This work was supported by&#160;the&#160;Royal Melbourne Hospital Neuroscience Foundation (A2087).

This study was approved by the Florey Animal Ethics Committee (The University of Melbourne, No. 22-025UM) in accordance with the Australian code for the care and use of animals for scientific purposes. C57BL/6 male mice (8 weeks), obtained from the Animal Resources Centre (Australia), were used for the present study.
1. Headstage design and fabrication
NOTE: The headstage PCB board is a compact 14 mm x 12 mm four-layer board designed to be placed directly on the animal&#39;s head. It utilizes a commercial amplifier chip (see Table of Materials), and all design and Gerber files are available online (GitHub link:&#160;https://github.com/dechuansun/Intan-headstage/tree/main/pcbway).
<ol>
	<li>Provide the following specifications to the manufacturer: Board thickness: 0.6 mm; Minimum tracking/spacing: 4 mils; Minimum hole size: 0.2 mm.</li>
	<li>During the PCB assembly process, follow this order:
	<ol>
		<li>Solder the amplifier chip onto the board using a hot air gun set at 350 &#176;C.</li>
		<li>Solder the passive components.</li>
		<li>Solder the SPI connector and the electrode connector (see Table of Materials).</li>
	</ol>
	</li>
	<li>Inspect the soldering under a microscope for quality assurance. Secure the SPI connector in place using epoxy for added stability.</li>
	<li>Use third-party recording software and a control board (see Table of Materials) for signal acquisition. Refer to the software user guide for detailed instructions.</li>
	<li>The designed headstage supports 8 channels. In the software, enable channels 8, 9, 12, 13, 20, 21, 22, and 23 for recording.</li>
</ol>
2. Electrode fabrication
<ol>
	<li>Cut PFA-coated tungsten wires (see Table of Materials) to specific lengths for different electrode types: prefrontal cortex electrode (12 mm), hippocampus electrode (10 mm), and ground electrode (6 mm).</li>
	<li>Cut the brass tube (see Table of Materials) into 3 mm segments.</li>
	<li>Remove 2 mm of the coating at the end of each wire using a lighter, then securely solder the electrode wire to the brass tube. The brass tube has an inside diameter of 0.45 mm and an outside diameter of 0.60 mm.</li>
	<li>For the ground electrode, solder an M1.2 stainless steel screw (see Table of Materials) to the electrode. Apply phosphoric acid-based flux to the screw to enhance soldering. After soldering, clean the screw using alcohol. 
	&#8203;NOTE: Wear gloves for protection during the soldering process.</li>
</ol>
3. Surgical procedure
<ol>
	<li>Anesthetize the mouse in an anesthesia chamber with 3% isoflurane and 1 L/min oxygen flow.</li>
	<li>Place the anesthetized mouse on a heating pad and secure it in a stereotaxic frame (see Table of Materials).</li>
	<li>Adjust the maintenance rate of isoflurane to 2.5-3% and reduce the oxygen flow to 500 mL/min. Using the toe pinch, verify if the animal is still under deep anesthesia.</li>
	<li>Subcutaneously inject carprofen at 0.5 mg/kg and apply eye ointment for eye protection.</li>
	<li>Shave and sterilize the mouse&#39;s head using povidone-iodine and 80% ethanol.</li>
	<li>Make an 8 mm incision along the midline of the scalp, removing connective tissue in the incision area.</li>
