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 be necessary to determine if the choice of anesthetic influences neuronal properties.
Remove the brain from the mouse and immediately place in cold (4-8 &deg;C) cutting artificial cerebrospinal fluid (ACSF) (see Table 1 for solution compositions) bubbled to saturation with carbogen (95% O2, 5% CO2).
After a maximum of three minutes, carefully remove the brain from the cutting ACSF using a spatula and gently place it in an upright position (dorsal side facing upwards) onto filter paper that has been moistened with the cutting ACSF (Figure 1A).
To facilitate accurate mounting of the hemispheres, use a razor or scalpel to remove as much of the cerebellum as possible without impacting the MEC (located at the caudal extreme of the cerebrum) and remove the rostral third of the cerebrum by sectioning in the coronal plane (Figure 1B).
Hemisect the brain, taking care that the section is exactly along the vertical plane of the midline (Figure 1C).
Return the hemispheres to the bubbled cutting ACSF for one and a half minutes.
1.2 Mount the hemispheres on a vibratome
Before mounting, ensure that the cutting edge of the vibratome blade is angled at 20 degrees from horizontal (Figure 1D). On the mounting surface make a shallow strip of superglue parallel to the vibratome blade, approximately the width of a hemisphere and long enough to accommodate the two hemispheres end-to-end.
Taking care to minimize physical impact, remove each hemisphere from the cutting ACSF with a spatula and position so that its medial surface rests on the spatula and its dorsal extent faces towards the microtome blade. Gently slide each hemisphere onto the strip of superglue, taking care to ensure that the medial surface of each hemisphere is parallel to the microtome base. We have found that for best results the dorsal surface of each hemisphere should be parallel to and face the vibratome blade (Figure 1D).
1.3 Maintenance of the preparation during slicing
Following mounting immediately submerge the hemispheres in cold cutting ACSF (4 - 8 &deg;C). Maintain the temperature and carbogen saturation throughout the slicing procedure. If direct cooling and bubbling of the solution in the cutting chamber is not practical, periodically replenish the cutting ACSF solution in the chamber with fresh chilled and bubbled solution.
1.4 Cut sections
Using a vibratome, remove cortex from both hemispheres in the sagittal plane until you identify the lateral most extent of the MEC (typically ~1mm down from the lateral surface) (Figure 1F).
Between cuts lift the vibratome blade up ~200 &mu;m to avoid dragging the blade back over and damaging the exposed tissue. Simultaneously cut 400 &mu;m parasagittal sections from both hemispheres (if they are lined up as shown in Figure 1D they will both be cut at the same time) until the medial extent of the MEC is reached. This can be identified by the absence of the thick white band around the ventro-caudal curve of the hippocampus (the external capsule), the non-convex shape of its rostral boundary and the angular dorso-caudal 'corner'. Figure 1E illustrates the circular appearance of the hippocampus in the parasagittal plane lateral to the MEC. Figures 1F-G illustrate how sections within the MEC appear at different lateral-medial positions when slicing. Note the progressively more bean-shaped appearance of the hippocampus at more medial positions. Each hemisphere typically yields two or three 400 &mu;m thick slices containing the MEC.
1.5 Incubate slices
After each cut immediately place the slices in carbogen-saturated standard ACSF maintained in a water bath at 35 &deg;C. Allow slices to incubate at 35 &deg;C for approximately 15 minutes in the ACSF after slicing is complete.
Remove the slice holder from the water bath and continue bubbling with carbogen at room temperature for at least 45 minutes.
2. Example Parasagittal Slice Experiment
A typical experiment using this preparation is to make electrophysiological recordings from stellate cells in layer II of the MEC.
2.1 Optimize optics
Before recording, ensure that the condenser is in focus on the slice plane (Koehler illumination) and is centered under the high magnification (e.g. 40x) objective.
2.2 Identify cells of interest
Use a low magnification (e.g. 4x) objective to identify an approximate recording region within the MEC (Figure 2A-B). Switch to a high magnification objective to identify viable cells within this region (Figure 2C-D).
As an example, putative layer II stellate cells are visually identified by their polygonal or ovoid shape and multiple primary dendrites with similar diameter, and absence of a single large diameter apical dendrite2,13,14. They are reliably identified on the edge of the Layer I/II border, where they are abundant and often appear in small groups2,9 (Figure 2C-D). Other cell types, such as interneurons and pyramidal cells should also be identifiable.
