Name: simultaneous monitoring of wireless electrophysiology and memory behavioral test as a tool to study hippocampal neurogenesis
Uploaded: 2020-08-20T13:00:00
Duration: 7 min 25 s
Description: Watch this Scientific Journal Video about Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis Video at JoVE.com

We want to thank Mr. Miguel Burgos, and Mr. Gustavo Lago for providing technical assistance. We are grateful to the Stoelting Co. for covering the video production costs, Jinga-hi, Inc. for providing technical assistance, and Divisi&#243;n de Investigaci&#243;n y Posgrado of the Universidad Iberoamericana Ciudad de M&#233;xico for granting funds to this work.

<ol>
	<li>H&#229;nell, A., Marklund, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structured+evaluation+of+rodent+behavioral+tests+used+in+drug+discovery+research.">Structured evaluation of rodent behavioral tests used in drug discovery research.</a> Frontiers in Behavioral Neuroscience. 8, 1-13 (2014).</li><li>Buenrostro-J&#225;uregui, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEXRAT+MALE:+A+smartphone+and+tablet+application+to+annotate+and+process+live+sexual+behavior+in+male+rodents.">SEXRAT MALE: A smartphone and tablet application to annotate and process live sexual behavior in male rodents.</a> Journal of Neuroscience Methods. 320, 9-15 (2019).</li><li>Jun, J. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erratum:+Neurogenesis+in+the+adult+is+involved+in+the+formation+of+trace+memories+(Nature+(2001)+410+(372-376)).">Erratum: Neurogenesis in the adult is involved in the formation of trace memories (Nature (2001) 410 (372-376)).</a> Nature. 414 (6866), 938 (2001).</li><li>Song, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+neurons+in+the+adult+mammalian+brain:+Synaptogenesis+and+functional+integration.">New neurons in the adult mammalian brain: Synaptogenesis and functional integration.</a> Journal of Neuroscience. 25 (45), 10366-10368 (2005).</li><li>Zhao, C., Teng, E. M., Summers, R. G., Ming, G. L., Gage, F. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-specific+functional+modification+of+the+dentate+gyrus+through+adult+neurogenesis:+A+critical+period+during+an+immature+stage.">Experience-specific functional modification of the dentate gyrus through adult neurogenesis: A critical period during an immature stage.</a> Journal of Neuroscience. 27 (12), 3252-3259 (2007).</li><li>Moreno-Jim&#233;nez, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+hippocampal+neurogenesis+is+abundant+in+neurologically+healthy+subjects+and+drops+sharply+in+patients+with+Alzheimer's+disease.">Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer's disease.</a> Nature Medicine. 25 (4), 554-560 (2019).</li><li>Cohen, S. J., Stackman, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+rodent+hippocampal+involvement+in+the+novel+object+recognition+task.+A+review.">Assessing rodent hippocampal involvement in the novel object recognition task. A review.</a> Behavioural Brain Research. 285, 105-117 (2015).</li><li>Fagan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+in+the+infant.">Memory in the infant.</a> Journal of Experimental Child Psychology. 9 (2), 217-226 (1970).</li><li>Baxter, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I've+seen+it+all+before"+Explaining+age-related+impairments+in+object+recognition.+Theoretical+comment+on+Burke+et+al.">I've seen it all before" Explaining age-related impairments in object recognition. Theoretical comment on Burke et al.</a> Behavioral Neuroscience. 124 (5), 706-709 (2010).</li><li>Antunes, M., Biala, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+object+recognition+memory:+Neurobiology,+test+procedure,+and+its+modifications.">The novel object recognition memory: Neurobiology, test procedure, and its modifications.</a> Cognitive Processing. 13 (2), 93-110 (2012).</li><li>Ennaceur, A., Delacour, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+one-trial+test+for+neurobiological+studies+of+memory+in+rats.+1:+Behavioral+data.">A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data.</a> Behavioural Brain Research. 31 (1), 47-59 (1988).</li><li>Winters, B. D., Forwood, S. E., Cowell, R. A., Saksida, L. M., Bussey, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+dissociation+between+the+effects+of+peri-postrhinal+cortex+and+hippocampal+lesions+on+tests+of+object+recognition+and+spatial+memory:+Heterogeneity+of+function+within+the+temporal+lobe.">Double dissociation between the effects of peri-postrhinal cortex and hippocampal lesions on tests of object recognition and spatial memory: Heterogeneity of function within the temporal lobe.</a> Journal of Neuroscience. 24 (26), 5901-5908 (2004).</li><li>Forwood, S. E., Winters, B. D., Bussey, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+lesions+that+abolish+spatial+maze+performance+spare+object+recognition+memory+at+delays+of+up+48+hours.">Hippocampal lesions that abolish spatial maze performance spare object recognition memory at delays of up 48 hours.</a> Hippocampus. 15 (3), 347-355 (2005).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rat brain in stereotaxic coordinates. , (1997).</li><li>. Getting started with ANY-maze Setting up and starting work with ANY-maze Available from: <a target="_blank" href="https://www.braintreesci.com/images/ANYMaze.pdf">https://www.braintreesci.com/images/ANYMaze.pdf</a> (2006)</li><li>. A detailed description of the ANY-maze measures Available from: <a target="_blank" href="https://www.braintreesci.com/images/ANYMaze.pdf">https://www.braintreesci.com/images/ANYMaze.pdf</a> (2010)</li></ol>