	<li>Apply hydrogen peroxide to clean the surface of the skull, being cautious not to touch the surrounding skin.</li>
	<li>Align the bregma and lambda landmarks to the same level for accurate electrode placement (bregma and lambda are where the sagittal suture intersects the coronal and lambdoid sutures).</li>
	<li>Drill holes for reference/ground electrode, anchor screws (0.9 mm drill burr), and active electrodes (0.3 mm drill burr) at specified coordinates.</li>
	<li>Attach the custom-made electrode (step 2) to the stereotaxic frame arm and ensure it is perpendicular to the brain.</li>
	<li>Implant the electrode in the hippocampal CA1 area (AP - 1.8 mm, ML - 1.3 mm, DV - 1.4 mm). 
	NOTE: AP, anteroposterior; ML, mediolateral; DV, dorsoventral.</li>
	<li>Repeat electrode implantation in the prefrontal cortex (AP - 2.0 mm, ML - 0.3 mm, DV - 1.7 mm).</li>
	<li>Secure electrodes with a commercially available powerful adhesive and dental cement (see Table of Materials).</li>
	<li>Implant two 1.2 mm anchor screws (AP - 1.8 mm, ML -1.6 mm) to prevent movement.</li>
	<li>Position the reference/ground electrode in direct contact with the dura mater, 2 mm posterior and 2 mm unilateral to the lambda landmark.</li>
	<li>Connect the brass tube side of the electrodes to a multi-channel socket connector (see Table of Materials) with the ground electrode in the middle.</li>
	<li>Use 0.8 mm heat shrink tubing on the exterior of the middle pin for isolation.</li>
	<li>Secure the electrodes, anchor screws, and connector with adhesive and dental cement.</li>
</ol>
4. Postoperative care
<ol>
	<li>To alleviate postoperative pain, inject carprofen at a dose of 5-10 mg/kg subcutaneously every 12-24 hours based on an assessment of pain for a period of three days.</li>
	<li>Provide the animal with a one-week recovery period before starting any recording or experimental procedures.</li>
</ol>
5. Recording procedure
<ol>
	<li>Handle the animal for 15 min, twice daily, for three consecutive days.</li>
	<li>Pick up the mice by gently closing the hand around them without applying excessive pressure.</li>
	<li>Place the headstage board on the animal&#39;s head for 30 min once a day for three consecutive days.</li>
	<li>On the recording day, acclimate the animal to the recording room for 30 min.</li>
	<li>Place the animal in a small recording chamber within a Faraday cage to reduce external electrical interference. Attach the custom headstage for recording.</li>
	<li>Open the recording software and select a 2.00 kHz sampling rate. Disable all channels except for 13 and 20 by selecting each channel and pressing the space bar.</li>
	<li>In the hardware bandwidth window, set the lower bandwidth to 2 Hz and the upper bandwidth to 100 Hz.</li>
	<li>In the software filtering window, adjust the low-pass filter to 100 Hz and the high-pass filter to 2 Hz.</li>
	<li>Choose the storage path by clicking on Select File Name, and then click on Record.</li>
	<li>Start each recording session with a 10 min habituation period followed by a 15 min baseline EEG recording.</li>
	<li>After the baseline recording, administer the drug via intraperitoneal injection and continue recording for an additional 30 min without delay. 
	NOTE: See the Results section for the details on the drugs used.</li>
</ol>