At this point experiments can be carried out, for example using whole-cell patch-clamp to record membrane potential or membrane current from identified neurons, and electrical or optogenetic methods to activate synaptic inputs to the recorded neuron. Neuron identity can be verified from the electrophysiological properties of the recorded neuron and by including fluorescent labels within the intracellular solution.
3. Measure Location Along the Dorso-ventral Axis
3.1 Image location of interest and surrounding slice
To determine the position of a recorded neuron along the dorsal-ventral axis, first use a low magnification objective to image the MEC region of the slice and surrounding areas. Mark the location of interest by, for example, including the recording electrode in an image (Figure 3A) or by stepping down the field iris diaphragm to leave a bright circle around the location of interest in a duplicate image. To pinpoint the recording location within the image the duplicate can then be superimposed on the initial image of the recording location and its surroundings (Figure 3B). Up to 3 separate low magnification (4x) images may be required to cover the area from a ventral recording location to the dorsal border of the MEC. These images can then be stitched together using image-manipulation software to provide an image that distance measurements can be taken from (Figure 3). Further verification of neuron identity and location can be carried out following recording by including inactive markers such as biocytin or Alexa dyes within the intracellular solution and then using appropriate processing of the tissue following recording2.
3.2 Establish the dorsal border of MEC
The dorsal border of the MEC provides a convenient landmark from which to measure dorsal-ventral position. The ventral border of the MEC is not as well defined.
Figure 4 illustrates how the MEC cellular landmarks defined in Nissl stained slices at different medial-lateral positions appear under DIC illumination.
Figure 4 A shows the circular hippocampus (Figure 4 (i)) and lack of a parasubicular protrusion into layer I (Figure 4 (iv)) associated with parasagittal slices containing Lateral Entorhinal Cortex (LEC).
Figure 4 B-C show typical slices that contain the MEC. The dorsal part of the prominent dark dorsal Entorhinal/Parasubicular boundary region in the DIC illuminated slices contains an area of the parasubiculum. The parasubiculum area is clearly revealed by Nissl staining in the corresponding images in column (iii). The dorsal border of the MEC (black arrows in 4B and 4C column (iv)) is ventral to the group of parasubicular cells that protrudes far out into layer I (red arrows in 4B and 4C column (iv)).
Comparison of Figures 4B and 4C columns (ii) and (iii) shows that the dorsal border of the MEC in the Nissl sections corresponds to a location that is ventral to the dorsal edge of the dark Entorhinal/Parasubicular boundary region in the DIC slice (black arrows). The location of the border can be estimated from DIC images and comparison to reference images (see also ref. 14). Future validation of the dorsal MEC border with molecular markers will improve the accuracy of this estimate. We note that stained reference sections, and sections that are processed for morphological identification of labeled neurons, may be subject to considerable shrinkage. Comparison of absolute distances relative to landmarks in DIC and reference sections therefore first requires measurement and correction for shrinkage (see e.g. ref 2).
3.3 Calibrate and measure distances
To facilitate easy measurement of distance in images, use the same low magnification objective to image a reference grid to establish a pixel:distance conversion ratio. Measure the pixel distance from the dorsal border of the MEC to the location of interest along the contour of the MEC using a graphics program and convert pixel distance to distance in &mu;m (Figure 5A). If neurons are filled with a marker such as biocytin, then location of the recorded neuron can then also be recovered by appropriate processing (see e.g. ref 2).
4. Representative Results
Figure 5A shows an example low magnification (4x) composite image of a parasagittal slice after recording, with the locations of recorded neurons and measurement guides superimposed. Recordings from the marked dorsal and ventral stellate cells are shown in Figure 5B. These recordings help to establish the identity of the cells and illustrate how electrical properties of MEC layer II stellate cells differ at dorsal and ventral locations.
<img alt="Figure 1" src="/files/ftp_upload/3802/3802fig1.jpg" /> 
