The authors wish to thank Anna Binaschi, Paolo Roncon and Eleonora Palma for their contribution to manuscripts published in precedence.

All experimental procedures have been approved by the University of Ferrara Institutional Animal Care and Use Committee and by the Italian Ministry of Health (authorization: D.M. 246/2012-B) in accordance with guidelines outlined in the European Communities Council Directive of 24 November 1986 (86/609/EEC). This protocol is specifically adjusted for glutamate and aspartate determination in rat brain dialysates obtained under EEG control of microdialysis sessions in epileptic and non-epileptic rats. Many of the materials...

In this work, we show how a continuous video-EEG recording coupled with microdialysis can be performed in an experimental model of TLE. Video-EEG recording techniques are used to correctly diagnose the different phases of the disease progression in animals and the microdialysis technique is used to describe the changes in glutamate release that occur in time (no changes have been found for aspartate in a previously published study2). We strongly recommend the use of a single device/implant to perf...

To provide insight into the functional impairment of glutamate-mediated excitatory and GABAergic inhibitory neurotransmission resulting in spontaneous seizures in temporal lobe epilepsy (TLE),we systematically monitored extracellular concentrations of GABA1 and later the levels of glutamate and aspartate2 by microdialysis in the ventral hippocampus of rats at various time-points of the disease natural course, i.e., during development and progression of epilepsy. We took advantage of the TLE pilocarpine model in rats, which mimics the disease very accurately in terms of behavioral, electrophysiological and histopathological changes3,4 and we correlated the dialysate concentration of amino acids to its different phases: the acute phase after the epileptogenic insult, the latency phase, the time of the first spontaneous seizure and the chronic phass5,6,7. Framing the disease phases was enabled by long-term video-EEG monitoring and the precise EEG and clinical characterization of spontaneous seizures. Application of the microdialysis technique associated with long-term video-EEG monitoring allowed us to propose mechanistic hypotheses for TLE neuropathology. In summary, the technique described in this manuscript allows the pairing of neurochemical alterations within a defined brain area with the development and progression of epilepsy in an animal model.
Paired devices, made up of a depth electrode juxtaposed to a microdialysis cannula, are often employed in epilepsy research studies where changes in neurotransmitters, their metabolites, or energy substrates should be correlated to neuronal activity.In the vast majority of cases, it is used in freely behaving animals, but it can be also conducted in a similar way in human beings, e.g., in pharmaco-resistant epileptic patients undergoing depth electrode investigation prior to surgery8. Both EEG recording, and dialysate collection may be performed separately (e.g., implanting the electrode in one hemisphere and the microdialysis probe in the other hemisphere or even performing the microdialysis in one group of animals while performing the sole EEG in another group of animals). However, coupling the electrodes to probes may have multiple advantages: it simplifies stereotaxic surgery, limits tissue damage to only one hemisphere (while leaving the other, intact, as a control for histological studies), and homogenizes the results as these are obtained from the same brain region and the same animal.
On the other hand, the preparation of the coupled microdialysis probe-electrode device requires skills and time if it is home-made. One could spend relatively high amounts of money if purchased from the market. Moreover, when microdialysis probes (probe tips are typically 200-400 &#181;m in diameter and 7-12 mm long)9, and EEG electrodes (electrode tips are usually of 300-500 &#181;m in diameter, and long enough to reach the brain structure of interest10) are coupled, the mounted device represents a bulky and relatively heavy object on one side of the head, which is troublesome for animals and prone to be lost especially when it is connected to the dialysis pump and the hard-wire EEG recording system. This aspect is more relevant in epileptic animals that are difficult to handle and less adaptive to the microdialysis sessions. Proper surgical techniques and appropriate post-operative care can result in long-lasting implants that cause minimal animal discomfort and should be pursued for combinatory microdialysis-EEG experiments10,11,12.
