neurons.</a> Journal of Neurophysiology. 69, 992-995 (1993).</li><li>Prinz, A. A., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp+comes+of+age.">The dynamic clamp comes of age.</a> Trends in Neuroscience. 27, 218-224 (2004).</li><li>Barnett, W., Cymbalyuk, G., Jung, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+systems+analysis:+real-time+systems+for+design+and+prototyping+of+neural+interfaces+and+prostheses.">Hybrid systems analysis: real-time systems for design and prototyping of neural interfaces and prostheses.</a> Biohybrid systems: nerves, interfaces, and machines. , 115-138 (2011).</li><li>Sorensen, M., DeWeerth, S., Cymbalyuk, G., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+hybrid+neural+system+to+reveal+regulation+of+neuronal+network+activity+by+an+intrinsic+current.">Using a hybrid neural system to reveal regulation of neuronal network activity by an intrinsic current.</a> Journal of Neuroscience. 24, 5427-5438 (2004).</li><li>Olypher, A., Cymbalyuk, G., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+systems+analysis+of+the+control+of+burst+duration+by+low-voltage-activated+calcium+current+in+leech+heart+interneurons.">Hybrid systems analysis of the control of burst duration by low-voltage-activated calcium current in leech heart interneurons.</a> Journal of Neurophysiology. 96, 2857-2867 (2006).</li><li>Calabrese, R. L., Prinz, A. A., DeSchutter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Realistic+modeling+of+small+neuronal+networks.">Realistic modeling of small neuronal networks.</a> Computational Modeling Methods for Neuroscientists. , 285-316 (2010).</li><li>Rybak, I. A., Molkov, Y. I., Jasinski, P. E., Shevtsova, N. A., Smith, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+bursting+in+the+pre-B&#246;tzinger+complex:+mechanisms+and+models.">Rhythmic bursting in the pre-B&#246;tzinger complex: mechanisms and models.</a> Progress in Brain Research. 209, 1-23 (2014).</li><li>Picton, L. D., Nascimento, F., Broadhead, M. J., Sillar, K. T., Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+pumps+mediate+activity-dependent+changes+in+mammalian+motor+networks.">Sodium pumps mediate activity-dependent changes in mammalian motor networks.</a> Journal of Neuroscience. 37, 906-921 (2017).</li></ol>

Modeling, dynamic clamp, and the resulting analyses that they enable are useful techniques for exploring how individual and groups of ionic conductance/currents contribute to the electrical activity of neurons (Figure 1, Figure 2,Figure 4, and Figure 5). The use of these techniques shows how the Na+/K+ pump current (Ipump) interacts with voltage-gated currents, particularly the persistent Na+ current (IP), to promote robust bursting in the leech heartbeat pattern generator&#39;s core HNs. By combining dynamic clamp experiments and modeling, it is possible to test models more directly than is possible with ordinary voltage recording and current clamp techniques. The results gathered from the dynamic clamp experiments (Figure 5) will be used to further refine the HN model. The basic method of dynamic clamping demonstrated here can be customized to reflect the properties of any neuron under study if a mathematical model of neuronal currents can be determined with voltage clamp experiments.
Successful completion of the experiments of the type shown here requires careful impalement of an HN or other neuron when using a sharp microelectrode, because strong bursting is curtailed by electrode penetration1. (Whole-cell patch recording techniques, which minimize introduced leak, are also applicable to other neurons, but do not work well on leech neurons.) It is critical that the impalement of the HN neuron causes minimal damage to the neuron (added leak), and input resistance should be monitored and must be in the range of 60-100 MOhms for successful experiments4.
Dynamic clamp is a powerful technique, but it has limitations imposed by neuronal geometry because the artificial conductances are implemented at the site of the recording electrode-usually the cell body-not at the site where rhythm-generating currents are usually localized5,6,10. In leech HN neurons, the cell body is electrically close to the integration zone (main neurite) of the neuron where most active currents are localized, and spikes are initiated.

