This work was supported by National Institutes of Health (National Institute of Neurological Disorders and Stroke) Grant 1R01NS060757-01 and by the E.L. Lindseth endowed chair to Dominique M. Durand. We thank Dr. Andrew M. Rollins&#8217; laboratory for the help on the OCT imaging.

NOTE: Animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the university. CD1 mice of either sex at the age of P10 to P20 are used in this study.
1. Solutions for Surgery and Experimental Recording
<ol>
	<li>Prepare normal artificial cerebrospinal fluid (aCSF) buffer containing (mM): NaCl 124, KCl 3.75, KH2PO4 1.25, MgSO4 2, NaHCO3 26, Dextrose 10, and CaCl2 2. Use this normal aCSF for tiss...

<ol>
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I., Ben-Ari, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+propagation+of+4-AP-induced+epileptiform+activity+in+neonatal+intact+limbic+structures+in+vitro.">Generation and propagation of 4-AP-induced epileptiform activity in neonatal intact limbic structures in vitro.</a> Eur J Neurosci. 12 (8), 2757-2768 (2000).</li><li>Grinvald, A., Manker, A., Segal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+spread+of+electrical+activity+in+rat+hippocampal+slices+by+voltage-sensitive+optical+probes.">Visualization of the spread of electrical activity in rat hippocampal slices by voltage-sensitive optical probes.</a> J Physiol. 333, 269-291 (1982).</li><li>Gloveli, T., 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=Orthogonal+arrangement+of+rhythm-generating+microcircuits+in+the+hippocampus.">Orthogonal arrangement of rhythm-generating microcircuits in the hippocampus.</a> Proc Natl Acad Sci USA. 102 (37), 13295-13300 (2005).</li><li>Amaral, D. G., Witter, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three-dimensional+organization+of+the+hippocampal+formation:+a+review+of+anatomical+data.">The three-dimensional organization of the hippocampal formation: a review of anatomical data.</a> Neuroscience. 31 (3), 571-591 (1989).</li><li>Albani, S. H., McHail, D. G., Dumas, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+studies+of+the+hippocampus+and+hippocampal-dependent+behaviors:+insights+from+interdisciplinary+studies+and+tips+for+new+investigators.">Developmental studies of the hippocampus and hippocampal-dependent behaviors: insights from interdisciplinary studies and tips for new investigators.</a> Neurosci Biobehav Rev. 43, 183-190 (2014).</li><li>Zhang, 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=Propagation+of+Epileptiform+Activity+Can+Be+Independent+of+Synaptic+Transmission,+Gap+Junctions,+or+Diffusion+and+Is+Consistent+with+Electrical+Field+Transmission.">Propagation of Epileptiform Activity Can Be Independent of Synaptic Transmission, Gap Junctions, or Diffusion and Is Consistent with Electrical Field Transmission.</a> J Neurosci. 34 (4), 1409-1419 (2014).</li><li>Kibler, A. B., Durand, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonal+wave+propagation+of+epileptiform+activity+in+the+planar+mouse+hippocampus+in+vitro.">Orthogonal wave propagation of epileptiform activity in the planar mouse hippocampus in vitro.</a> Epilepsia. 52 (9), 1590-1600 (2011).</li><li>Wang, D., McMahon, S., Zhang, Z., Jackson, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+voltage+sensor+imaging+of+electrical+activity+from+neurons+in+hippocampal+slices+from+transgenic+mice.">Hybrid voltage sensor imaging of electrical activity from neurons in hippocampal slices from transgenic mice.</a> J Neurophysiol. 108 (11), 3147-3160 (2012).</li><li>Wingenfeld, K., Wolf, O. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+,+memory,+the+hippocampus.">Stress , memory, the hippocampus.</a> Front Neurol Neurosci. 34, 109-121 (2014).</li><li>Liu, J. S., 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=Spatiotemporal+dynamics+of+high-K+-induced+epileptiform+discharges+in+hippocampal+slice+and+the+effects+of+valproate.">Spatiotemporal dynamics of high-K+-induced epileptiform discharges in hippocampal slice and the effects of valproate.</a> Neurosci Bull. 29 (1), 28-36 (2013).</li><li>Oka, H., Shimono, K., Ogawa, R., Sugihara, H., Taketani, M. <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+planar+multielectrode+array+for+extracellular+recording:+application+to+hippocampal+acute+slice.">A new planar multielectrode array for extracellular recording: application to hippocampal acute slice.</a> J Neurosci Methods. 93, 61-68 (1999).</li><li>Kibler, A. B., Jamieson, B. G., Durand, D. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potassium+channel+blockers+4-aminopyridine+and+tetraethylammonium+increase+the+spontaneous+basal+release+of+[3H]5-hydroxytryptamine+in+rat+hippocampal+slices.">The potassium channel blockers 4-aminopyridine and tetraethylammonium increase the spontaneous basal release of [3H]5-hydroxytryptamine in rat hippocampal slices.</a> J Pharmacol Exp Ther. 282 (1), 262-270 (1997).</li><li>Perreault, P., Avoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-aminopyridine-induced+epileptiform+activity+and+a+GABA-mediated+long-lasting+depolarization+in+the+rat+hippocampus.">4-aminopyridine-induced epileptiform activity and a GABA-mediated long-lasting depolarization in the rat hippocampus.</a> J Neurosci. 12 (1), 104-115 (1992).</li><li>Chesnut, T. J., Swann, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptiform+activity+induced+by+4-aminopyridine+in+immature+hippocampus.">Epileptiform activity induced by 4-aminopyridine in immature hippocampus.</a> Epilepsy Res. 2 (3), 187-195 (1988).</li><li>Nam, Y., Wheeler, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Microelectrode+Array+Technology+and+Neural+Recordings.">In Vitro Microelectrode Array Technology and Neural Recordings.</a> Crit Rev Biomed Eng. 39 (1), 45-62 (2011).</li><li>Gonzalez-Sulser, A., 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=Hippocampal+neuron+firing+and+local+field+potentials+in+the+in+vitro+4-aminopyridine+epilepsy+model.">Hippocampal neuron firing and local field potentials in the in vitro 4-aminopyridine epilepsy model.</a> J Neurophysiol. 108 (9), 2568-2580 (2012).</li></ol>