The advantages and limitations of the microdialysis technique have been reviewed in detail by many neuroscientists. Its primary advantage over other in vivo perfusion techniques (e.g., fast flow push-pull or cortical cup perfusion) is a small diameter of the probe which covers a relatively precise area of interest13,14,15. Second, the microdialysis membrane creates a physical barrier between the tissue and the perfusate; therefore, high-molecular weight substances do not cross and do not interfere with the analysis16,17. Moreover, the tissue is protected from the turbulent flow of the perfusate18. Another important advantage is the possibility to modify the perfusate flow for maximizing the analyte concentration in the perfusate (i.e., the process of microdialysis can be well defined mathematically and can be modified to yield high concentrations of the analyte in the sample)19. Finally, the technique may be used to infuse the drugs or pharmacologically active substances into the tissue of interest and to determine their effect at the site of intervention20. On the other hand, microdialysis has a limited resolution time (typically more than 1 min due to the time needed for collecting samples) in comparison to electrochemical or biological sensors; it is an invasive technique that causes tissue damage; it compromises the neurochemical balance within the space around the membrane due to the continuous concentration gradient of all soluble substances which enters the perfusate together with the analyte of interest. Finally, the microdialysis technique is highly influenced by the limits of the analytical techniques employed for the quantification of substances in the perfusate9,21,22,23. The high-performance liquid chromatography (HPLC) after derivatization with orthophthaldialdehyde for glutamate and aspartate analysis in biological samples has been well validated24,25,26,27 and its extensive discussion is out of the scope of this manuscript, but the data produced by using this method will be described in detail.
When performed properly and without modifications of the perfusate composition, microdialysis can provide reliable information about the basal levels of neurotransmitter release. The largest portion of the basal levels is likely the result of the transmitter spillover from the synapses9. Because in many instances the simple sampling of the neurotransmitter in the extra synaptic space is not sufficient to pursue the goals of an investigation, the microdialysis technique can be also employed to stimulate neurons or to deprive them of important physiological ions such as K+ or Ca2+, in order to evoke or prevent the release of the neurotransmitter.
High K+ stimulation is often used in neurobiology to stimulate neuronal activity not only in awake animals but also in primary and organotypic cultures. The exposure of a healthy central nervous system to solutions with high concentrations of K+ (40-100 mM) evokes the efflux of neurotransmitters28. This ability of neurons to provide an additional release in response to high K+ may be compromised in epileptic animals1 and in other neurodegenerative diseases29,30. Similarly, the Ca2+ deprivation (obtained by perfusing Ca2+ free solutions) is used to establish calcium-dependent release of most neurotransmitters measured by microdialysis. It is generally believed that Ca2+ dependent release is of neuronal origin, whereas Ca2+ independent release originates from glia, but many studies raised controversy over the meaning of Ca2+-sensitive measurements of e.g. glutamate or GABA9: thus, if possible, it is advisable to support microdialysis studies with microsensor studies, as these latter have higher spatial resolution and the electrodes allows to get closer to synapses31.
Regarding microdialysis studies in epileptic animals, it is important to stress that the data obtained from most of them rely upon video or video-EEG monitoring of seizures, i.e., of the transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain32. There are some specifics of electrographic seizures in pilocarpine treated animals which should be considered when preparing the experiment. Spontaneous seizures are followed by depressed activity with frequent EEG interictal spikes3 and occur in clusters33,34. Sham operated non-epileptic animals may exhibit seizure-like activity35 and therefore the parameters for EEG recordings evaluation should be standardized36 and, if possible, the timing of microdialysis sessions should be well defined. Finally, we highly recommend following the principles and methodological standards for video-EEG monitoring in control adult rodents outlined by experts of International League Against Epilepsy and American Epilepsy Society in their very recent reports37,38.
Here, we describe microdialysis of glutamate and aspartate in parallel with the long-term video-EEG recordings in epileptic animals and their analysis in the dialysate by HPLC. We will emphasize the critical steps of the protocol that one should take care of for best result.

