This work was supported by grants from the Fondation de France, the Institut National de la Sant&#233; Et de la Recherche M&#233;dicale, the Pierre &#38; Marie Curie University and the program &#39;Investissements d&#39;avenir&#39; ANR-10-IAIHU-06.

All procedures were carried out in accordance with the guidelines of the European Union (directive 2010/63/EU) and approved by the Charles Darwin Ethical Committee on Animal Experimentation. We describe here the procedure we routinely use in our laboratory, however most steps can be adapted to match everyone&#39;s specific needs.
1. Surgical Preparation
Note: All incision and pressure points should be repeatedly infiltrated with local anesthetic (lidocaine or bupivacaine). The present procedure is terminal, if an aseptic preparation is required several modifications should be implemented.
<ol>
	<li>Anesthetize a rat with sodium pentobarbital (40 mg/kg) and ketamine (50 mg/kg) in two locally separated intraperitoneal (IP) injections.</li>
	<li>Let the animal go under general anesthesia and repeatedly verify that a surgical plane of anesthesia is attained (no reaction to toe pinching). Place the rat on a feedback heating blanket and insert a rectal probe to maintain core temperature around 37 &#176;C.</li>
	<li>Place a catheter in the peritoneal cavity to facilitate subsequent injection of anesthetic agents and to avoid perforating organs by repeated needle punctures 30.
	<ol>
		<li>Clip the hair over a small (~2 - 3 mm) area above a region located within the stomach&#39;s lower right or left quadrant. 
		Note: Depilatory cream can also be used.&#160;</li>
		<li>Make a 2 - 3 mm incision on the skin with sharp scissors or a scalpel. Using blunt dissection, remove fat and muscle layers until the peritoneal cavity is observed.</li>
		<li>Insert about 1 - 2 cm of a small catheter in the cavity and close the wound with surgical glue. 
		Note: The diameter and the total length of the catheter should be minimal (e.g., 2 French - corresponding to 0.043 mm inside diameter) to reduce dead volumes. Polyurethane tubes are most suited.</li>
		<li>Make a loop and suture the catheter to the skin to secure it in place.</li>
	</ol>
	</li>
	<li>Install a tracheal tube to control ventilation during artificial respiration.
	<ol>
		<li>As for the previous step, prepare the area of interest (1 - 2 cm over the trachea right above the manubrium), remove the hair and incise the skin.</li>
		<li>Blunt dissect the first layers of fat and muscles, then move the salivary gland aside and expose the trachea by gently dissecting the last layer of muscles.</li>
		<li>Carefully remove the tissues over the trachea and slide a thread below it using small forceps. Make a surgeon&#39;s knot with the thread but do not tight it yet.</li>
		<li>Incise the trachea transversally between two cartilaginous rings. Swab blood in the trachea if any.</li>
		<li>Insert a tracheal tube with the appropriate diameter and tighten the thread&#39;s knot to secure the tube steady. For additional stability the thread can further be attached at a higher point of the trachea tube.</li>
		<li>Suture the wound close or use surgical staples. Avoid surgical glue here if the tracheal tube is intended to be reused.</li>
	</ol>
	</li>
	<li>Install the animal in a stereotaxic frame and carefully monitor the following physiological variables: ECoG, SpO2, EtCO2, heart rate (via an electrocardiogram, ECG) and internal temperature. Monitor and adjust these variables to keep a proper depth of anesthesia and physiological state. Specifically, supplement anesthesia with a small dose (10 mg/kg) of sodium pentobarbital if necessary.</li>
	<li>Apply eye ointment on both eyes to avoid desiccation. Clip the hair over the scalp, make a longitudinal incision (~2 cm) and resect the connective tissues overlying the skull using a scalpel or a curette.</li>
	<li>Make a small (~1.5 mm diameter) craniotomy over the region of interest with a dental drill. 
	Note: Here, the barrel field of the primary somatosensory cortex is targeted ( 7 - 8 mm anterior to the interaural line, 4.5 - 5.5 mm lateral to the midline 31). Rinse repeatedly to dissipate heat.</li>
	<li>Use extra fine forceps to gently make a small hole in the dura. Reserve a ~0.5 mm region within the cranial trepanation to place the ECoG electrode (see next step). Permanently keep the cortex moist with 0.9% NaCl solution (or artificial cerebro-spinal fluid).</li>