LFP Recording in Mouse Brain for Cognitive Research and Drug Analysis

<ol>
	<li>Einevoll, G. T., Kayser, C., Logothetis, N. K., Panzeri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+and+analysis+of+local+field+potentials+for+studying+the+function+of+cortical+circuits.">Modelling and analysis of local field potentials for studying the function of cortical circuits.</a> Nat Rev Neurosci. 14 (11), 770-785 (2013).</li><li>Buzsaki, G., Anastassiou, C. A., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+extracellular+fields+and+currents-EEG,+ECOG,+LFP+and+spikes.">The origin of extracellular fields and currents-EEG, ECOG, LFP and spikes.</a> Nat Rev Neurosci. 13 (6), 407-420 (2012).</li><li>Sigurdsson, T., Stark, K. L., Karayiorgou, M., Gogos, J. A., Gordon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+hippocampal-prefrontal+synchrony+in+a+genetic+mouse+model+of+schizophrenia.">Impaired hippocampal-prefrontal synchrony in a genetic mouse model of schizophrenia.</a> Nature. 464 (7289), 763-767 (2010).</li><li>Witton, 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=Disrupted+hippocampal+sharp-wave+ripple-associated+spike+dynamics+in+a+transgenic+mouse+model+of+dementia.">Disrupted hippocampal sharp-wave ripple-associated spike dynamics in a transgenic mouse model of dementia.</a> J Physiol. 594 (16), 4615-4630 (2016).</li><li>Englot, D. J., Konrad, P. E., Morgan, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+and+global+connectivity+disturbances+in+focal+epilepsy,+related+neurocognitive+sequelae,+and+potential+mechanistic+underpinnings.">Regional and global connectivity disturbances in focal epilepsy, related neurocognitive sequelae, and potential mechanistic underpinnings.</a> Epilepsia. 57 (10), 1546-1557 (2016).</li><li>Pievani, M., De Haan, W., Wu, T., Seeley, W. W., Frisoni, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+network+disruption+in+the+degenerative+dementias.">Functional network disruption in the degenerative dementias.</a> Lancet Neurol. 10 (9), 829-843 (2011).</li><li>Sigurdsson, T., Duvarci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal-prefrontal+interactions+in+cognition,+behavior+and+psychiatric+disease.">Hippocampal-prefrontal interactions in cognition, behavior and psychiatric disease.</a> Front Syst Neurosci. 9, 190 (2015).</li><li>Sun, D., 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=Effects+of+antipsychotic+drugs+and+potassium+channel+modulators+on+spectral+properties+of+local+field+potentials+in+mouse+hippocampus+and+pre-frontal+cortex.">Effects of antipsychotic drugs and potassium channel modulators on spectral properties of local field potentials in mouse hippocampus and pre-frontal cortex.</a> Neuropharmacology. 191, 108572 (2021).</li><li>Bokil, H., Andrews, P., Kulkarni, J. E., Mehta, S., Mitra, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronux:+A+platform+for+analyzing+neural+signals.">Chronux: A platform for analyzing neural signals.</a> J Neurosci Methods. 192 (1), 146-151 (2010).</li><li>Bozkurt, A., Lal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+flexible+printed+circuit+technology+based+microelectrode+array+for+extracellular+stimulation+of+the+invertebrate+locomotory+system.">Low-cost flexible printed circuit technology based microelectrode array for extracellular stimulation of the invertebrate locomotory system.</a> Sens Actuator A Phys. 169 (1), 89-97 (2011).</li><li>Du, 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=High-resolution+mapping+of+in+vivo+gastrointestinal+slow+wave+activity+using+flexible+printed+circuit+board+electrodes:+Methodology+and+validation.">High-resolution mapping of in vivo gastrointestinal slow wave activity using flexible printed circuit board electrodes: Methodology and validation.</a> Ann Biomed Eng. 37, 839-846 (2009).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience.+Histological+Staining+of+Neural+Tissue.">Neuroscience. Histological Staining of Neural Tissue.</a> JoVE. , (2023).</li></ol>

The authors have nothing to disclose.

The protocol presented here outlines the procedure for constructing a customized headstage specifically designed for the simultaneous recording of dual local field potentials (LFPs) in the hippocampus (HIP) and the prefrontal cortex (PFC). The detailed steps provided in this protocol offer sufficient information for researchers to thoroughly examine signal communication both within each region and between the HIP and PFC.
The custom-designed headstage utilizes a commercial amplifier chip. Whil...