<table>
	<tbody>
		<tr>
			<td>3-channel two-twisted electrode</td>
			<td>Invivo1, Plastic One, Roanoke, Virginia, USA</td>
			<td>MS333/3-B/SPC</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>guide cannula</td>
			<td>Agn Tho&#39;s, Lindig&#246;, Sweden</td>
			<td>MAB 4.15.IC</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Resin KK2 Plastik</td>
			<td>Elettra Sport, Lecco, Italy</td>
			<td>KK2</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Super Attack gel Loctite</td>
			<td>Henkel Italia Srl, Milano, Italy</td>
			<td>2047420_71941</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Imalgene-Ketamine</td>
			<td>Merial, Toulouse, France</td>
			<td>221300288 (AIC)</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma, Milano, Italy</td>
			<td>X1251</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Isoflurane-Vet</td>
			<td>Merial, Toulouse, France</td>
			<td>103120022 (AIC)</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Altadol 50 mg/ ml - tramadol</td>
			<td>Formevet, Milano, Italy</td>
			<td>103703017 (AIC)</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Gentalyn 0.1% crm - gentamycine</td>
			<td>MSD Italia, Roma, Italy</td>
			<td>20891077 (AIC)</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>simplex rapid dental cement</td>
			<td>Kemdent, Associated Dental Products Ltd, Swindon, United Kingdom</td>
			<td>ACR811</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>GlasIonomer CX-Plus Cement</td>
			<td>Shofu, Kyoto, Japan</td>
			<td>PN1167</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>probe clip holder</td>
			<td>Agn Tho&#39;s, Lindig&#246;, Sweden</td>
			<td>p/n 100 5001</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Histoacryl&#174; Blue Topical Skin Adhesive</td>
			<td>TissueSeal, Ann Arbor, Michigan, USA</td>
			<td>TS1050044FP</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Valium 10 mg/2 ml - diazepam</td>
			<td>Roche, Monza, Italy</td>
			<td>019995063 (AIC)</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>1 mL syringe with 25G needle</td>
			<td>Vetrotecnica, Padova, Italy</td>
			<td>11.3500.05</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>rat flexible feeding needle 17G</td>
			<td>Agn Tho&#39;s, Lindig&#246; Sweden</td>
			<td>7206</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Grass Technology apparatus</td>
			<td>Grass Technologies, Natus Neurology Incorporated, Pleasanton, California, USA</td>
			<td>M665G08</td>
			<td>Equipment (AS40 amplifier, head box, interconnecting cables, telefactor model RPSA S40)</td>
		</tr>
		<tr>
			<td>modular data acquisition and&#160;analysis system MP150</td>
			<td>Biopac, Goleta, California, USA</td>
			<td>MP150WSW</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>digital video surveillance system</td>
			<td>AverMedia Technologies, Fremont, California, USA</td>
			<td>V4.7.0041FD</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>microdialysis probe</td>
			<td>Agn Tho&#39;s, Lindig&#246; Sweden</td>
			<td>MAB 4.15.1.Cu</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>microdialysis probe</td>
			<td>Synaptech, Colorado Springs, Colorado, USA</td>
			<td>S-8010</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>block heater</td>
			<td>Grant Instruments, Cambridge, England</td>
			<td>QBD2</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>stirrer</td>
			<td>Cecchinato A, Aparecchi Scientifici, Mestre, Italy</td>
			<td>711</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>infusion pump</td>
			<td>Univentor, Zejtun, Malta</td>
			<td>864</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>fine bore polythene tubing</td>
			<td>Smiths Medical International Ltd., Keene, New Hampshire, USA</td>
			<td>800/100/100/100</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>blue tubing adapters</td>
			<td>Agn Tho&#39;s, Lindig&#246; Sweden</td>
			<td>1002</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>red tubing adapters</td>
			<td>Agn Tho&#39;s, Lindig&#246; Sweden</td>
			<td>1003</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>2.5 mL syringe with 22G needle</td>
			<td>Chemil, Padova, Italy</td>
			<td>S02G22</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>vial cap</td>
			<td>Cronus, Labicom, Olomouc, Czech Republic</td>
			<td>VCA-1004TB-100</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>septum</td>
			<td>Thermo Scientific, Rockwoood, Tennessee, USA</td>
			<td>National C4013-60 8 mm TEF/SIL septum</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>glass insert with bottom spring</td>
			<td>Supelco, Sigma, Milano, Italy</td>
			<td>27400-U</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>autosampler vial</td>
			<td>National Scientific, Thermo Fisher Scientific, Monza, Italy</td>
			<td>C4013-2</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Smartline manager 5000 system controller and degasser unit</td>
			<td>Knauer, Berlin, Germany</td>
			<td>V7602</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Smartline 1000 quaternary gradient pump</td>
			<td>Knauer, Berlin, Germany</td>
			<td>V7603</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>spectrofluorometric detector</td>
			<td>Shimadzu, Kyoto, Japan</td>
			<td>RF-551</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>chromatogrphic column</td>
			<td>Knauer, Berlin, Germany</td>
			<td>25EK181EBJ</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>chromatogrphic pre-column</td>
			<td>Knauer, Berlin, Germany</td>
			<td>P5DK181EBJ</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>mobile phase solution A</td>
			<td>0.1 M sodium phosphate buffer, pH 6.0</td>
			<td></td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>mobile phase solution B</td>
			<td>40% 0.1 M sodium phosphate buffer, 30% methanol, 30% acetonitrile, pH 6.5</td>
			<td></td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Ringer solution</td>
			<td>composition in mM: MgCl2 0.85, KCl 2.7, NaCl 148, CaCl2 1.2, 0.3% BSA</td>
			<td></td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>modified Ringer solution</td>
			<td>composition in mM: MgCl2 0.85, KCl 100, NaCl 50.7, CaCl2 1.2, 0.3% BSA</td>
			<td></td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>saline</td>
			<td>0.9% NaCl, ph adjusted to 7.0</td>
			<td></td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>sucrose solution</td>
			<td>10% sucrose in distilled water</td>
			<td></td>
			<td>Solution</td>
		</tr>
	</tbody>
</table>