Dr. Sylvia Ortega-Martinez works as an employee of Stoelting Co., a company that provided and sponsored the production and open access to this article.

Behavioral and electroencephalography research is difficult and challenging by nature. Therefore, the combination of both techniques presents significant critical steps. Thus, both concurrent techniques are not widely used. In real practice, every group around the world performs behavioral tests with special conditions, such as animals, analyzed parameters, or treatments. The above creates significant controversies in the field and the need for developing standard procedures available to everybody. Here, we have prepared this detailed procedure with all the critical steps and methodological considerations that are not usually described or mentioned in most of the published articles. These are discussed below.
Production of the needed materials is a fundamental step in the success of this technique. In this regard, the electrode needs to be built from scratch using stainless steel screws, copper cables, and silver welder. These materials are difficult to weld together permanently, in such a manner that the conductivity and strength of each electrode must be verified before use. It is possible to use another type of wire for the electrode assembly; however, the copper is flexible enough to manipulate the electrode to insert it into the amplifier connector. In this regard, the use of commercial electrodes is desirable, but their acquisition could be complicated and expensive. The surgery is one of the most critical steps in this protocol. It is highly recommended and even necessary to have an experienced surgeon, particularly for electrodes implantation. Since the surgery frequently requires lengthening anesthesia time and sometimes a welding application during surgery, each laboratory must perform the necessary tests with the appropriate anesthesia (different cocktails can be used) for each strain of rodents, particularly under vivarium conditions, differences between litters, and even individual differences among animals. Proper planning and consideration could prevent losing animals during surgeries. The electrodes implantation is another crucial step. It requires great care to avoid punching the skull and damage meninges or brain tissue. Screws should be placed correctly, that is, completely fixed in the skull otherwise, noise and artifacts will be presented on signals, like those related to a lousy colocation or movement that does not use the EEG recording. Pre- and post-operative treatment and conditions must always be performed and observed to avoid the rodent&#8217;s suffering. Subcutaneous lidocaine can be used on the head skin before making the incision with the scalpel. A drop of saline to the animal&#8217;s eyes will help prevent dryness. Also, a saline solution must be administered in the mouth, and after the surgery, 1 mL must be administrated either subcutaneous or intraperitoneally to compensate the animal&#39;s fluid balance and prevent dehydration. Immediately after surgery, an anti-inflammatory medication (to reduce pain), as well as antibiotics via subcutaneous or topical antibiotics, must be administrated directly on the periphery of the scalp where the dental cement cap is located (to decrease the probability of infection). Repeat the above procedure 24 h after the surgery. The positioning of the EEG amplifier on the animal&#39;s back is the main difficulty for the simultaneous recording. The design and manufacture of a vest are specifically based on the animals&#8217; size. The vest must allow the natural movement of the rodent (see Figure 5). This latter will guarantee the main advantage of the technique, which is the recording of free movements. Since the animals did not attempt to remove the vest, the head connector, or cables after surgery and during subsequent days, it was presumed that the setup did not generate movement restriction significantly or caused pain or discomfort. For a correct EEG segmentation in epochs based on events marked by the BTS is mandatory to write down a well-defined protocol. The temporary marks could be merged by time series manipulation because both systems use the same clock to set up their timestamps. The above extends the possibilities for animal experimentation incorporating electrophysiological data for analysis.