An erratum was issued for:&#160;<a href="https://jove.unicartagenaproxy.elogim.com/t/61473">Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses</a>. An author name was updated.
The&#160;name of the first author was updated from:
Ricardo Javier Erazo Toscano
to:
Ricardo Javier Erazo-Toscano

An erratum was issued for:&#160;<a href="https://jove.unicartagenaproxy.elogim.com/t/61473">Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses</a>. An author name was updated.

Erratum: Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses

Heartbeat in leeches is driven by a CPG consisting of 9 bilateral pairs of heart interneurons (HNs) distributed across as many mid-body segmental ganglia. At the core of the CPG are mutually inhibitory pairs of interneurons located in the 3rd and 4th segmental ganglia that form half-center oscillators (HCOs) (Figure 1A). These neurons continue to burst when synaptically isolated pharmacologically using bicuculline1. Others, such as the pair in the 7th segmental ganglia (the focus of this protocol), are also bursters, capable of producing bursting activity when synaptically isolated. They are not mutually connected and receive only descending input, and thus are easily isolated by severing the ganglion from the rest of the nerve cord. This independent bursting activity is sensitive to introduced leak current caused by penetration with sharp microelectrodes for recording&#160;but&#160;vigorously burst when recorded with loose patch methods1.
Both individual HN neurons and HN HCOs&#160;have been modeled (Hodgkin-Huxley-based single isopotential compartment models of HN neurons containing all experimentally identified voltage-gated and synaptic currents), and all the burst characteristics of the living system have been successfully captured2. Myomodulin, an endogenous neuropeptide in leeches, markedly decreases the period (T) of the burst rhythm of isolated HN neurons and HN HCOs. This modulator acts to increase h-current (hyperpolarization-activated inward current, Ih) and to decrease Ipump3. This observation led to the exploration of how Ipump interacts with Ih, and how their co-modulation contributes to the rhythmic activity of HN neurons. Activation of the pump by increasing [Na+]i (using the ionophore monensin) speeds the HN burst rhythm in both HN HCOs and isolated HN neurons4. This speed-up was dependent on Ih.&#160;When Ih was blocked (2 mM Cs+), the burst period was not altered by this method of pump activation; however, the burst duration (BD) was curtailed, and the interburst interval (IBI) increased in both HN HCOs and isolated HN neurons4.
For this protocol, all the currents of a living HN(7) neuron, including the pump current, Ipump, are incorporated in the HN model as follows:
<img alt="Equation 1" src="/files/ftp_upload/61473/61473eq01v2.jpg" /> (1)
where C is the membrane capacitance (in nF), V is the membrane potential (in V), t is time (in s). Detailed ionic current descriptions and equations have been described elsewhere2,4. The complete HN model neuron runs in real time (Figure 2). The software will be made available on GitHub upon publication and will be suitable to run on the digital signal processing board described in the Table of Materials. Here, the focus of enquiry is the Na+/K+ pump current (Ipump) and the voltage-gated currents contributing significant Na+ flux: a fast Na+ current (INa) and a persistent Na+ current (IP). The maximal conductances of these currents are&#160;<img alt="Equation 1a" src="/files/ftp_upload/61473/61473eq1a.jpg" /> <img alt="Equation 1b" src="/files/ftp_upload/61473/61473eq1b.jpg" /> respectively. The Na+/K+ pump exchanges three intracellular Na+ ions for two extracellular K+ ions, thus producing a net outward current. Importantly, it pumps 3 times as much Na+ out of the neuron as this current indicates, which is important for calculating the intracellular Na+ concentration.