	<li>Place a low impedance (~60 k&#937;) silver electrode (the ECoG electrode) on the dura, avoiding the cortical region not covered by the meninges, and place the reference electrode on a scalp muscle on the other side of the head.</li>
	<li>At this stage (30 min after the last sodium pentobarbital injection), maintain the anesthesia by repeated injections of sodium pentobarbital (10-15 mg/kg/h) or fentanyl (3-6 &#181;g/kg/h) via the IP catheter. The former will result in a slow oscillatory, sleep-like, ECoG pattern whereas the latter will result in a desynchronized, waking-like, cortical profile.</li>
	<li>Adjust the artificial ventilation system so that the respiratory frequency and volume are similar to those of the rat&#39;s spontaneous breathing (normal range 70 - 115 breath/min 32). Then, connect the mechanical ventilation to the tracheal tube and verify the proper thoracic cage inflation (on both sides). If not, adjust the position of the ventilation tubing in the tracheal axis. If necessary, suck up the secretion present in the trachea with a catheter connected to a syringe or a vacuum pump.</li>
	<li>If all physiological variables 32&#8211;35 and ECoG 29,36 patterns reflect a stable surgical plane of anesthesia, do an intramuscular injection of gallamine triethiodide in each leg to paralyze the rat, 40 mg/kg for the first injection and then 20 mg/kg, every 2hr&#160;19,29,36,50.</li>
</ol>
2. Intracellular Recordings
<ol>
	<li>Pull a glass micropipette (sharp microelectrode) with a ~0.2 &#181;m tip such as its resistance ranges between 50 and 80 M&#8486; once filled with 2 M potassium acetate (KAc).</li>
	<li>Place the pipette in a specific holder with a silver/silver-chloride (Ag/AgCl) wire to connect the pipette solution to an intracellular amplifier (via a head stage). The holder should be attached to a micromanipulator. Place a Ag/AgCl reference electrode on the rat&#39;s neck muscles.</li>
	<li>Slowly insert the pipette in the brain down to the region of interest and verify its resistance by monitoring the voltage drops in response to current steps. Use the buzz (or zap) button of the amplifier to clear the pipette if needed.</li>
	<li>Use cotton swabs or synthetic absorption triangles to dry the craniotomy (be careful not to touch the ECoG or intracellular electrodes) before covering it with silicone elastomer or 4% agarose to reduce brain movements.</li>
	<li>Lower the pipette in 1 - 2 &#181;m steps until its resistance increases when approaching a cell. Then use the buzz function of the amplifier to penetrate into the neuron.</li>
</ol>
3. Induce the Isoelectric State
<ol>
	<li>Perform the appropriate experimental protocol (e.g., firing responses to intracellularly injected currents, current-voltage relations, sensory stimuli responses) at this stage while monitoring simultaneously the neuron&#39;s membrane potential, the ECoG and the physiological variables.</li>
	<li>Ensure that the intracellular electrode is stable in the cell by analyzing the steadiness of both the membrane potential and the action potential properties throughout the recording session. If not, do not go to the next step and wait until the recording becomes stable again or search for another neuron.</li>
	<li>Inject a high but infra-lethal dose of sodium pentobarbital (~90 mg/kg, can be as low as 35 mg/kg from the pentobarbital initial condition and up to 155 mg/kg from the fentanyl initial condition) via the IP line. 
	Note: Within 15 - 20 min&#160;the intracellular and ECoG waveforms should slow down with intermittent electrical silences to transiently reach the so called &#34;burst-suppression&#34; profile 37,38, which progressively collapses to a complete isoelectric state. It is expected that the heart rate significantly slows down (by ~10 - 20%) but the SpO2 and EtCO2 should stay relatively steady.</li>
	<li>If the isoelectric state is not reached, inject a small amount of sodium pentobarbital (~10% of the step 3.1 dose). Wait 15 min&#160;before adding more anesthetic if the isoelectric state is still not achieved.</li>
	<li>Repeat the experimental protocol to compare the impact of the network dynamics on the recorded neuron&#39;s integrative properties. 
	Note: After cessation of anesthetic injections, the electrical brain activity should fully recover within 3 to 4 hr.</li>
	<li>At the end of the experiment, inject a lethal dose of sodium pentobarbital (200 mg/kg, IP) to euthanize the rat.</li>
</ol>