microdialysis of excitatory amino acids during eeg recordings in freely moving rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Probe recovery
The mean recovery (i.e., the mean amino acid content in the perfusate as a percentage of the content in an equal volume of the vial solution) was 15.49 &#177; 0.42% at a flow rate of 2 &#956;L/min and 6.32 &#177; 0.64 at 3 &#956;L/min for glutamate and 14.89 &#177; 0.36% at a flow rate of 2 &#956;L/min and 10.13 &#177; 0.51 at 3 &#956;L/min for aspartate when using the cuprophane membrane probe. If using the...

Watch this Scientific Journal Video about Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats at JoVE.com

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

This method can help answer key questions in the neurosciences such as the alterations in neurochemistry during the natural history of neurological diseases. The main advantage of proposed microdialysis EEG technique is that such combined approach allows the pairing of changes in the neurotransmitter release within specific stages of the disease development and progression. Begin by switching on the amplifier positioned outside of the Faraday Cage.Open the EEG software and start the EEG acquisition and observe the EEG signal produced by unconnected cables. Next, connect the animal to the tethered EEG recording system by holding the animal's head between two stretched fingers of one hand and screwing down the connectors to the electrode pedestal using the other free hand. Set an amplification factor on each channel of the amplifier according to the electrode signal of a single animal so the EEG signal is in scale.Then let the animal explore the new cage for at least one hour under the direct observation of the researcher. Adjust the cable according to the animal's commodity, and ensure that the cables do not interfere with the animal's movements and lying posture. Finally, check for the correct image framing on the video cameras and then start the video EEG recording.Begin by preparing the microdialysis probes for the first use according to the manufacturer user's guide and fill them with Ringer's Solution. Then, cut 10 centimeter long pieces of FEP Tubing and connect them to the inlet and outlet cannulas of the probe using the tubing adapters of different colors. Ensure that tubing touches the adapters with no dead space in all connections.Remove the dummy cannula from its guide using the tweezers by holding the animal's head firmly. Insert the microdialysis probe into the guide cannula and use modeling clay to further firm up the cannula. Next, connect the animal to the tethered EEG recording system.Then put the animal into the plexiglass cylinder and let it explore the new environment. Follow the awake and freely moving rat movements. Then, connect the inlet of the probe to the 2.5 milliliter syringe with a blunted 22 gauge needle containing Ringer's Solution using the tubing adapters.Push one milliliter of Ringer's Solution into the probe in 10 seconds by pushing the piston of the 2.5 milliliter syringe continuously. Check for the drop of the liquid appearing on the outlet to signify when the probe is ready for use. Finally, fill up the 2.5 milliliter syringes connected to FEP tubing by tubing adapters with Ringer's Solution and mount them onto the infusion pump.Start the pump at two microliters per minute and let it run overnight. After verifying via video EEG recordings the absence of seizures in the three hours preceding sample collection, stop the pump carrying the FEP tubing cannulated syringes filled up with Ringer's Solution. Mount another set of 2.5 milliliter syringes connected to FEP tubing with tubing adapters filled up with a modified Ringer's Solution containing 100 millimolar potassium solution.Start the pump at two microliters per minute and let it