Name: microdialysis of excitatory amino acids during eeg recordings in freely moving rats
Uploaded: 2018-11-08T13:30:00.000+00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats at JoVE.com

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

The authors have nothing to disclose.

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 perform them both in each animal for the reasons discussed in the Introduction.
Whenever available, radiotelemetry should be preferred to tethered systems for chronic EEG recording as it minimizes interferences with behavior and reduces harm risk and distress for the animals54. However, the tethered EEG recording is much less expensive than telemetry.
In our laboratory, we use the connectors to the EEG recording system and microdialysis tubing in parallel, such that wires and tubings are attached to two different swivels. This is the most critical issue for these experiments: the wires and tubing tend to cross frequently due to the animal&#39;s movements. Therefore, we use connectors and tubing long enough to let the animal chase its own tail (a behavior that is typically observed with potassium stimulation) or fall down and roll during generalized epileptic seizures. It is advisable to firm further the microdialysis cannulas inserted in their guide by modeling clay, in order to strengthen their contact with the guide (sometimes, microdialysis cannulas are bumped against the walls of the cage during generalized seizures and may slip off). On the other hand, it is advisable to keep the tubing as short as possible, to minimize the delay between neurochemical time and collection time. This is particularly important when collection periods are short. In general, the microdialysis tubing should be of adequate length and capacity to ensure that the sampling time does not exceed the time between dialysis outlet and collection. It was observed that the solutes tend to diffuse more between some plugs if the tubing dead time is superior to the sampling rate55. Therefore, the experimental dead time/volume of microdialysis tubing should be reduced as much as possible and determined very precisely in order to correlate the neurochemistry data with the animal&#39;s behavior. Finally, it is important to note that both swivels and electrodes coupled with cannula for combined EEG and microdialysis studies are commercially available. Therefore, whenever possible, set up the EEG system with the option to perform the microdialysis experiments.
The minor recommendations are: (i) before beginning any experiment, check that the EEG recording system and/or microdialysis set up are functioning properly and troubleshoot any problem; we suggest that having one reserve set up ready (another pump with syringes mounted on and completed of tubing filled up with working solutions) when performing the experiment, as well as a sufficient number of ready to use microdialysis probes for changing broken ones; (ii) when transferring animal into the working EEG-microdialysis cage it is helpful to have a second person assisting and starting the acquisitions; (iii) make sure that the column and autosampler reached the appropriate temperatures before chromatography; in addition, use standards and construct the calibration plots before any dialysate samples are injected on the chromatographic column; (v) whenever needed, try to develop the chromatographic or other analytical method to measure multiple analytes at the same time.
Alterations in neurotransmission have implications in many CNS disorders (including epilepsy) and there has been a great interest over the decades to quantify these changes during the progression from a healthy to a diseased phenotype. Today, only a few techniques allow the measurement of changes in neurotransmitter levels over days or months. Microdialysis is one of these techniques. In a large number of cases, like that described here, it is performed in freely moving animals and coupled to conventional offline analytical assays like high performance liquid chromatography (HPLC) or capillary electrophoresis (CE), with which it reaches 5-30 min temporal resolution31,56. Clearly, these sampling intervals do not reflect the rapid neurotransmitter dynamics in the vicinity of synapses, but may be convenient for some long