The technique presented here can be used in any neuroscience research area and with the most common murine species and even other species. The versatility of the Behavioral Tracking Software is one of the most significant advantages since it could be used in a great versatility of mazes as Morris water maze, open-field, novel object recognition, conditioned place preference, hole board, elevated plus maze, Y-maze, radial arm maze, Barnes maze, and others. It can be used up to 16 cameras simultaneously. Additionally, hundreds of different measures (for more detailed information see the manuals31,32) can be reported. Consider that this work describes experimentation for EEG recordings, some other techniques like Local Fields Potentials or single-unit recording are possible. However, users must consider that the general setup and several preparatory steps need to change for other purposes. So, when this technique is used together with EEG Wi-Fi recording, the possibilities are extended, because it adds new perspectives to animal studies like those performed on human beings to evaluate several characteristics of the EEG integration and dynamics, like connectivity, EEG band power, or evoked responses. Unlike human beings, animal experimentation is possible to evaluate drug administration, gene modifications, or expression, among many other experimental paradigms. For EEG analysis, consider that some protocols have a very low number of repetitions of the desired behaviors, which restrains the possibility to average responses and obtain reliable results. Therefore, be careful to design the recording and analysis protocols that it is considered to perform before beginning the experiment. Nevertheless, it must be contemplated that working in animal experimentation is not possible to prevent movement, increasing the complexity of the experimental protocol and considerations for signal analysis and behavioral tasks. Currently, equipment for full tracking systems and EEG recordings are not standardized or modular, which means that their setup is intended to a single protocol and adaptations to explore other behavioral tasks, implying/suggesting higher costs to a large number of laboratories. This situation could be solved by following the options explained in this study. Nevertheless, several improvements could be realized for more reliable experiments. The work can be improved at several steps starting from electrodes fabrication through behavioral and signals processing. Nonetheless, it is demonstrated that animal tracking and EEG acquisition are possible using an affordable high-tech but inexpensive setup.
In summary, the present work is an attempt to help scientists, particularly in the neurosciences field, to be able to use these two techniques that are not commonly used in combination. The simultaneous recording technique of EEG and behavioral testing using Behavioral Tracking Software has many advantages, and it can be particularly useful in many fields of Neuroscience, particularly in learning and memory areas. Considering this equipment has other capabilities as a deep recording of subcortical structures as the hippocampus, but as mentioned, several preparatory steps will change. Wireless equipment solves almost all the limitations of a conventional wire approach, such as animals&#39; mobility problems from one cage to another, hindered or entangled animals with the cables. This setup technique is user-friendly, as described above, and an almost untrained or non-specialized group of experts or individuals can use this software. The price for the EEG equipment is lower than a regular EEG amplifier. Behavioral Tracking Software is also one of the most affordable software for video tracking in the market. This software requires annual licenses. The equipment can be used in more than one experimental setup, different animals, and the type of versatility. We hope that this effort will help the scientific community and provide an easy access to simultaneously study the behavior and electroencephalography.

Behavioral test is crucial in neuroscience research for a large amount of information generated in an in vivo context. In this regard, researchers have been widely using different behavioral tests to analyze sensory-motor function, social interactions, anxiety-like and depressive-like behavior, substance dependence and various forms of cognitive functions1. Manual recording of behavioral tests might be difficult, exhausting, and inaccurate even for most expert observers. Even though some efforts have been made to develop free and open-source software for behavior registration (e.g., sexrat male2 app for sexual behavior), several alternatives allow the automatic and real-time behavioral recording of different animal species from fish3 to rodents4,5,6. Video tracking is a valuable method for quick and accurate behavior recording used in a wide variety of applications7. A more potential feature in the behavioral recording area is to explore neural activity during the behavioral manifestation. Simultaneous recording of neuronal activity (from single cells to the major brain areas) and behavioral tasks could show us how the brain generates specific behavioral patterns8. Behaviors are a sequence of minor components that could reveal correlates between the neural activity and movements or actions. If neuronal activity and behavioral patterns could be simultaneously recorded through multiple timescales, they could explain how each brain state correlates to each particular behavior (for a more in-depth examination of behavioral recording, see Datta et al., 2019 review8). Therefore, synchronized recording of behavioral and neuronal activity at the desired scale (from neurons to large areas of the brain) is considered as an extremely useful tool. There are several systems intended to integrate behavioral recordings with other measurements as neural activity4,5.