The Na+/K+ pump current depends on intracellular Na+ concentrations and is expressed by the following sigmoidal function:
<img alt="Equation 2" src="/files/ftp_upload/61473/61473eq02.jpg" /> (2)
where [Na]i&#160;is the intracellular Na+ concentration,&#160;<img alt="Equation 4" src="/files/ftp_upload/61473/61473eqimp.jpg" /> is the maximal Na+/K+ pump current, [Na]ih is the intracellular Na+ concentration for the half-activation of the Na+/K+ pump, and [Na]is the sensitivity of the Na+/K+ pump to [Na]i. [Na]i&#160;builds as a result of the Na+ influxes carried by IP and INa and is diminished by the Na+ efflux of the Na+/K+ pump. The contribution of Ih and ILeak to the total Na+ flux is small and is not considered in the real-time model.
<img alt="Equation 3" src="/files/ftp_upload/61473/61473eq03.jpg" /> (3)
where, v is the volume (~6.7 pL) of the intracellular Na+ reservoir, F is Faraday&#39;s constant, and the extracellular Na+ concentration is kept constant.
Voltage-gated and leak conductances have been differentiated-these respond to membrane potential - from the pump current, which is regulated by the calculated intracellular Na+ concentration ([Na+]i). [Na+]i is built up through Na+ entry via the fast Na+ current (INa) that produces action potentials (spikes) and the persistent Na+ current (IP) that provides the depolarization to support spiking. [Na+]i is, in turn, reduced by the action of the pump through the extrusion of Na+. Baseline living HN values of <img alt="Equation 1a" src="/files/ftp_upload/61473/61473eqgp.jpg" />(5nS) and <img alt="Equation 1a" src="/files/ftp_upload/61473/61473eqgna.jpg" />(150 nS) have been assumed, and we take account of any added dynamic clamp <img alt="Equation 1a" src="/files/ftp_upload/61473/61473eqgp.jpg" />.
The goal of the protocol described here is to manipulate Ipump precisely and reversibly in real time to discover how it interacts with voltage-gated currents (persistent Na+ current in the current protocol) to control rhythmic bursting in single HNs. To accomplish this goal, dynamic clamp was used, which artificially introduces, upon command, a precise amount of any current that can be calculated as the model is running. This method has advantages over pharmacological manipulation of the pump, which affects the entire tissue, can have off-target effects that are often hard to reverse, and cannot be precisely manipulated. Dynamic clamp5,6 reads the voltage of a recorded neuron in real time (Figure 1B) and calculates and injects, in real time, the amount of any current based on model equations and the set values of any <img alt="Equation 1a" src="/files/ftp_upload/61473/61473eqgx.jpg" /> or <img alt="Equation 1a" src="/files/ftp_upload/61473/61473eqimx.jpg" />. Similar methods can easily be applied to any neuron that can be recorded intracellularly. However, parameters will have to be rescaled to the neuron chosen, and the neuron should be isolated from synaptic inputs, e.g., pharmacologically.

