We are thank the following for their support: Commonwealth Scholarship Commission UK funding (HP), EPSRC (HP), BBSRC (MFN) and European Union Marie Curie Actions (MFN).

1. Parasagittal Slice Preparation
1.1 Dissect out cerebral hemispheres
All animal experimentation should follow local ethical review and national regulations. In the case of the experiments described here, the work conforms to the United Kingdom Animals (Scientific Procedures) Act 1986. We routinely use cervical dislocation without anesthetic to euthanize the mouse before removing the brain. Alternatively the mouse can be terminally anesthetized, but in this case it may ...

<ol>
	<li>O'Donnell, C., Nolan, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+of+synaptic+responses:+an+organizing+principle+for+optimization+of+neural+circuits.">Tuning of synaptic responses: an organizing principle for optimization of neural circuits.</a> Trends Neurosci. 34, 51-60 (2011).</li><li>Garden, D. L. F., Dodson, P. D., O'Donnell, C., White, M. D., Nolan, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+of+synaptic+integration+in+the+medial+entorhinal+cortex+to+the+organization+of+grid+cell+firing+fields.">Tuning of synaptic integration in the medial entorhinal cortex to the organization of grid cell firing fields.</a> Neuron. 60, 875-889 (2008).</li><li>Kuba, H., Yamada, R., Fukui, I., Ohmori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonotopic+specialization+of+auditory+coincidence+detection+in+nucleus+laminaris+of+the+chick.">Tonotopic specialization of auditory coincidence detection in nucleus laminaris of the chick.</a> Journal of Neuroscience. 25, 1924-1934 (2005).</li><li>Hafting, T., Fyhn, M., Molden, S., Moser, M. -. B., Moser, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructure+of+a+spatial+map+in+the+entorhinal+cortex.">Microstructure of a spatial map in the entorhinal cortex.</a> Nature. 436, 801-806 (2005).</li><li>Sargolini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjunctive+representation+of+position,+direction,+and+velocity+in+entorhinal+cortex.">Conjunctive representation of position, direction, and velocity in entorhinal cortex.</a> Science. 312, 758-762 (2006).</li><li>Fyhn, M., Hafting, T., Witter, M. P., Moser, E. I., Moser, 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=Grid+cells+in+mice.">Grid cells in mice.</a> Hippocampus. 18, 1230-1238 (2008).</li><li>Giocomo, L. M., Zilli, E. A., Frans&#233;n, E., Hasselmo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+frequency+of+subthreshold+oscillations+scales+with+entorhinal+grid+cell+field+spacing.">Temporal frequency of subthreshold oscillations scales with entorhinal grid cell field spacing.</a> Science. 315, 1719-1722 (2007).</li><li>Giocomo, L. M., Hasselmo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+constants+of+h+current+in+layer+II+stellate+cells+differ+along+the+dorsal+to+ventral+axis+of+medial+entorhinal+cortex.">Time constants of h current in layer II stellate cells differ along the dorsal to ventral axis of medial entorhinal cortex.</a> Journal of Neuroscience. 28, 9414-9425 (2008).</li><li>Burgalossi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcircuits+of+functionally+identified+neurons+in+the+rat+medial+entorhinal+cortex.">Microcircuits of functionally identified neurons in the rat medial entorhinal cortex.</a> Neuron. 70, 773-786 (2011).</li><li>Dodson, P. D., Pastoll, H., Nolan, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+organization+of+theta-like+activity+intrinsic+to+entorhinal+stellate+neurons+is+mediated+by+differences+in+stochastic+current+fluctuations.">Dorsal-ventral organization of theta-like activity intrinsic to entorhinal stellate neurons is mediated by differences in stochastic current fluctuations.</a> J. Physiol. (Lond). 589, 2993-3008 (2011).</li><li>Nolan, M., Dudman, J., Dodson, P., Santoro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN1+channels+control+resting+and+active+integrative+properties+of+stellate+cells+from+layer+II+of+the+entorhinal+cortex.">HCN1 channels control resting and active integrative properties of stellate cells from layer II of the entorhinal cortex.</a> Journal of Neuroscience. 27, (2007).</li><li>Boehlen, A., Heinemann, U., Erchova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+range+of+intrinsic+frequencies+represented+by+medial+entorhinal+cortex+stellate+cells+extends+with+age.">The range of intrinsic frequencies represented by medial entorhinal cortex stellate cells extends with age.</a> Journal of Neuroscience. 30, 4585-4589 (2010).</li><li>Klink, R., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+characteristics+of+layer+II+projection+neurons+in+the+rat+medial+entorhinal+cortex.">Morphological characteristics of layer II projection neurons in the rat medial entorhinal cortex.</a> Hippocampus. 7, 571-583 (1997).</li><li>van Groen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entorhinal+cortex+of+the+mouse:+cytoarchitectonical+organization.">Entorhinal cortex of the mouse: cytoarchitectonical organization.</a> Hippocampus. 11, 397-407 (2001).</li><li>Dolorfo, C. L., Amaral, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entorhinal+cortex+of+the+rat:+organization+of+intrinsic+connections.">Entorhinal cortex of the rat: organization of intrinsic connections.</a> The Journal of Comparative Neurology. 398, 49-82 (1998).</li></ol>