Although electroencephalography is considered one of the most widely used techniques in the field of clinical and research neuroscience, the relatively high mobility, as well as the size of the EEG recording device, makes this technique unique and challenging for detection in case of in vivo models9. Some solutions to this problem have been developed e.g., the use of cables and swivel-devices that allow animals to move freely in the arena. Nevertheless, cable-based systems often impose problems to conduct studies, e.g., during the transfer of an animal from one cage to another, hindrance or entanglement of the animal with the cables is observed. Telemetric devices have been developed for wireless electrophysiological recordings to increase the flexibility of the recording situation10,11. However, such systems have shown considerable limitations due to their low number of recording channels and low sampling rates11. In this study, we used a commercially available wireless system that sends EEG signals from the animal through a Wi-Fi connection with a freely-moving rodents system12. The apparatus weighs 6 grams and stands up to 16 channels recorded at 1 kSps. This system allows EEG or spike recording in the animal environment, with a reduced disturbance, serving as an economical solution compared to the traditional electrophysiological systems in the market. In addition, we have synchronized this data using a video tracking software to provide correlation between EEG and behavioral patterns. This synchronization is done offline by alignment and interpolation of data and events based on timestamps generated by both systems and is processed on MATLAB.
Adult neurogenesis is defined as the proliferation, survival, and differentiation in neurons of newly generated cells in the dentate gyrus of animals13,14. This process is known to be associated with memory and learning improvement which increases adult neurogenesis in rodents through enriched environment (EE) conditions15. EE consists of housing rodents in small groups inside a large cage provided with toys and tubes, where animals have novel and complex but no biological relevance15. Although EE stimulates hippocampal neurogenesis, it also varies in many factors such as age, animal strain, specific stimulation conditions, or neurogenesis detection procedure. In middle-aged mice exposed to EE housing for seven days, the birth of new granular cells (GC) in the hippocampal dentate gyrus (DG) has been reported16. Studies attempting to ablate adult neurogenesis in adult rats selectively have suggested that new granular cells of about 1 - 2 weeks of age are required in the learned response17. Around 2 or 3 weeks after the GC are born in adults DG, several characteristic features such as dendritic spines, which are essential for excitatory synaptic transmission18, begin to appear. Zhao et al. performed a quantitative analysis to show that the peak of spine growth occurs during the first 3 - 4 weeks19. Several electrophysiological in vivo studies suggest that only three weeks of EE housing conditions produces alterations in the DG&#39;s synaptic transmission and increase cell excitability20. Also, it has been reported that exposure to an enriched environment at 1&#8211;4 weeks after BrdU injections significantly increased the density of BrdU/NeuN cells in the DG granular layer in mice21. These authors suggest that a critical period exist between one and three weeks after EE exposure since a substantial increase in the number of new neurons was observed21. Studies of adult hippocampal neurogenesis (AHN) in humans has been controversial since there was no direct evidence. However, a recent report described the developmental stages of AHN in the human adult brain, identifying thousands of immature neurons in the DG, and thereby demonstrating the importance of AHN during aging in humans22. Based on the evidence mentioned previously, the study of AHN in animal models is more important than ever (for a more in-depth examination of AHN, see Leal-Galicia et al., 2019 review15).