term microdialysis applications (e.g., disease development or drug effect studies) which require coupling neurochemical, EEG and behavioral data. However, other studies are primarily concerned with measuring real-time or close to real-time changes in neurotransmitter release. For these, the microdialysis technique must be refined to increase the speed of sampling (therefore decreasing sample volumes). Indeed, the classic microdialysis technique is often criticized for its poor temporal (minutes) and spatial resolution (the conventional probe is much larger than the synaptic cleft)9,21,56,57. However, it is the mass sensitivity of the analytical method coupled to microdialysis what determines the microdialysis time resolution (i.e., its resolution is equal to the time required to have enough sample to be detected by an analytical technique56). Thus, when the microdialysis produces tiny amounts of samples, the sensitivity of quantification techniques must be increased. To date, such improvements in temporal resolution of the microdialysis technique followed 3 different lines. One of these is represented by miniaturization of the columns and/or detection cells of classic HPLC methods; these are called UHPLC (ultra-performant HPLC) techniques and allow to achieve 1-10 min time resolution58,59,60. Another approach is to couple a classic HPLC to mass spectrometry (MS) or tandem (MS/MS) for multiplex analysis of neurotransmitters in brain dialysates. Combined HPLC-MS assays have an excellent sensitivity and reach about 1-5 min time resolution56,61,62,63. A third line of improvement exists in modifications of capillary electrophoresis (CE). If CE uses laser induced fluorescence detection (CE-LIFD), it enables the determination of submicromolar concentrations of various neurotransmitters in nanoliter fractions obtained every 5 min55,64,65 or even at 10 s intervals56. A clear advantage that emerges from UHPLCs or advanced CEs analytical approaches is that the sampling may be done in freely moving animals, not compromising experiments in which spontaneous behavior must be observed and analyzed. On the other hand, there are methods that permits the brain dialysate sampling at even hundred milliseconds temporal resolution (e.g., enzyme reactor based on-line assays or droplet collection of dialysate coupled to MS techniques), but these are typically used in restrained animals66 or under general anesthesia67,68,69, not allowing to couple microdialysis with behavioral studies.
When considering the second most important weakness of microdialysis, i.e., relatively low spatial resolution due to the membrane dimensions (often about 0.5 mm in diameter and 1-4 mm long), an alternative may be the microprobes developed with low-flow push-pull sampling. These probes consist of two silica capillaries (of 20 &#181;m ID and 200 &#181;m OD) fused side-by-side and sheathed with a polymeric tubing. During the experiment, these capillaries are perfused at very low flow rates, such that fluid is pulled out of one capillary and a sample is retrieved from the other at the same flow rate. Because the sampling occurs only at the probe tip, the spatial resolution is greater than with the probe for microdialysis70. Another possibility is to switch from miniaturized probes to microelectrode arrays (biosensors) for real-time neurotransmitter evaluation. Different electrochemical techniques (based principally on voltammetry or amperometry) permit analyte sampling very close to the synapse (micron scale) and in less than 1 s31,70,71. These devices can measure the concentration of multiple analytes from multiple brain regions. However, they also require some refinements, for example to avoid artifacts and a relatively rapid deterioration.
Considering the latest advances in in vivo neurochemical monitoring, it seems likely that the different transmitter sampling methods will be combined in one sensor in the near future. The work on microfabricated sampling probes has already started, and we believe that further progress in microfabrication technologies together with analytical advances will further facilitate in vivo neurochemical monitoring investigation. At this time, however, the conventional microdialysis correlated to EEG remains a valid method for many neuroscience applications.