<table><tbody><tr><td>&lt;strong&gt;ANIMALS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Hirudo verbana&lt;/em&gt;</td><td>Leech.com, https://www.leech.com/collections/live-leeches</td><td></td><td>live leeches 2-3 grams</td></tr><tr><td>&lt;strong&gt;CHEMICALS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ARTIFICIAL POND WATER</td><td></td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C5670-100G</td><td>1.8 mM add last after adjusting pH</td></tr><tr><td>glucose</td><td>Sigma Aldrich</td><td>G7021-100G</td><td>10 mM</td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H4034-100G</td><td>10 mM</td></tr><tr><td>Instant Ocean (sea salt )</td><td>Spectrum Brands Inc., Madison, WI</td><td></td><td>0.05% (w/v) diluted in deionized water</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9333-500G</td><td>4 mM</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653-250G</td><td>115 mM</td></tr><tr><td>NaOH 0.1 N Solution</td><td>Sigma Aldrich</td><td>2105-50ML</td><td>Adjust to pH 7.4 with NaOH</td></tr><tr><td>&lt;strong&gt;MICROELECTRODES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>K Acetate</td><td>Sigma Aldrich</td><td>P1190-100G</td><td>2 M</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9333-500G</td><td>20 mM</td></tr><tr><td>SALINE</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>#5 Forceps</td><td>Fine Science Tools Dumont</td><td>11251-30 OR 11251-20</td><td>For general leech dissection</td></tr><tr><td>AxoClamp 2A/2B DCC electrometer</td><td>Axon Instruments Molecular Devices</td><td>2A/2B</td><td>For recording of neuronal membrane potential and discontinuous current clamp</td></tr><tr><td>Black resin</td><td>Dow Sylguard</td><td>170</td><td>Lines general dissect dish</td></tr><tr><td>Capilary glass 1 mm outer diameter, 0.75 mm inner diameter</td><td>A-M Systems</td><td>615000</td><td>For fabricating sharp microelectrodes</td></tr><tr><td>Clear resin</td><td>Dow Sylguard</td><td>184</td><td>Lines Petri dish used to mount ganglion for electrophysilogy</td></tr><tr><td>Dark field condenser</td><td>Nikon</td><td>Dry 0.95-0.80 MBL 1210</td><td>For illuminating the ganglion preparation during cell impalement</td></tr><tr><td>Digidata 1440A</td><td>Axon CNS Molecular Devices</td><td>1440A</td><td>Performs A to D and D to A for data acquisition and stimulation during electrophysiology</td></tr><tr><td>Digital signal processing board</td><td>dSpace</td><td>CLP1104</td><td>Our software implements all the conductances/currents in our model HN neuron on a DS1103 dSPACE PPC Controller Board in real-time at a rate of 20 kHz with a ControlDesk GUI (dSPACE, Paderborn, Germany)9.&amp;nbsp;</td></tr><tr><td>Falming/Brown Microelectrode Puller</td><td>Sutter Instruments</td><td>P-97</td><td>For fabricating sharp microelectrodes</td></tr><tr><td>Fiber-Lite high intensity illuminator</td><td>Dolan Jenner Industries</td><td>170D</td><td>For illuminating the general dissection and for illuminating the ganglion preparation during cell impalement</td></tr><tr><td>Headstage amplifier for AxoClamp 2A</td><td>Axon Instruments</td><td>HS-2A Gain:0.1LU</td><td>Now part of Molecular Devices for recording of neuronal membrane potential and discontinuous current clamp</td></tr><tr><td>Light guide</td><td>Dolan Jenner Industries</td><td>Rev R 38 08 3729107</td><td>For illuminating the general dissection and for illuminating the ganglion preparation during cell impalement</td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>MPC-385</td><td>Micromanipulator for cell impalement with microelectrodes</td></tr><tr><td>Micromanipulator controller</td><td>Sutter Instruments</td><td>MPC-200</td><td>Controls micromanipulators for cell impalement with microelectrodes</td></tr><tr><td>Minuten pins</td><td>BioQuip</td><td>0.15 mm diameter 1208SA</td><td>Should be shortened by curtting to ~5 mm</td></tr><tr><td>Optical Breadboard 3&#039; x 5&#039; x 8&quot;</td><td>Newport</td><td>Obsolete</td><td>With the 4 pneumatic Isolators below used to construct a vibration free workspace for electrophysiology</td></tr><tr><td>Oscilloscope</td><td>HAMEG Instruments</td><td>HM303-6</td><td>To monitor electrode setteling during DCC</td></tr><tr><td>Pascheff-Wolff spring scissors</td><td>Moria</td><td></td><td>Supplied by Fine Science Tools (Foster City, CA) catalog # 15371-92</td></tr><tr><td>pClamp 9 Software</td><td>Axon Instruments</td><td>9</td><td>Now part of Moleculear Devices uses the Digidata 1440 for data acquisition and stimulation during electrophysiology</td></tr><tr><td>Pneumatic Isolators 28&quot;</td><td>Newport</td><td>Obsolete</td><td>With optical breadboard used to construct a vibration free workspace for electrophysiology</td></tr><tr><td>Simulink / MATLAB software</td><td>MathWorks</td><td>2006 (Obsolete)</td><td>Implements dynamic clamp on the digital signal processing board</td></tr><tr><td>Stereomicroscope</td><td>Wild</td><td>M5A</td><td>10x Eye Pieces used for dissecting the leech and removingand desheathing ganglia</td></tr><tr><td>Steromicroscope</td><td>Wild</td><td>M5</td><td>20x Eye Pieces used in electrophysiologcal station to visualize neuron for microelectrode penetration</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91500-09</td><td>For general leech dissection</td></tr></tbody></table>