Nothing to disclose.

To facilitate investigation of MEC circuit properties that follow a dorsal-ventral organization we have described here in detail a procedure for producing a parasagittal slice preparation that preserves the dorsal-ventral extent of the MEC.
Critical steps
Removing the brain from the animal. Take particular care to avoid exerting pressure on the brain. This is more important than rapid removal of the brain.
Slicing.</...

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td>Cutting ACSF(mM)</td>
 <td>Standard ACSF(mM)</td>
 <td>Internal solution (mM)</td>
 <td>CASNumber</td>
 <td>Supplier Catalogue Number</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>86</td>
 <td>124</td>
 <td></td>
 <td>7647-14-5</td>
 <td>Sigma S9888</td>
 </tr>
 <tr>
 <td>NaH2PO4</td>
 <td>1.2</td>
 <td>1.2</td>
 <td></td>
 <td>13472-35-0</td>
 <td>Sigma 71505</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>2.5</td>
 <td>2.5</td>
 <td>10</td>
 <td>7447-40-7</td>
 <td>Sigma P3911</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>25</td>
 <td>25</td>
 <td></td>
 <td>144-55-8</td>
 <td>Fischer S/4240</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>25</td>
 <td>20</td>
 <td></td>
 <td>50-99-7</td>
 <td>Sigma G5767</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>75</td>
 <td></td>
 <td></td>
 <td>57-50-1</td>
 <td>Sigma S5016</td>
 </tr>
 <tr>
 <td>CaCl2</td>
 <td>0.5</td>
 <td>2</td>
 <td></td>
 <td>10043-52-4</td>
 <td>VWR 190464K</td>
 </tr>
 <tr>
 <td>MgCl2</td>
 <td>7</td>
 <td>1</td>
 <td>2</td>
 <td>7786-30-3</td>
 <td>Sigma 63020</td>
 </tr>
 <tr>
 <td>K Gluconate</td>
 <td></td>
 <td></td>
 <td>130</td>
 <td>299-27-4</td>
 <td>Sigma G4500</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td></td>
 <td></td>
 <td>10</td>
 <td>7365-45-9</td>
 <td>Sigma H3375</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td></td>
 <td></td>
 <td>0.1</td>
 <td>67-42-5</td>
 <td>Sigma E4378</td>
 </tr>
 <tr>
 <td>Na2ATP</td>
 <td></td>
 <td></td>
 <td>2</td>
 <td>34369-07-8</td>
 <td>Sigma A7699</td>
 </tr>
 <tr>
 <td>Na2GTP</td>
 <td></td>
 <td></td>
 <td>0.3</td>
 <td>36051-31-7</td>
 <td>Sigma G8877</td>
 </tr>
 <tr>
 <td>NaPhospho-Creatine</td>
 <td></td>
 <td></td>
 <td>10</td>
 <td>19333-65-4</td>
 <td>Sigma P7936</td>
 </tr>
 <tr>
 <td>Biocytin (optional)</td>
 <td></td>
 <td></td>
 <td>2.7</td>
 <td>576-19-2</td>
 <td>Sigma B4261</td>
 </tr>
 </tbody> 
</table>
Table 1. Cutting ACSF, standard ACSF and K-Gluconate internal solution recipes.

preparation of parasagittal slices for the investigation of dorsal-ventral organization of the rodent medial entorhinal cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

Watch this Scientific Journal Video about Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex at JoVE.com

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

preparation-parasagittal-slices-for-investigation-dorsal-ventral

Research

JoVE Journal

Neuroscience

15.5K visualizações.  University of Edinburgh. Nós descrevemos processos de preparação e de gravação electrofisiológico de fatias do cérebro que mantêm o eixo dorso-ventral do córtex entorrinal medial (MEC). Devido codificação neural da localização seguinte uma organização dorsal-ventral dentro do MEC, estes procedimentos facilitar a investigação dos mecanismos celulares importantes para navegação e de memória.