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&#039;s, Lindig&amp;ouml;, 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&#039;s, Lindig&amp;ouml;, Sweden</td><td>p/n 100 5001</td><td>Equipment</td></tr><tr><td>Histoacryl&amp;reg; 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&#039;s, Lindig&amp;ouml; 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&amp;nbsp;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&#039;s, Lindig&amp;ouml; 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&#039;s, Lindig&amp;ouml; Sweden</td><td>1002</td><td>Material</td></tr><tr><td>red tubing adapters</td><td>Agn Tho&#039;s, Lindig&amp;ouml; 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: MgCl&lt;sub&gt;2&lt;/sub&gt; 0.85, KCl 2.7, NaCl 148, CaCl&lt;sub&gt;2 &lt;/sub&gt;1.2, 0.3% BSA</td><td></td><td>Solution</td></tr><tr><td>modified Ringer solution</td><td>composition in mM: MgCl&lt;sub&gt;2&lt;/sub&gt; 0.85, KCl 100, NaCl 50.7, CaCl&lt;sub&gt;2 &lt;/sub&gt;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 polyacrylonitrile membrane probe, the mean recovery was 13.67 &#177; 0.42% at a flow rate of 2 &#956;L/min and 6.55 &#177; 1.07 at 3 &#956;L/min for glutamate and 14.29 &#177; 0.62% at a flow rate of 2 &#956;L/min and 8.49 &#177; 0.15 at 3 &#956;L/min for aspartate (Figure 4A-4B). As it can be clearly seen in Figure 4A, the slower flow rate (2 &#956;L/min) enhances the dialyzing performance of both probes. For the following experiments the cuprophane membrane endowed probe perfused at a flow rate 2 &#956;L/min was chosen, because its mean recovery was higher (even if not significantly) at this flow rate for both analytes and because of experimental continuity (these probes were used for analyzing GABA in precedence1).
Seizures development and progression of the disease after status epilepticus
The behavioral and EEG monitoring of seizures, their evaluation, was done in all the animals employed in this study to confirm the development and progression of TLE disease in these.
The robust convulsive SE, that was interrupted after 3 h using diazepam, occurred 25&#177;5 min after the pilocarpine injection. Then, all the animals entered a latency state in which they were apparently well and they were continuously video-EEG monitored in order to verify that no spontaneous seizures occurred in the first 9 days or to identify the first spontaneous seizure, respectively for the latency and the first seizure group. The first spontaneous seizure occurred 11.3 &#177; 0.6 days after SE (mean &#177; SEM, n=21). Thereafter, seizures occurred in clusters, and aggravated in time. In late chronic phase (days 55-62 after SE) the epileptic rats experienced 3.3&#177;1.2 (mean &#177; SEM, n=12) generalized seizures daily. There was a clear progression of the disease. Many, but not all EEG seizures, corresponded with behavioral seizure activity. Figure 5B shows the recorded paroxysmal epileptiform activity that was observed about 500 ms before and during behavioral seizures. Figure 5A shows control traces in non- epileptic rats.
Representative basal values of amino acids found in microdialysis perfusate and potassium stimulated release of glutamate
Basal glutamate concentration found in chronic epileptic rats (0.87 &#177; 0.06 &#181;M) was significantly higher than in control animals (0.59 &#177; 0.03 &#181;M; p &#60; 0.05 vs. controls; one-way ANOVA and post hoc Dunnett&#39;s test). There was no statistically significant difference between chronic epileptic (0.31 &#177; 0.04 &#181;M) and non-epileptic animals (0.30 &#177; 0.05 &#181;M) in basal or high K+ evoked aspartate concentrations. See the original article for details2. The reported basal levels of glutamate in control rats are in line with those found by others in similar studies (i.e., about 0.75 &#181;M when using a 2 &#181;L/min flow and membranes of 2 mm effective length)46,47,48,49,50,51,52,53. However, many different factors can influence the results of microdialysis, for example the effective length of the probe and the membrane cut off.
High K+-evoked an additional release of glutamate for about 30 min in control rats and for about 60 min in chronic epileptic rats (Figure 6). See the original article for details2. As can be seen from the depicted time course, the 10 min time resolution of microdialysis was sufficient to capture the variances in glutamate release found in both groups of animals.
HPLC calibration and limits