Figure 1. Preparation of parasagittal slices. A Whole brain resting with dorsal side facing upwards on filter paper moistened with cutting ACSF. B Brain after removal of cerebellum and rostral third of frontal cortex. C Hemisected brain ready for mounting. D mounted hemispheres on glue strip parallel to cutting edge of vibratome blade prior to submersion in cutting ACSF and slicing. E Appearance of section containing LEC during the slicing procedure after the removal of lateral tissue and not yet sufficiently medial for a standard parasagittal MEC slice. F Appearance of 'lateral' part of the MEC after removing a further 400 &mu;m of tissue (the surface is 400 &mu;m medial to that shown in (F)). G Appearance of 'medial' part of the MEC after a 400 &mu;m parasagittal slice has been cut. H-I Schematics of E-F respectively indicating anatomical landmarks, c: cerebellum, L: lateral entorhinal cortex (LEC), m: Medial Entorhinal Cortex (MEC), h: hippocampus, orange: external capsule, blue: corpus callosum, cyan: dentate gyrus, green: CA3 &amp; CA1. The appearance of rostral boundary of the hippocampus in slices that contain LEC is concave (black arrow in H) whereas in slices that contain only MEC, the boundary can be linear (red arrow in I) or concave (red arrow in J). The approximate shapes of colored anatomical landmarks were obtained from the annotations in the Allen Brain Atlas (<a href="http://mouse.brain-map.org/atlas/ARA/Sagittal/browser.html">http://mouse.brain-map.org/atlas/ARA/Sagittal/browser.html</a>).
<img alt="Figure 2" src="/files/ftp_upload/3802/3802fig2.jpg" /> 
Figure 2. Identifying MEC layer II cells under DIC illumination. A Example composite image of a parasagittal brain slice at low magnification (4x). Separate images have been aligned and blended to remove distracting edges and vignetting. Important landmark areas h: hippocampus, m: MEC. B Crop from an individual low magnification (4x) image that contains layer II of the MEC, visible as the dark vertical stripe near the right hand edge. C MEC layer II at high magnification (40x) with DIC illumination. The image is an aligned and blended composite of several images. The dense central 'column' of cells is Layer II. D Single high magnification DIC image showing a group of healthy putative stellate cells and interneurons in layer II. As shown here, stellate cells often occur in groups on the border of layer I/II. Pyramidal cells tend to be found closer to the Layer II/III border. The dotted outlines in A-C indicate the extent of B-D respectively. Scale bars: in A and B 500 &mu;m, in C and D 100 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3802/3802fig3.jpg" /> 
Figure 3. Measuring the dorsal-ventral position of locations of interest. A Aligned low magnification (4x) images that include the entire dorso-ventral extent of the MEC between the location of interest (marked by the tip of a recording electrode) and the dark Entorhinal / Parasubicular boundary region landmark (used to establish the dorsal border of the MEC - see Figure 4). B As in A but including a superimposed image with a stopped down field iris diaphragm (overlaid with reduced opacity) instead of a recording electrode to mark the location of interest.
<img alt="Figure 4" src="/files/ftp_upload/3802/3802fig4.jpg" /> 
Figure 4. Estimating the dorsal border of the MEC from DIC images of parasagittal slices. A-C Parasagittal sections (lateral to medial) from the Entorhinal Cortex. DIC and Nissl sections are from different mice. A-C column (i): Whole parasagittal slices (400 &mu;m thick) (aligned and blended composites of low magnification (4x) images). A-C column (ii): Close up of entorhinal cortex with characteristic dark Entorhinal / Parasubicular boundary region in the top area of each image. A-C column (iii) Nissl stained slices (40 &mu;m thick) from a different mouse aligned with images in A-C (ii). The darker, dense layer of cells is layer II. Comparing columns (ii) and (iii) shows how the Nissl stained cells appear in DIC images. A-C column (iv): Detail of Nissl stained slices. B and C column (iv) include cells from the parasubiculum (dorsal part of the dark Entorhinal/Parasubicular boundary region in the DIC images) and cells from dorsal MEC. In B (iv) and C (iv) the large dorsal patch of parasubicular cells that extends deep into layer I is easily visible (red arrows). The ventral edge of these patches corresponds to the dorsal border of the MEC (black arrows). In A (iv) the parasubicular patch is absent, indicating that the slice is too lateral for a standard parasagittal MEC slice preparation. In all panels, black arrows indicate the dorsal border of MEC. Grey arrows indicate the approximate ventral border of the MEC.
<img alt="Figure 5" src="/files/ftp_upload/3802/3802fig5.jpg" /> 
Figure 5. Representative results. A Blended and cropped portion of the image in Figure 3A. The positions of a dorsal cell and a ventral cell are indicated by blue and green filled circles respectively. The black arrow marks the estimated dorsal border of the MEC and is extended into the deep layers by the white dotted line. The solid white line is a guide showing the contour path along which the pixel measurement of distance from the dorsal border of the MEC to the ventrally located cell was taken. Scale bar: 500 &mu;m. B Electrophysiological traces from the cells indicated in A. From left to right - subthreshold responses to current steps used to calculate input resistance, spiking response to a large positive current step, spike detail.