As previously mentioned, the hippocampus has been linked to a fundamental function in learning and memory capacities. The formation of memories goes through three distinct processes: encoding (memory acquisition), consolidation (memory storage), and retrieval (memory recognition)23. Recognition memory in humans is tested using the visual paired comparisons task24. The fundamentals of human and animal models of memory and amnesia are the behavioral tests that assess the ability to recognize a previously presented stimuli25,26, as the visual paired comparisons task does in humans. Therefore, one of the most used behavioral tests for assessing the ability of a rodent to recognize a previously presented stimulus, that is to say, the learning and memory capacity is the spontaneous novel object recognition task (NORT)23,27. NORT protocol consists of two identical novel objects in a familiar arena for 10 min in the acquisition trial. After a specific time between 028 and 48 hours29 (variable time according to each protocol), the animal is returned to the same arena containing one of the same familiar objects, and one novel object. The animal spontaneously explores the novel object if the familiar object was memorized26. The preference ratio is commonly used in assessing exploration performance. It is determined by dividing the total object exploration time from the exploration time of the novel or familiar object. The NORT has some advantages over other recognition memory tests. Most importantly, it requires no external motivation, reward, or punishment. It does not generate stressful conditions. Finally, no training is needed to evoke the behavior of exploring the objects (for a more in-depth examination of NORT, see ref.23).
Therefore, simultaneous recording of multiple data modalities and their integration in the study of learning and memory, as an effect of adult hippocampal neurogenesis is highly attractive and provides a compelling solution for researchers in the field. The present work will expose all processes involved in the simultaneous behavioral video-tracking assessment (novel object recognition task) and wireless electroencephalography recording. Here we have reviewed the electrode manufacturing process, epidural (skull screw) electrodes implantation surgery, environmental enrichment protocol (for hippocampal neurogenesis induction), following NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG and behavior data analysis executed on MATLAB computing environment.

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		</tr>
		<tr>
			<td>8 pin Receptacle Housing Male</td>
			<td>Amphenol FCI</td>
			<td>10147603-00008LF</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic Resin</td>
			<td>MDC Dental</td>
			<td>NicTone</td>
			<td>For fixating the screws to the skull</td>
		</tr>
		<tr>
			<td>ANY-maze video tracking software</td>
			<td>Stoelting, Co.</td>
			<td>version 6.1</td>
			<td>http://www.anymaze.co.uk/)</td>
		</tr>
		<tr>
			<td>benzalkonium chloride antiseptic solution</td>
			<td>Benzal</td>
			<td>Benzal</td>
			<td></td>
		</tr>
		<tr>
			<td>Bulldog clamps</td>
			<td>Cientifica VelaQuin</td>
			<td></td>
			<td>For retracting the skin</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Logitech</td>
			<td>c920</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crimp contact</td>
			<td>Amphenol FCI</td>
			<td>10147604-01LF</td>
			<td></td>
		</tr>
		<tr>
			<td>DELL PC</td>
			<td>DELL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>JAGA16</td>
			<td>Jinga-Hi, Inc.</td>
			<td>JAGA16</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>PiSA Agropecuaria</td>
			<td>ANESKET</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>R2020a</td>
			<td>MathWorks</td>
			<td>Script was develop ped in collaboration with Jinga-Hi, Inc.</td>
		</tr>
		<tr>
			<td>Monomer</td>
			<td>MDC Dental</td>
			<td>NicTone</td>
			<td>For fixating the screws to the skull</td>
		</tr>
		<tr>
			<td>Neurophys software</td>
			<td>Jinga-Hi, Inc./ Neurosys, LLC</td>
			<td>Neurosys 3.0.0.7</td>
			<td></td>
		</tr>
		<tr>
			<td>Screwdrive</td>
			<td></td>
			<td></td>
			<td>For inserting the screws into the skull</td>
		</tr>
		<tr>
			<td>Screws</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Screws equiped with electrode</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic instrument</td>
			<td>KOPF</td>
			<td></td>
			<td>For the surgery</td>
		</tr>
		<tr>
			<td>Variable speed rotary tool</td>
			<td>Dremel 3000</td>
			<td>Dremel</td>
			<td>For making the holes where the screws will be inserted</td>
		</tr>
		<tr>
			<td>Voltmeter</td>
			<td>PROAM</td>
			<td>MUL-040</td>
			<td>For confirming that the electrode conducts electricity</td>
		</tr>
		<tr>
			<td>Xilazine</td>
			<td>PiSA Agropecuaria</td>
			<td>PROCIN</td>
			<td>For anesthesia</td>
		</tr>
	</tbody>
</table>