The data were calculated based on calibration curves obtained with standard solutions of glutamate and aspartate and the internal standard L-homoserine. The concentration of the neurotransmitters glutamate and aspartate in the perfusates was expressed in absolute values (&#181;mol/L). Each calibration plot was constructed by analysis of solutions of glutamate and aspartate at four concentration levels (five replicates at each level).Regression coefficients were calculated for calibration plots: y = kx + q, where x was the concentration ratio of aspartate or glutamate to L-homoserine (IS) and y was the corresponding peak-area ratio of aspartate or glutamate to L- homoserine (IS). The coefficient of determination (r2) was calculated. The applicability of HPLC method was within the limits; the lower limit of quantification was determined as the lowest concentration in the standard calibration curve and the upper limit of quantification as the highest used concentration of amino acid analytes for calibration, respectively. Limit of detection (LOD) was also calculated. Some of these values are delineated in Table 1. A model chromatogram of blank sample, standards sample and collected dialysis sample obtained with above described method are shown in Figure 7.
Probe localization
Microdialysis probe and recording electrode were implanted into the right ventral hippocampus and their correct placement was verified. Only those animals where the implantation was maximally in 500 &#956;m distant from stabilized coordinates (see Figure 8) were included in analysis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58455/58455fig1.jpg" /> 
Figure 1. Step-by-step preparation of the device to be implanted. (A) 3-channel electrode with 10 cm long grounding electrode in its protective sleeve on the left and guide cannula for microdialysis on the right needed to assemble the device. (B). The bare electrode and guide cannula in detail. The first step is to remove (C) and insert (D) the metal guide cannula from and into its plastic dummy few times to ease its release once implanted into the animal&#39;s head. The second step is to bend two times the twisted registering electrode to be aligned to dialysis guide cannula (E, F). (G) The electrode tip should be cut to be 0.5 mm longer than the tip of the metal guide cannula. (H) Check for the precision of the cut using the digital caliper. Subsequently, about 1 mm long silicon circlet should be used to fix the alignment of electrode to guide cannula foot (I). (J) The photograph showing how to ring the electrode and guide cannula shaft. The final step is to put a drop of resin or glue onto the guide cannula pedestal fixing the silicon circlet to it (K). (L) Assembled device ready to be sterilized. <a href="https://www.jove.com/files/ftp_upload/58455/58455fig1large.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/58455/58455fig2.jpg" /> 
Figure 2. Photographs of different types of devices for microdialysis-EEG in rats used (A) and the photograph of the probe clip holder (B) used to implant these devices. (A) The guide cannula (in green) is replaced by a microdialysis cannula typically 24 h before the experiment. The electrical connector of the device (first left was used for the recordings described in this manuscript) permits the attachment of wires that conduct electrical signal to amplifier and data collection equipment. The device is surgically attached to the skull of anesthetized rats and recordings may be obtained later without causing pain or discomfort in freely behaving rats. <a href="https://www.jove.com/files/ftp_upload/58455/58455fig2large.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/58455/58455fig3.jpg" /> 
Figure 3. Experimental design. The week before status epilepticus (SE) induction, the rats are implanted with the device. SE is induced by pilocarpine and animals (if not dialyzed and killed at 24 h after SE; the rats from acute group) are video monitored for 5 days (blue line), then video-EEG monitored to assess the seizure frequency and duration in their home cages (green line). For the microdialysis experiment, the epileptic and respective non-epileptic control rats are transferred to another EEG set up equipped with cylinder cages in 24 h before the microdialysis session and still video-EEG monitored (light green line). The vertical red lines represent the dialysis sessions at different time-points of epileptic disease development. The horizontal red lines represent the different groups of epileptic animals (and respective non-epileptic animals), where the arrow indicates the last day of the microdialysis and the day of animal&#39;s death. <a href="https://www.jove.com/files/ftp_upload/58455/58455fig3large.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/58455/58455fig4.jpg" /> 