The development of the unfolded hippocampus preparation, where the longitudinal and transverse axes of the hippocampus are preserved in combination with a penetrating microelectrode array, provides a powerful tool to investigate the anatomy connections or neural propagation in the hippocampus 7. This unfolding procedure is also applicable for studying hippocampus in adult mice. Recent studies with this preparation showed that the 4-AP-induced epileptiform activity could propagate with a diagonal wave front acr...

Understanding the neural conduction or propagation of neural signals is crucial for determination of the mechanism of neural communication in both the normal function and pathological conditions in the brain 1-3. The hippocampus is one of the most extensively studied structures in the brain since it plays fundamental role in several brain functions such as memory, and spatial tracking and is involved in several pathological changes that dramatically impact behavior as well 1,6 . Although, the hippocampus exhibits a complex organization, the different elements of its structure can be readily identified and accessed in the slice preparation4-6. In the transverse direction of the hippocampus, neural activity is known to propagate through the tri-synaptic pathway that comprise the Dentate Gyrus (DG), CA3, CA1 andsubiculum 4,5. It is believed that synaptic transmission and axonal conduction play a major role for communication in this transverse circuit 4,6. However, propagation of neural signal takes place in both transverse and longitudinal directions 4,6. This implies that the hippocampus cannot be fully investigated by using slice preparations which limit the observation to a particular direction of propagation 4. The longitudinal slice was developed to investigate the axonal pathways along the longitudinal axis 5. Researchers have observed behavior-specific gamma and theta oscillations predominantly along the transverse and longitudinal axes respectively 6.&#160;These behaviors have been studied separately, yet simultaneous access to both directions is crucial to understand these behaviors. Even with the development of the intact hippocampus preparation, it is difficult to monitor the propagation throughout the entire tissue due to the folded-structure of the hippocampus 4. The unfolded hippocampus provides access to the packed neurons in a form of a flat two-dimensional cell layer 7,8.
By unfolding the dentate gyrus (DG) (Figure 1), the hippocampus adopts a flattened shape with a rectangular configuration in which both transverse and longitudinal connections remain intact with the pyramidal cell layer arranged in a two-dimensional sheet containing both CA3 and CA1, leaving a flat piece of neural tissue that can be used to investigate neural propagation (Figure 2) 8. Neural activity can then be monitored with individual glass pipettes, microelectrode arrays, stimulating electrodes, as well as voltage sensitive dyes (VSD) 3,7,8. In addition, genetically encoded voltage indicator from transgenic mice can be used to track the propagation pattern 9.
The flat configuration of the unfolded hippocampal network is well suited for optical method recording but also for a microelectrode array. Most of the commercially available arrays are fabricated with flat or low profile electrodes and can record neural activity in both tissue slices and cultured neurons 10-12. However, the signal-to-noise ratio (SNR) decreases when the signals are obtained from an intact tissue since the soma of the neurons are located deeper into the tissue. Microelectrode electrode arrays with high aspect ratios are required to improve the SNR.
To this effect, a penetrating microelectrode array (PMEA) has been developed in our laboratory, and provides the ability to directly probe into the tissue by inserting 64 spikes with a diameter of 20 &#181;m and height of 200 &#181;m into the unfolded hippocampus 7,13. This microelectrode array has higher SNR compared to the voltage sensitive dye imaging and the SNR remains stable during an experiment 7,13. The combination of the unfolded hippocampal preparation and the PMEA provides a new way to investigate the neural propagation over a two-dimensional plane. Experiments using this technique have already yielded significant results about the mechanisms of neural signal propagation in the hippocampus whereby neural activity can propagate independently of synaptic or electric synapses 7.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>desiccator jar</td>
			<td>LABRECYCLERS Inc.</td>
			<td>5410</td>
			<td>Place regular paper towels at the bottome of the jar for animal anesthesia use.&#160;</td>
		</tr>
		<tr>
			<td>A blade and Custome made surgical stage for unfolding hippocampus</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>A petri dish is place upside down (in the center) in the ice with a wet filter paper place on top of it.&#160;</td>