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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.

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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 ...

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15.5K Views.  University of Edinburgh. Мы описываем процедуры подготовки и электрофизиологические записи с срезах мозга, которые поддерживают спинно-вентральной оси медиальное энторинальной коры (MEC). Поскольку нейронная кодирования место следующим спинно-вентрально организации в MEC, эти процед?...

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

Brain Imaging Investigation of the Neural Correlates of Emotion Regulation

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress has been made in understanding conscious/deliberate emotion regulation (ER), less is known about non-conscious/automatic ER and the associated neural correlates. This is in part due to the problems inherent in the unitary concepts of automatic and conscious processing1. Here, we present a protocol that allows investigation of the neural correlates of both deliberate and automatic ER using functional magnetic resonance imaging (fMRI). This protocol allows new avenues of inquiry into various aspects of ER. For instance, the experimental design allows manipulation of the goal to regulate emotion (conscious vs. non-conscious), as well as the intensity of the emotional challenge (high vs. low). Moreover, it allows investigation of both immediate (emotion perception) and long-term effects (emotional memory) of ER strategies on emotion processing. Therefore, this protocol may contribute to better understanding of the neural mechanisms of emotion regulation in healthy behaviour, and to gaining insight into possible causes of deficits in depression and anxiety disorders in which emotion dysregulation is often among the core debilitating features.

We present a protocol that allows investigation of the neural correlates of deliberate and automatic emotion regulation, using functional magnetic resonance imaging. This protocol can be used in healthy participants, both young and older, as well as in clinical patients.

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress ...

Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the neural mechanisms underlying this enhancing effect of emotion on memory. A method that has proven particularly influential in the 
investigation of the memory-enhancing effect of emotion is the so-called subsequent memory paradigm (SMP). This method was originally used to investigate the 
neural correlates of non-emotional memories3, and more recently we and others also applied it successfully to studies of emotional memory (reviewed in4, 5-7). 
Here, we describe a protocol that allows investigation of the neural correlates of the memory-enhancing effect of emotion using the SMP in conjunction with 
event-related functional magnetic resonance imaging (fMRI). An important feature of the SMP is that it allows separation of brain activity specifically 
associated with memory from more general activity associated with perception. Moreover, in the context of investigating the impact of emotional stimuli, 
SMP allows identification of brain regions whose activity is susceptible to emotional modulation of both general/perceptual and memory-specific processing. 
This protocol can be used in healthy subjects8-15, as well as in clinical patients where there are alterations in the neural correlates of emotion perception 
and biases in remembering emotional events, such as those suffering from depression and post-traumatic stress disorder (PTSD)16, 17.

We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations.

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the ...

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...