Local field potentials (LFPs) refer to the electrical activity recorded from the extracellular space, reflecting the collective activity of a localized group of neurons. They exhibit a diverse range of frequencies, spanning from slow waves at 1 Hz to fast oscillations at 100 Hz or 200 Hz. Specific frequency bands have been associated with cognitive functions such as learning, memory, and decision making1,2. Changes in LFP properties have been used as biomarkers for various neurological disorders, including dementia and schizophrenia3,4. Analyzing LFP recordings can offer valuable insights into the underlying pathological mechanisms associated with these conditions and potential therapeutic strategies.
Dual LFP recording is a technique used to measure localized electrical activity within and between two specific brain regions. This technique provides a valuable opportunity to investigate the intricate neural dynamics and signal communication occurring within and between distinct brain regions. Prior studies have revealed that detecting alterations in the neuronal properties of individual brain regions can be complex, but changes in interregional cortical communication can be observed5,6. Therefore, the utilization of dual LFP recording offers a potent means to address this issue.
Hippocampal-prefrontal connectivity plays a crucial role in modulating cognitive functions, and dysfunction has been linked to various neurological disorders7,8. Dual electrode recordings of these regions can provide information about these interactions. Unfortunately, there is limited information available on methods for performing dual electrode LFP recordings between these areas. Moreover, commercially available recording devices are generally expensive and lack adaptability to specific experimental designs. The conventional method for recording LFPs involves using a shielded cable to connect the recording device to electrodes implanted in an animal's brain. However, this approach is susceptible to motion artifacts and environmental noise, impacting the quality and reliability of the recorded signals.
This protocol describes a comprehensive procedure for performing dual-electrode LFP recordings in the mouse hippocampus and prefrontal cortex, using a low-cost custom-designed headstage that can be placed on the animal's head. These methods enable researchers to investigate region-specific oscillatory patterns within two discrete cerebral regions and explore interregional information exchange and connectivity between these areas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brass tube&#160;</td>
			<td>Albion Alloys, USA</td>
			<td>Inside diameter of 0.45 mm</td>
		</tr>
		<tr>
			<td>Carprofen&#160;</td>
			<td>Rimadyl, Pfizer Animal Health&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial amplifier chip</td>
			<td>Intantech</td>
			<td>RHD 2132</td>
		</tr>
		<tr>
			<td>Control board</td>
			<td>Intantech</td>
			<td>RHD recording system</td>
		</tr>
		<tr>
			<td>Dental cement&#160;</td>
			<td>Paladur</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat shrinks</td>
			<td>Panduit</td>
			<td>0.8 mm diameter</td>
		</tr>
		<tr>
			<td>M1.2 stainless steel screw</td>
			<td>Watch tools</td>
			<td>Clock and watch screw</td>
		</tr>
		<tr>
			<td>Multichannel socket connector&#160;</td>
			<td>Harwin, AU</td>
			<td>1.27 mm pitch, PCB socket</td>
		</tr>
		<tr>
			<td>PFA-coated tungsten wires&#160;</td>
			<td>A-M SYSTEMS, USA</td>
			<td>Inside diameter of 150 &#181;m&#160;</td>
		</tr>
		<tr>
			<td>Phosphoric acid-based flux</td>
			<td>Chip Quik</td>
			<td>CQ4LF-0.5</td>
		</tr>
		<tr>
			<td>Recording software</td>
			<td>Intantech</td>
			<td>RHX recording software</td>
		</tr>
		<tr>
			<td>Stereotactic Frame</td>
			<td>World Precision Instruments</td>
			<td>Mouse stereotactic instrument</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>UHU</td>
			<td>Ultra fast</td>
		</tr>
	</tbody>
</table>

dual extracellular recordings in the mouse hippocampus and prefrontal cortex

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal populations. It serves as a crucial tool in cognitive research, particularly in brain regions like the hippocampus and prefrontal cortex. Dual LFP recordings between these areas are of particular interest as they allow the exploration of interregional signal communication. However, methods for performing these recordings are rarely described, and most commercial recording devices are either expensive or lack adaptability to accommodate specific experimental designs. This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of antipsychotic drugs and potassium channel modulators on LFP properties in these areas. The technique enables the measurement of LFP properties, including power spectra within each brain region and coherence between the two. Additionally, a low-cost, custom-designed recording device has been developed for these experiments. In summary, this protocol provides a means to record signals with high signal-to-noise ratios in different brain regions, facilitating the investigation of interregional information communication within the brain.

The results shown here demonstrate the effects of several drugs on local field potentials (LFPs) properties tested in four cohorts of C57BL/6 male mice (n = 8 for each cohort; age: 8 weeks; weight: 24.0 &#177; 0.42 g). The drugs tested included the antipsychotic drug clozapine, the potassium channel modulators 4-Aminopyridine (4-AP), and retigabine, as well as the control vehicle saline.
As shown in Figure 1, the mouse was placed in a small recording chamber and L...

Watch this Scientific Journal Video about Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex at JoVE.com

Author Spotlight: Studying Drug Impacts on Brain Signals Using Dual LFP Recording Protocol in Mice

This protocol outlines the use of a custom-designed recording device and electrodes to record local field potentials and investigate information flow in the mouse hippocampus and prefrontal cortex.

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal ...

This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of drugs on LFP properties in these areas. This protocol provides a low-cost and ultra-light brain signal recording device that is fully optimized for cognitive research in mice. This study provides a means to record signals with high signal-noise ratios in different brain regions, facilitating the investigation of interregional information communication within the brain.

dual-extracellular-recordings-mouse-hippocampus-prefrontal

Research

JoVE Journal

Neuroscience

1.3K Views.  The University of Melbourne. This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of drugs on LFP properties in these areas. This protocol provides a low-cost and ultra-light brain signal recording device that is fully optimized for cognitive research in mice. This study provides a means to record signals with high signal-noise ratios in different brain regions, facilitating the investigation of interregiona...

Video: Author Spotlight: Studying Drug Impacts on Brain Signals Using Dual LFP Recording Protocol in Mice

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

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

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

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

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

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

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.