run. Check for the absence of air bubbles in the system and ensure that the tubing touches the adapters with no dead space in all connections. Then, check for the drop of the liquid appearing on the outlet to signify when the probe is ready for use.Next, connect the FEP tubing of syringes filled up with Ringer's Solution to the inlet cannula of the probe in each animal and wait for the appearance of the liquid drop on the tip of the outlet. Connect the outlet of the probe to the FEP tubing which leads to collection in the test tube. Insert the FEP tubing into the closed 0.2 milliliter test tube with a perforated cap and ensure that the tube stays in place by fixing it with a piece of modeling clay.After running the pump at two microliters per minute for 60 minutes without collecting samples to equilibrate the system, collect five consecutive 30 minute dialysate samples under baseline conditions, and store samples on ice. After 10 minutes, switch the tubing from the syringes containing 100 millimolar potassium Ringer's Solution to normal Ringer's Solution and let the pump run. After collection of the fifth post-equilibration dialysate, collect 20 microliter dialysate fractions every 10 minutes for one hour.Then collect three additional 30 minute dialysate samples and stop the pump. Lastly, store the samples at minus 80 degrees Celsius after the experiment until HPLC analysis. After the experiment is complete, rinse the used microdialysis probes with distilled water and store them in a vial filled with clean distilled water until next use.Finally, rinse the entire microdialysis setup with distilled water followed by 70 per cent ethanol. Replace ethanol with air, and store the setup in a sterile environment. These are the representative EEG traces recorded from the hippocampus of control and epileptic rats during the chronic phase of the disease.No EEG alteration was observed in control rats, whereas a synchronized paroxysmal epileptiform activity was observed about 500 milliseconds before and during the behavioral seizures in epileptic rats. Basal glutamate concentration found in chronic epileptic rats was significantly higher than in control animals. High potassium evoked an additional release of glutamate for about 30 minutes in control rats and for about 60 minutes in chronic epileptic rats.While attempting this procedure, it is important to remember that animals implanted with the device are prone to lose it over a course of prolonged experiment, so use a light head set and sufficiently long cables to avoid in interfering with animal mobility. Depending on the availability of an appropriate analytical essay, the method may be further used to test different soluble molecules in the brain when employing EEG recording at the same time. After its development, this technique allowed the researchers in the field of epileptology to provide an insight into the alterations in excitatory neurotransmission in the case of a temporal lobe epilepsy.After watching this video, we trust you will have a better understanding of how to perform in vivo microdialysis together with depth electrode EEG recording in a freely moving epileptic rat.

microdialysis-excitatory-amino-acids-during-eeg-recordings-freely

Research

JoVE Journal

Neuroscience

11.1K vistas.  University of Ferrara and National Institute of Neuroscience. Este método puede ayudar a responder preguntas clave en las neurociencias como las alteraciones en la neuroquímica durante la historia natural de las enfermedades neurológicas. La principal ventaja de la técnica de EEG de microdiálisis propuesta es que este enfoque combinado permite el emparejamiento de cambios en la liberación de neurotransmisores dentro de etapas específicas del desarrollo y progresión de la enfermedad. Comience encendiendo el amplificador situado fuera de la jaula ...