contribution of the na+/k+ pump to rhythmic bursting, explored with modeling and dynamic clamp analyses

The Na+/K+ pump, often thought of as a background function in neuronal activity, contributes an outward current (Ipump) that responds to the internal concentration of Na+ ([Na+]i). In bursting neurons, such as those found in central pattern generator (CPG) neuronal networks that produce rhythmic movements, the [Na+]i and therefore the Ipump, can be expected to vary throughout the burst cycle. This responsiveness to electrical activity, combined with independence from membrane potential, endow Ipump with dynamical properties not common to channel-based currents (e.g., voltage- or transmitter-gated or leak channels). Moreover, in many neurons, the pump&#39;s activity is modulated by a variety of modulators, further expanding the potential role of Ipump in rhythmic bursting activity. This paper shows how to use a combination of modeling and dynamic clamp methods to determine how Ipump and its interaction with persistent Na+ current influence rhythmic activity in a CPG. Specifically, this paper will focus on a dynamic clamp protocol and computational modeling methods in heart interneurons of medicinal leeches.

Modeling with the addition of Ipump4 brought the experimental findings presented in the introduction section into sharper focus and began to explain the pump-assisted mechanism of bursting. The real-time model demonstrated here has been tuned (<img alt="Equation 30" src="/files/ftp_upload/61473/61473eqgx.jpg" />&#160;and <img alt="Equation 31" src="/files/ftp_upload/61473/61473eqimx.jpg" /> parameters chosen) so that it produces regular rhythmic activity falling within the bounds of normal activity as observed in experiments - f, IBI, BD, T&#160;- and continues to produce such activity when the myomodulin-modulated parameters <img alt="Equation 32" src="/files/ftp_upload/61473/61473eqimp.jpg" /> (the maximal pump current) and <img alt="Equation 33" src="/files/ftp_upload/61473/61473eqgh.jpg" /> (maximal conductance of h-current) are varied or co-varied in the model. The parameter values determined can be used as a benchmark or canonical set for modeling experiments. In these model instances, Ipump oscillates throughout the burst cycle as [Na+]i around a baseline level. Ipump contributes to burst termination during the burst phase, and the hyperpolarization it produces activates Ih during the IBI; notice the maximal level of Ih near burst initiation (Figure 2).
Although the real-time HN model has all implemented currents2,4 available for dynamic clamping, the focus here was on <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimp.jpg" /> and <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" />, which are available for changes while the model is running in the dynamic clamp GUI (Figure 3). Dynamic clamp allows the experimenter to add (or subtract with a negative <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgx.jpg" /> or <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimx.jpg" />) any conductance or current into a neuron artificially that mimics the voltage and ionic dependence of a real conductance or current. Thus, it is possible to fully explore how a particular conductance/current interacts with the endogenous conductances/currents inside cells (Figure 1). The real-time HN model indicates that the persistent Na+ current (IP) in HN neurons contributes much of the Na+ entry strongly affecting [Na+]i (Figure 2) and thus, Ipump. Because IP is active at relatively negative membrane potentials, it opposes Ipump even during the IBI.
These observations indicate that it is instructive to explore interactions between <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimx.jpg" /> and <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" /> in isolated HN neurons with dynamic clamp as discussed previously8,9,10. These experiments (ongoing) are performed with sharp microelectrode recordings in single, synaptically isolated HN(7) neurons (seventh ganglion severed from the nerve cord). To date, these experiments show that robust bursting is restored in tonically active HN neurons (due to microelectrode penetration introduced leak) by co-addition of IP and Ipump with dynamic clamp (Figure 4). This is an important observation indicating that a bursting mechanism is available in these neurons (even when leak is compromised) that results from the interaction of Ipump and IP. Preliminary results indicate their strong complicated interaction, which can be explored in the model and experiments (Figure 5).
In conclusion, Ipump in response to periodic increases in [Na+]i during bursting activity contributes to the burst rhythm through burst termination (decreasing BD). The interaction of IP and Ipump constitutes a mechanism that is sufficient to support endogenous bursting activity; this mechanism can reinstate robust bursting in HN interneurons recorded intracellularly in ganglion 7. The interaction between IP and Ipump through [Na+]i affects the HN burst period non-monotonically and ensures robustness of autonomous bursting. These conclusions are in line with experiments and modeling in vertebrate systems11,12.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61473/61473fig01.jpg" /> 