The authors have nothing to disclose.

We describe here a new method to suppress in vivo spontaneous cerebral electrical activity at both network and cellular levels. This procedure leads to an extreme brain state, known as isoelectric comatose 41. From a clinical point of view, such an electrocerebral inactivity is the most severe abnormality that can be seen on the EEG. It is mostly associated with an irreversible coma, with all patients either dying or continuing in a persistent vegetative state 42, but can be at least partia...

In the absence of any environmental stimuli or behavioral tasks, the &#34;resting&#34; brain generates a continuous stream of electrical activity that can be recorded from the scalp, as electroencephalographic (EEG) waves. The intracellular correlate of this endogenous cerebral activity is characterized by background membrane voltage fluctuations (also known as &#34;synaptic noise&#34;), which are composed of a combination of excitatory and inhibitory synaptic potentials that reflect the ongoing activity of afferent networks 1,2. This spontaneous activity varies in frequency and amplitude with the different states of vigilance. Elucidating the impact of network activity on the excitability and responsiveness of single neurons is one of the major challenges of neurosciences 3,4.
Many experimental and computational studies have explored the functional impact of ongoing synaptic activity on the integrative properties of neurons. However, the role of the different neuronal parameters impacted by the background synaptic noise remains elusive. For instance, the mean level of membrane depolarization has been found positively 5,6 or negatively 7-9 correlated with the ability of sensory inputs to trigger action potentials. Moreover, whereas some investigations suggest that fluctuations of the membrane potential, resulting from a continuously varying stream of afferent synaptic inputs, strongly affect the responsiveness of single neurons by modulating the gain of their input-output relationship 3,10-13, others indicate that changes in membrane input conductance mediated by shunting inhibition are sufficient to modulate the neuronal gain regardless of the magnitude of membrane fluctuations 14,15. Finally, recent studies performed on awake animals stressed how the processing of sensory information in single neuron critically depends upon the state of vigilance and the current behavioral demand 16,17.
A straightforward strategy to elucidate the functional role of a given process in a highly interconnected system is to determine how its absence specifically alters the functioning of the system. This method has been extensively used in neuroscience research, for example using experimental lesions or inactivation of different brain areas 18-21, or pharmacological blockade of specific ion channels 22,23. Notably, it has been applied in vivo to unveil how functional connectivity and network dynamics affect single cell computation 24-27. However, to date local manipulations intended to block the firing of neurons and/or perturb their basic biophysical properties can be partially effective and are limited to relatively small brain volumes 28.
To overcome these limitations, we developed a new in vivo experimental approach in the rat to compare the electrophysiological properties of single neurons recorded in a given brain state, i.e., embedded in a particular network dynamic, to those obtained after complete suppression of the whole brain synaptic activity 29. In the control conditions, two distinct cortical dynamics could be generated. Sleep-like electrocorticographic (ECoG) patterns were induced by injection of moderate doses of sodium pentobarbital. Alternatively, fast ECoG waves of small amplitude comparable to the cortical activity underlying the waking state (waking-like pattern) could be produced by injection of fentanyl. Subsequently, while maintaining the same ECoG and intracellular recording, a complete silencing of endogenous brain electrical activity was obtained by systemic injection of a high dose of sodium pentobarbital, characterized by isoelectric ECoG and intracellular activities. Because the induction of such an extreme comatose could potentially have fatal consequences on biological functions, a careful and continuous monitoring of the physiological variables was essential. Therefore, we meticulously followed the heart beat frequency, the end-tidal CO2 concentration (EtCO2), the O2 saturation (SpO2) and core temperature of the rat throughout the experiments.
We evaluate single neurons properties during these different states using sharp microelectrodes, which are particularly suited for long and stable recordings in vivo. The procedure described here, can be combined with other electrophysiological and imaging approaches and could be extended to other animal models.