Figure 4. In vitro recovery of two dialysis probes. Mean in vitro recovery (%) of aspartate and glutamate using two different commercially available microdialysis probes (both endowed with 1 mm long dialyzing membrane) at (A) 2 &#956;L/min and (B) 3 &#181;L/min flow rate. Data are the mean &#177; SEM of 3 independent experiments run in triplicates. There are not statistically significant differences between the efficiency of various probes (Student&#39;s unpaired t-test, p&#60;0.05).Using a flow rate 2 &#956;L/min the glutamate recovery increased about 5% compared to 3 &#181;L/min flow rate, thus the slower flow rate was used for microdialysis experiments. <a href="https://www.jove.com/files/ftp_upload/58455/58455fig4large.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/58455/58455fig5.jpg" /> 
Figure 5. Illustrative EEG recordings from ventral hippocampus of paraoxystic activities as can be seen at chronic phase in control and epileptic rats. (A) Two representative traces recorded in two saline treated non-epileptic rats. (B) Traces recorded in two epileptic rats. Epileptiform discharges correspond with class 3 behavioral seizures in these rats. <a href="https://www.jove.com/files/ftp_upload/58455/58455fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58455/58455fig6.jpg" /> 
Figure 6. Time-course of the effect of potassium stimulation on glutamate release from the rat hippocampus. Representative result of the microdialysis experiment performed in 6 control (open circles) and 6 chronic epileptic rats (black circles). The graph shows the temporal changes of dialysate glutamate concentration in the course of microdialysis experiments and during high 100 mM K+ stimulation. The time of high K+ stimulus (10 min) is indicated by the black bar on bottom of the graph. The data are the means &#177; SEM of 6 animals per group. <a href="https://www.jove.com/files/ftp_upload/58455/58455fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58455/58455fig7.jpg" /> 
Figure 7. Illustration of chromatograms. Known peaks are labeled. Pink trace: chromatogram of Ringer&#39;s solution without intentionally added amines after OPA/5-ME derivatization (blank sample). Blue trace: chromatogram of dialysate sample after derivatization showing the peaks of amino acids: aspartate (tR 4.80 min), glutamate (tR 6.75 min) and glutamine (tR 9.19 min) and the peak of IS L-homoserine (2.5 &#181;M, retention time, tR 9.83 min). Red trace: Chromatogram of standard of aspartate (2.5 &#181;M) and glutamate (2.5 &#181;M) in Ringer&#39;s solution. Azure and yellow background of the picture stands for mobile phase A (azure) and mobile phase B (yellow) portion used for analytes elution. A red rectangle indicated area (tR 10.41 min and further) shows the peaks of unknown substances and OPA degradation products. All injection volumes were 20 &#181;L. The derivatives were separated at a flow rate of 0.8 mL/min. <a href="https://www.jove.com/files/ftp_upload/58455/58455fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58455/58455fig8.jpg" /> 
Figure 8. Representative image of combined electrode-probe placement within the ventral hippocampus. (A) Photograph shows the scare left by the device tip in detail (black arrow). (B) Schematic illustration of the electrode-probe tip positions within the implanted ventral hippocampus of 12 rats. The solid squares (some overlapping) indicate correctly localized probe-electrode tips. Open squares indicate incorrectly localized probe-electrode tips in animals excluded from the study (n=3). Coronal brain slices containing probes and recording sites were processed after experiments for histological analysis. The numbers above the illustration show the distance from Bregma (according to Pellegrino et al. 1979 atlas of rat brain; nose bar + 5.0 mm, co-ordinates used: A -3.4 mm, L+5.4 mm; P + 7.5 mm from dura). <a href="https://www.jove.com/files/ftp_upload/58455/58455fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analyte</td>
			<td>c (&#956;mol/l)</td>
			<td>k</td>
			<td>q</td>
			<td>r2</td>
			<td>LOD&#160; (pmol/l)</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>0.25-2.5</td>
			<td>5.215</td>
			<td>1043.79</td>
			<td>0.999</td>
			<td>19.4</td>
		</tr>
		<tr>
			<td>Aspartate</td>
			<td>0.25-2.5</td>
			<td>2.258</td>
			<td>1994.72</td>
			<td>0.998</td>
			<td>31.7</td>
		</tr>
	</tbody>
</table>
Table 1. Quantification characteristics of HPLC method used for amino acids determination. Concentration range of standards (c), slope (k), intercept (q), coefficient of determination (r2) and limit of detection (LOD) describing the calibration plots obtained with standard solutions of glutamate and aspartate (0.25, 0.5, 1.0 and 2.5 &#181;M) and internal standard L-homoserine (2.5 &#181;M) using the described HPLC method with spectrofluorometric detection.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.3K Views.  University of Ferrara and National Institute of Neuroscience. 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.