Figure 1: Leech heart interneuron electrical activity and implementation of Ipump and IP with dynamic clamp. (A) Normal bursting activity simultaneously recorded, extracellularly (top) and intracellularly (bottom), in a leech heartbeat HCO from a third ganglion, a schematic of the recorded neurons and their mutually inhibitory synaptic connections at right. (B) Dynamic clamp schematic when recording a HN(7) interneuron in an isolated ganglion 7; note there is no synaptic interaction between the two HN(7) interneurons. (Ci) Bursting in a leak-compromised HN(7) interneuron. (Cii) More robust bursting can be produced by adding dynamic clamp Ipump (<img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimp.jpg" />&#160;= 0.1 nA), which makes up for the microelectrode induced leak, but depresses excitability, and <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" /> (1 nS), which increases excitability. Black dashed lines indicate baseline values. Abbreviations: HN = heart interneuron; HCO = half-center oscillator; Ipump = outward current; IP = persistent Na+ current; <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimp.jpg" /> = maximal Na+/K+ pump current; <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" /> = maximal conductance of the persistent Na+ current; Vm = membrane potential; [Na+]i = internal concentration of Na+. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61473/61473fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61473/61473fig02.jpg" /> 
Figure 2: Single HN interneuron model showing traces for membrane potential (Vm), Ih, Ipump, [Na+]i, and IP. Outward hyperpolarizing currents are negative, and inward depolarizing currents are positive. Black dashed lines indicate baseline values. Abbreviations: HN = heart interneuron; Ipump = outward current; IP = persistent Na+ current; <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimp.jpg" /> = maximal Na+/K+ pump current;&#160;Ih =&#160;hyperpolarization-activated inward current; <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" /> = maximal conductance of the persistent Na+ current; <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgh.jpg" /> = maximal conductance of the hyperpolarization-activated inward current; Vm = membrane potential; [Na+]i = internal concentration of Na+. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61473/61473fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61473/61473fig03.jpg" /> 
Figure 3: Graphical user interface of real-time heart interneuron (HN) model and dynamic clamp implemented on a digital signal processing board. Upper Left: Red Math boxes are user-determined parameter boxes for the real-time model, whereas Blue Live boxes are user-determined parameter boxes used in the dynamic clamp. El = the reversal potential of the leak current; Gl = leak conductance; Gh = h-current maximal conductance; Gp = P current maximal conductance; GCaS = slow calcium current maximal conductance; PumpMax = pump maximal current; [GSyn2 maximal synaptic conductance to the respective neuron; ThreshSyn2 spike crossing threshold for mediating a synaptic potential - these used to make a hybrid (living/model) half-center oscillator not illustrated here.]. Lower Left for Dynamic Clamp. At the very left are 5 computed values of dynamic clamp variables: Ipump = pump current injected; Ih = h-current injected (not used here); IP = P current injected; NaI = calculated internal Na+ concentration; ENa = calculated sodium reversal potential. Lower Left for Dynamic Clamp. To the right of the computed variables are 6 user determined parameter boxes: GNa = assumed endogenous fast sodium maximal conductance use to calculate Na+ flux associated with action potentials; PumpMaxL = maximal pump current to be injected by dynamic clamp; Naih see equation (2); Gh = maximal conductance to determine h-current to be injected by dynamic clamp; Gp = assumed endogenous P current&#160;maximal conductance use to calculate Na+ flux associated with endogenous P current; GpinHNLive = maximal conductance to determine P current to be injected by dynamic clamp. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61473/61473fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61473/61473fig04.jpg" /> 
Figure 4: Dynamic clamp analysis of independent HN(7) bursting. Upregulation of <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" /> from (A) 4.0 nS to (B) 9.0 nS slows down the independent HN burst rhythm. Experimental traces show rhythmic bursting in isolated HN(7) neuron with dynamic clamp. The ranges of oscillation of [Na+]i and Vm increase with upregulated <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" />. Traces top to bottom: recorded Vm, injected Ipump, calculated [Na+]i, and injected IP. Black dashed lines indicate baseline values. Abbreviations: HN = heart interneuron; Ipump = outward current; IP = persistent Na+ current; <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimp.jpg" /> = maximal Na+/K+ pump current; <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" /> = maximal conductance of the persistent Na+ current; Vm = membrane potential; [Na+]i = internal concentration of Na+. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61473/61473fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61473/61473fig05.jpg" /> 
Figure 5: Dynamic clamp analysis of independent HN(7) bursting. Upregulation of <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" /> tends to decrease, followed by an increased HN burst period. In individual experiments (points connected by lines) using dynamic clamp, <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" /> values were swept while <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimp.jpg" /> was held constant. Colors represent different constant levels of added <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimp.jpg" /> used in different experiments. Abbreviations: HN = heart interneuron; <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqimp.jpg" /> = maximal Na+/K+ pump current; <img alt="Equation 18" src="/files/ftp_upload/61473/61473eqgp.jpg" /> = maximal conductance of the persistent Na+ current. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61473/61473fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses at JoVE.com

Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses

Presented here is a method for investigation of the roles of the Na+/K+ pump and persistent Na+ current in leech heart interneurons using dynamic clamp.

Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses

The Na+/K+ pump, often thought of as a background function in neuronal activity, contributes an outward current (Ipump) that responds to the internal ...

contribution-na-k-pump-to-rhythmic-bursting-explored-with-modeling

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

2.6K Views.  Emory University. Presented here is a method for investigation of the roles of the Na+/K+ pump and persistent Na+ current in leech heart interneurons using dynamic clamp.

Video: Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Neuroscience

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...

Biochemistry

Recapitulation of an Ion Channel IV Curve Using Frequency Components

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.
METHODS: Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages.
RESULTS AND CONCLUSIONS: In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|&lt;0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.

There are technical obstacles to measuring current flux through multiple ion channels simultaneously, and later discerning what portion of the transmembrane current is due to each channel type. To address this need, this method presents a way to generate the IV curve of individual channel types using specific frequency components.

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into ...

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV.
Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

The pedunculopontine nucleus (PPN) is located in the brainstem and its neurons are maximally activated during waking and rapid eye movement (REM) sleep brain states. This work describes the experimental approach to record in vitro gamma band subthreshold membrane oscillation in PPN neurons.

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is implicated in diseases and disorders including attention deficit hyperactivity disorder, Parkinson's disease, and traumatic brain injury. Thus, DA neurotransmission and the methods to study it are of intense scientific interest. In vivo fast-scan cyclic voltammetry (FSCV) is a method that allows for selectively monitoring DA concentration changes with fine temporal and spatial resolution. This technique is commonly used in conjunction with electrical stimulations of ascending DAergic pathways to control the impulse flow of dopamine neurotransmission. Although the stimulated DA neurotransmission paradigm can produce robust DA responses with clear morphologies, making them amenable for kinetic analysis, there is still much debate on how to interpret the responses in terms of their DA release and clearance components. To address this concern, a quantitative neurobiological (QN) framework of stimulated DA neurotransmission was recently developed to realistically model the dynamics of DA release and reuptake over the course of a stimulated DA response. The foundations of this model are based on experimental data from stimulated DA neurotransmission and on principles of neurotransmission adopted from various lines of research. The QN model implements 12 parameters related to stimulated DA release and reuptake dynamics to model DA responses. This work describes how to simulate DA responses using QNsim1.0 and also details principles that have been implemented to systematically discern alterations in the stimulated dopamine release and reuptake dynamics.

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Contribution of the Na⁺/K⁺ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses

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Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Recapitulation of an Ion Channel IV Curve Using Frequency Components

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

One-channel Cell-attached Patch-clamp Recording