		</tr>
		<tr>
			<td>Custom made tissue recovery chamber</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Plastic tubes were glued with plastic mesh at the bottom and bubbled with 95% O2/ 5% CO2 in the aCSF.</td>
		</tr>
		<tr>
			<td>Straight Operating&#160;Scissors</td>
			<td>Fisher Scientific</td>
			<td>S17336B&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Medco Instruments No.:81995&#160;</td>
			<td>This scissors is used to&#160;&#160; decapitate the mice.</td>
		</tr>
		<tr>
			<td>Integra Miltex Goldman-Fox&#160;Scissors</td>
			<td>Fisher Scientific</td>
			<td>12-460-517&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; MILTEX INC&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; No.:5-SC-320</td>
			<td>This scissors is used to cut the skull of the mice.&#160;</td>
		</tr>
		<tr>
			<td>Miltex 
			Hysterectomy Forceps</td>
			<td>Claflin Medical equipment</td>
			<td>CESS-722033-00001</td>
			<td>This Forceps is used to peel the cut skull to expose the brain</td>
		</tr>
		<tr>
			<td>Micro Spatula</td>
			<td>Cardinal Health</td>
			<td></td>
			<td>This micro spatula is used to tranfer the whole brain of a semisphere into the recorering chamber.&#160;</td>
		</tr>
		<tr>
			<td>Frey Scientific Stainless Steel Semi-Micro Spatula</td>
			<td>Cardinal Health</td>
			<td></td>
			<td>this semi micro spatula is used to tranfer the unfolded hippocampus into the glucose aCSF in the recovering chamber.</td>
		</tr>
		<tr>
			<td>small paint brush</td>
			<td>Lowe&#39;s</td>
			<td>tem #:&#160;105657&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Model #:&#160;90219</td>
			<td>The one with the smallest size in a normal paint brush package</td>
		</tr>
		<tr>
			<td>Fire polished glass help tool</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was fire polished and made from the regular Pasteur glass pipettes.</td>
		</tr>
		<tr>
			<td>Custom made glass needle</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was fire polished and made from the regular Pasteur glass pipettes.</td>
		</tr>
		<tr>
			<td>Custom made glass tool with a metal wire loop</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was fire polished and made from the regular Pasteur glass pipettes with a reshaped metal wire loop.</td>
		</tr>
		<tr>
			<td>Custom made glass solution dropper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was&#160; made from the regular Pasteur glass pipettes with its tips cut and a rubber head attached with the cut end.</td>
		</tr>
		<tr>
			<td>Custom made tissue anchor</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Nylon fiber mesh was glued on a insulated copper wire ring. The tissue anchor was hold by an micromanipulator.&#160;</td>
		</tr>
		<tr>
			<td>Custom fabricated microelectrode array</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>More detail about the array please refer to&#160; Kibler, et al, 2011.&#160;</td>
		</tr>
		<tr>
			<td>Custom made filter and amplifiers circuits for the array</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>More detail about the array please refer to&#160; Kibler, et al, 2011.&#160;</td>
		</tr>
		<tr>
			<td>Data acquisition processor 3400a</td>
			<td>Microstar Laboratories</td>
			<td>N/A</td>
			<td>This is a complete data acquisition system with A/D converter.</td>
		</tr>
	</tbody>
</table>

neural activity propagation in an unfolded hippocampal preparation with a penetrating micro-electrode array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

The data shown in the figures here were recorded in the unfolded hippocampus preparation with 4-AP (100 &#181;M) aCSF added during incubation of the tissue in the recording chamber at RT (25 &#176;C). Normally activity starts within 5 min, but in some hippocampal tissues from the older animals it may take longer. The 4-AP-induced neuronal firing observed with the PMEA is the same as previously reported 14,15. Since the electrodes have a height of 200 &#181;m, the electrode tips are located just below the cell ...

Watch this Scientific Journal Video about Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array at JoVE.com

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

neural-activity-propagation-an-unfolded-hippocampal-preparation-with

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JoVE Journal

Neuroscience

8.3K visualizações.  Case Western Reserve University.  Nós desenvolvemos uma hipocampo in vitro desdobrada que preserva CA1-CA3 matriz de neurónios. Combinada com a rede de micro-eléctrodo penetrante, actividade neuronal pode ser monitorizada em ambas as orientações longitudinais e transversais. Este método oferece vantagens em relação a preparações de fatias do hipocampo como a propagação em todo o hipocampo podem ser gravados simultaneamente. 