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

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Medicine

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

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) ...

Extracellular Electrophysiological Recording in the Motor Cortex of Mice

Multichannel Extracellular Recording in Freely Moving Mice

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

Author Spotlight: Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice 

The protocol describes the methodology of extracellular recording in the motor cortex (MC) to reveal extracellular electrophysiological properties in freely moving conscious mice, as well as the data analysis of local field potentials (LFPs) and spikes, which is useful for evaluating the network neural activity underlying behaviors of interest.

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by ...

Assessment of Excitatory Neurotransmitter Levels in Chronic Epilepsy-Induced Rats

<a href='https://app.jove.com/v/58455/microdialysis-excitatory-amino-acids-during-eeg-recordings-freely'>Source: Soukupov&#225;, M., et al. Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats. J. Vis. Exp. (2018)</a>This video demonstrates the assessment of extracellular excitatory neurotransmitter levels in rats with chronic epilepsy induction. After implanting a microdialysis probe into the brain and confirming the absence of seizures using electroencephalography (EEG), extracellular fluid is collected to evaluate elevated neurotransmitter levels in epileptic rats. A high-potassium solution is then infused to sustain neuronal excitability and trigger an increased release of neurotransmitters, with samples collected to assess the potassium-induced peak in excitatory neurotransmitters.

Source: Soukupov&#225;, M., et al. Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats. J. Vis. Exp. (2018)This video ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

Source: Haumesser, J. K., et al. <a href="https://app.jove.com/v/55940">Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats.</a> J. Vis. Exp.(2017).
This video demonstrates the detailed procedure for preparing an anesthetized rat for electrophysiological recordings of brain activity from multiple sites. The scalp is dissected to access the skull, and holes are drilled at precise locations to install and secure electrodes. Tungsten microwire electrodes are then inserted into the targeted location to record the data.

Elektrofizjologiczne zapisy in vivo aktywności mózgu z wielu miejsc u szczurów

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Neurophysiology

Electrophysiology Techniques

Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

Source: Soukupov&#225;, M., et al. <a href="https://app.jove.com/v/58455">Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.</a> J. Vis. Exp. (2018)
This video demonstrates electroencephalographic (EEG) recording of epileptic seizures in epilepsy-induced rats. A rat with electrodes implanted in the ventral hippocampus is connected to the EEG recording system and placed in a plexiglass chamber. The rat is allowed to move freely while the brain activity is recorded to monitor the occurrence of seizures.

Elektroencefalograficzny zapis napadów padaczkowych u szczurów z padaczką

Stimulation and Modulation Techniques

Concurrent Electroencephalographic and Local Field Potential Recordings in an Anesthetized Rat

In this procedure, clean and dry the skull surrounding the burr hole using a cotton swab. Carefully apply the conductive EEG paste on a flat side of an EEG spider electrode. Leave a small hole clear of EEG paste on the spider electrode to allow a multilaminar microelectrode to pass through the hole without contacting the paste and the spider electrode.
Align the spider electrode with the burr hole in the skull with the EEG paste facing the skull. Carefully press the spider electrode onto the skull, making firm contact with the skull via the EEG paste.
Remove any paste obscuring the burr hole using a needle on a syringe and remove excessive EEG paste beyond the periphery of the spider electrode, so that the contact between the spider electrode and the skull is spatially constrained to the size of the electrode. Then, smear EEG paste onto the reference electrode for the EEG, and place it securely inside the incision at the back of the rat&#39;s neck.
Next, connect the EEG electrodes to the pre-amplifier via a passive signal splitter for low-impedance signals. At this stage, test the resistance of the EEG probe to make sure it is below five kiloohms. If not, add more EEG paste and make sure the spider electrode makes a good contact with the skull.
Subsequently, mount a micromanipulator arm on the stereotactic frame. Connect a linear 16-channel microelectrode to a 16-channel acute head stage clipped securely onto the micromanipulator arm. Then, smear EEG paste onto the reference electrode for the microelectrode, and then place it securely inside the incision next to the reference electrode for the EEG. Adjust the angle of the micromanipulator arm so that the microelectrode is perpendicular to the cortical surface.
Now, lower the microelectrode under a microscope so that the tip of the microelectrode is aimed at the tiny opening at the bottom of the burr hole, until the uppermost electrode just penetrates the cortical surface. Care must be taken to avoid forcing the microelectrode onto the surface of the dura, as this would break the electrode.
Insert the microelectrode to the cortical surface at a depth of 1500 micrometers. Microadjust the depth by applying a train of stimulus to the whisker pad, and observing the 16-channel evoked LFP on a PC monitor. Carefully turn the z-axis knob on the micromanipulator until the highest amplitude of the evoked LFP occurs as this coincides with the layer 4 in the cortex.