<table><tbody><tr><td>Sodium Pentobarbital</td><td>Centravet</td><td>Pentobarbital</td><td></td></tr><tr><td>Ketamine 500</td><td>Merial</td><td>Imalg&amp;egrave;ne 500</td><td></td></tr><tr><td>Fentanyl&amp;nbsp;</td><td>Janssen-Cilag</td><td>Fentanyl</td><td></td></tr><tr><td>Xylocaine</td><td>Centravet</td><td>Xylovet</td><td></td></tr><tr><td>Gallamine triethiodide</td><td>Sigma</td><td>G8134</td><td></td></tr><tr><td>ECoG amplifier</td><td>A-M Systems</td><td>AC amplifier, Model 1700</td><td></td></tr><tr><td>Intracellular amplifier</td><td>Molecular Devices</td><td>Axoclamp 900A</td><td></td></tr><tr><td>Data acquisition interface</td><td>Cambridge Electronic Design</td><td>CED power 1401-3&amp;nbsp;</td><td></td></tr><tr><td>Data analysis software</td><td>Cambridge Electronic Design</td><td>Spike2 version 7</td><td></td></tr><tr><td>micromanipulator</td><td>Scientifica</td><td>IVM-3000</td><td></td></tr><tr><td>Capillary Puller</td><td>Narishige</td><td>PE-2</td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Harvard Apparatus</td><td>GC150F-10</td><td></td></tr><tr><td>Silver wire 0.125 mm (intracellular recording)</td><td>WPI</td><td>AGT0525</td><td></td></tr><tr><td>Ag-AgCl reference</td><td>Phymep</td><td>E242</td><td></td></tr><tr><td>Silver wire 0.25 mm (ECoG recording)</td><td>WPI</td><td>AGT1025</td><td></td></tr><tr><td>Artificial respiration system</td><td>Minerve</td><td>Alpha Lab</td><td></td></tr><tr><td>Physiological parameters monitoring</td><td>Digicare</td><td>LifeWindow Lite</td><td></td></tr><tr><td>Heating Blanket</td><td>Harvard Apparatus</td><td>507215</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>Scissors</td><td>FST</td><td>15005-08</td><td></td></tr><tr><td>Forceps Dumont #5</td><td>FST</td><td>11295-10</td><td></td></tr><tr><td>Forceps Dumont #5SF</td><td>FST</td><td>11252-00</td><td></td></tr><tr><td>IP Polyurethane catheter - 0.43x0.69 mm&amp;nbsp;&amp;nbsp;</td><td>Instech</td><td>BTPU-027</td><td></td></tr><tr><td>Silicon elastomere</td><td>WPI</td><td>KWIK-CAST</td><td></td></tr><tr><td>Dental drill</td><td>NSK</td><td>Y1001151 and P496</td><td></td></tr><tr><td>Surgical glue</td><td>3M</td><td>vetbond</td><td></td></tr></tbody></table>

induction of an isoelectric brain state to investigate the impact of endogenous synaptic activity on neuronal excitability in vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Inducing and maintaining an isoelectric brain state is a delicate in vivo experimental procedure. It has been proved to be a powerful tool to directly study the impact of cortical network activity on neuronal excitability and transfer function 29. Figure 1 shows the multi-parameter monitoring, including ECoG and vital constants, of the animal&#39;s physiological state before (Figure 1A) and after (Figure 1B) induction ...

Watch this Scientific Journal Video about Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo at JoVE.com

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

induction-an-isoelectric-brain-state-to-investigate-impact-endogenous

Research

JoVE Journal

Neuroscience

8.0K Views.  Inserm U1127.  Deze procedure voert langdurige in vivo intracellulaire recordings uit één neuronen in fysiologisch relevante cerebrale staten en na volledige afschaffing van lopende elektrische activiteiten, waardoor een isoelektrisch hersentoestand. De fysiologische constanten van het dier worden zorgvuldig gecontroleerd tijdens de overgang naar de kunstmatige comateuze toestand. 

Video: Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

Mapping Cerebral Blood Flow and Electrical Fields: Electrode Placement in Mice

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Author Spotlight: Stimulation-Based Approach to Improve Cerebral Blood Flow in Alzheimer's Model 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

Inducing Long-Term Plasticity of Intrinsic Neuronal Excitability in Neurons of the Dorsal Lateral Geniculate Nucleus

The dorsal lateral geniculate nucleus (dLGN) has long been held to act as a basic relay for visual information traveling from the retina to cortical areas, but recent findings suggest largely underestimated functional plasticity of dLGN principal cells. However, the cellular mechanisms supporting these changes have not been fully explored. Here, we report a protocol to induce long-term potentiation of intrinsic neuronal excitability (LTP-IE) in dorsal dLGN relay cells from acute brain slices of young rats. Intrinsic plasticity is generally induced in parallel with synaptic plasticity. However, in dLGN neurons, LTP-IE is reliably induced by spiking activity at a frequency of 40 Hz for 10 min. LTP-IE in dLGN relay neurons is long-lasting as it can be followed up to 40 min after the induction protocol. In conclusion, the results of this study provide the first evidence for the induction of intrinsic plasticity in dLGN relay cells, thus further pointing to the role of thalamic neurons in activity-dependent visual plasticity.

Here, we describe a protocol for inducing long-term plasticity of neuronal intrinsic excitability in relay neurons from the dorsal lateral geniculate nucleus maintained in ex vivo brain slices.

The dorsal lateral geniculate nucleus (dLGN) has long been held to act as a basic relay for visual information traveling from the retina to cortical ...

In Vivo Recording of Intracellular Signals from a Single Neuron

Source: Altwegg-Boussac, T., et al. <a href="https://app.jove.com/v/53576">Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo.</a> J. Vis. Exp. (2016).
This video demonstrates the recording of electrical signals from single brain neurons using a fine-tipped glass micropipette and a specialized setup. The micropipette is carefully inserted into the rat&#39;s brain and is buzzed to penetrate the neuron to record internal neuronal electrical signals.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

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