simultaneous monitoring of wireless electrophysiology and memory behavioral test as a tool to study hippocampal neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The methods described above were applied to record EEG and rat activity simultaneously after the environmental enrichment treatment. Three-month-old male Wistar rats were under a medium-term environmental enrichment treatment protocol for 20 days, and they were operated to fix six skull screw electrodes paired on frontal, central and parietal regions referenced to a seventh electrode located at NZ. Animals were maintained under natural dark-light conditions, with ad libitum access to food and water. This work shows the integration between the EEG system and the behavioral tracking software for a simultaneous live recording. We only used animals treated under EE protocol since we do not pretend to compare the effectiveness of the treatment, but only exemplify the advantages of the equipment. As evidence that the 20 days environmental enrichment housing protocol used stimulates the adult neurogenesis, we present BrdU positive cell count data from animals under EE and animals housed under standard conditions from unpublished data from our lab. Three-month-old male Wistar rats were used. They were injected three times with BrdU with 12 h between each other. Animals were anesthetized (pentobarbital (50 mg/kg, i.p.) and euthanized by transcardial perfusion (see Figure 5). To ensure that the vest attached to the EEG device does not limit animal movements, we performed the open field test (OFT) into two groups, one group underwent surgery while wearing the equipment (vest and EEG amplifier), and the other group of animals remained intact without wearing the hardware. We did not find significant differences in the distance traveled by the animals in 10 min of testing (see Figure 5). The typical NORT protocol consists of the presentation of two objects, and the replacement of one of them with a new object. The behavioral tracking software monitored exploration time.
The Behavioral Tracking Software recorded a group of animals to evaluate their key performance parameters. Therefore, we used three parameters to evaluate exploration performance. The preference ratio was calculated using the animals&#39; head time spent in the object zone, which reports the total amount of time that the animals&#39; head spent in each object. Also, we calculated a preference ratio for the time spent moving towards the objects, which shows the total amount of time spent on every animal that was moving towards each object zone. Additionally, the spent time per visit to each object was calculated. Figure 6 shows the three-parameter results mentioned above. In the acquisition trial, there were no distinctions between objects in the three assessed parameters: head time in the object zone for the three trials, time moving towards the objects for the three trials, and time per visit in each object. There were no differences in the STM trial. Meanwhile, in the LTM trial, an exploration preferent ratio significantly higher for the novel object was seen. Additionally, in the LTM trial, a preference for the novel object in the time spent per visit (panel C) could be seen, as well. Video 1 shows a representative example of a rat recorded in the experiment while Video 2 shows a representative example of simultaneous EEG and behavioral recording.
It was possible to match time events tracked with the Behavioral Tracking and the EEG software recording using the computer&#8217;s clock. Figure 7 and Figure 8 show the changes in EEG relative power over alpha and beta bands. These are related to motor control, concentration, and memory, suggesting that exploration is just related to these functions. The results of animal 3 show that alpha power tends to reduce on STM regarding ACQ and LTM, suggesting a desynchronization related to exploration or memory retrieval. The number of object recognition (processed epochs) was low. By this point, it is not possible to determine if a statistical test would validate if such difference is real, or an artifact was able to produce such experimental conditions. Nevertheless, epochs segmentation, labeling, and analysis have become possible by a timeline of simultaneous marking events in animals and EEG outcomes produced for future research projects. Combining these systems prevents a wrongful identification of events by a manual marking process, which has become a significant issue in animal experimentation purposes. The combination of the BTS and electrophysiological (EP) activity could be accurately associated with animal behavior; nevertheless, experimental conditions require the use of advanced signal processing techniques to eliminate motion artifacts and make improvements in the experimental setup effectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61494/61494fig01.jpg" /> 
Figure 1: Examples of enriched environment (EE) conditions cage.&#160;Housing was provided with toys and tubes, in which animals find novel and complex but no biological relevance. <a href="https://www.jove.com/files/ftp_upload/61494/61494fig01large.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/61494/61494fig02.jpg" /> 
Figure 2: Positions of epidural electrodes in the rat skull.&#160;The screws were simultaneously used as anchor for the headset and as electrodes. F = frontal; C = frontoparietal; P = parietal; 3 = left; 4 = right; NZ = as the ground reference. <a href="https://www.jove.com/files/ftp_upload/61494/61494fig02large.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/61494/61494fig03.jpg" /> 
Figure 3: Representative images of an epidural (skull screw) electrodes implantation surgery.&#160;Image showing implanted intracranial electrodes screws in rats at different stages of the surgery. Ensure aseptic techniques are followed while performing this procedure.&#160;<a href="https://www.jove.com/files/ftp_upload/61494/61494fig03large.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/61494/61494fig04.jpg" /> 
Figure 4: Representative images of a rat along with the experimental setup.&#160;The rat was made to wear the vest attached to the EEG device with an embedded battery, inside the arena used for the NORT protocol. The image shows the headset and the cable connector installed on the head&#39;s rat. <a href="https://www.jove.com/files/ftp_upload/61494/61494fig04large.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/61494/61494fig05.jpg" /> 
Figure 5: Evidence of movement capability, and adult neurogenesis stimulation by EE protocol.&#160;(A) Representative images of the animal activity for 10 min in the Open Field Test (OFT) and the mean distance that animals wearing the equipment/surgery traveled, and animals without the equipment/No surgery. (B-E) Representative DG section with BrdU labeled cells (intense dark) for EE and standard housing groups. Panels B and D show a low magnification of the DG, and panels C and E show the box area at higher magnification. Panels B and C are tissue from the EE housing group, panels D and E are from the standard housing group. The inset illustrates the average numbers of labeled cells in both groups. ML - molecular layer; GCL &#8211; granular cell layer; SGZ &#8211; subgranular zone; arrows - BrdU+ cells. The graphs show the mean &#177; SEM. The T-student test was used to compare groups. * p&#8804;0.05. No significant differences were found between groups in the Open Field Test. <a href="https://www.jove.com/files/ftp_upload/61494/61494fig05large.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/61494/61494fig06.jpg" /> 
Figure 6: Exploration performance in NORT assessment.&#160;(A) Head time in the object zone for the three trials. (B) Time moving towards the objects for the three trials. (C) Time per visit in each object. The graphs show the mean &#177; SEM. Two-way repeated-measures ANOVA with Sidak&#39;s multiple comparisons test was used in all parameters. * p&#8804;0.05, ** p&#8804;0.01 between the objects in the respective trial. <a href="https://www.jove.com/files/ftp_upload/61494/61494fig06large.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/61494/61494fig07.jpg" /> 
Figure 7: Changes over alpha EEG band power associated with exploration.&#160;This figure show changes in relative alpha power, from half second to 2.5 after animal begins the exploration of the objects. The six graphs corresponded to Frontal, Central, and Parietal electrodes (from top to bottom) and left and right sides. Boxplots show the distribution of such time series for each condition combination of an Object: &#8220;Familiar&#8221; and &#8220;Novel,&#8221; and stage: &#8220;ACQ,&#8221; &#8220;STM&#8221; and &#8220;LTM.&#8221; <a href="https://www.jove.com/files/ftp_upload/61494/61494fig07large.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/61494/61494fig08.jpg" /> 
Figure 8: Changes over beta EEG band power associated with exploration.&#160;This figure show changes on relative beta power, from half second to 2.5 after animal begins the exploration of the objects. The six graphs corresponded to Frontal, Central, and Parietal electrodes (from top to bottom) and left and right sides. Boxplots show the distribution of such time series for each condition combination of an Object: &#8220;Familiar&#8221; and &#8220;Novel,&#8221; and stage: &#8220;ACQ,&#8221; &#8220;STM&#8221; and &#8220;LTM.&#8221; <a href="https://www.jove.com/files/ftp_upload/61494/61494fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Representative video showing a rat recorded in the experiment. The rat was inside the arena used for the NORT protocol. The rat was wearing the vest attached to the EEG device with an embedded battery. <a href="https://cloudflare.jove.com/files/ftp_upload/61494/VIDEO_1.mp4" target="_blank">Please click here to download this video.</a>
Video 2: Representative video showing simultaneous EEG and behavioral recording. EEG signal was displayed on the left side while the behavioral test (NORT) was displayed on the right side of the video. <a href="https://cloudflare.jove.com/files/ftp_upload/61494/VIDEO_2.mp4" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis Video at JoVE.com

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis Video (Video) | JoVE

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

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