Name: generation and on-demand initiation of acute ictal activity in rodent and human tissue
Uploaded: 2019-01-19T12:30:00
Description: Watch this Scientific Journal Video about Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue at JoVE.com

This work was supported by the Canadian Institutes of Health Research (MOP 119603 to Peter L. Carlen and Taufik A. Valiante), the Ontario Brain Institute (to Taufik A. Valiante), and the Mightex Student Research Grant (to Michael Chang). We would like to thank Liam Long for his assistance in filming the video manuscript. We would like to acknowledge Paria Baharikhoob, Abeeshan Selvabaskaran, and Shadini Dematagoda for their assistance in compiling the figures and tables in this manuscript. Figures 1A, 3A, 4A, and 6A are all original figures made from data published in Chang et al.16.

All research involving patients was performed under a protocol approved by the University Health Network Research Ethics Board in accordance with the Declaration of Helsinki. Procedures involving animals were in accordance with guidelines by the Canadian Council on Animal Care and approved by the Krembil Research Institute Animal Care Committee.
1. Protocol I: Acute In vitro Seizure Model
<ol>
	<li>Preparation of dissection solutions and artificial cerebrospinal fluid</stron...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+definitions+and+classifications+of+seizure+emergencies.">Overview: definitions and classifications of seizure emergencies.</a> Journal of Child Neurology. 22 (5_suppl), 9S-13S (2007).</li><li>L&#246;scher, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+review+of+current+animal+models+of+seizures+and+epilepsy+used+in+the+discovery+and+development+of+new+antiepileptic+drugs.">Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs.</a> Seizure. 20 (5), 359-368 (2011).</li><li>Jones, R. S., da Silva, A. B., Whittaker, R. G., Woodhall, G. L., Cunningham, M. 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The brain slices are treated with a proconvulsant drug or an altered ACSF perfusate to increase the neural network&#39;s excitability and promote a precipitation of ictal events (electrographic seizure-like events). For mice, the preferred coronal slices of the somatosensory-motor area should contain the cingulate cortex, area 2 (CG), but not the retrosplenial area (RS); these anatomical markers help identify the range of coronal slices that are best for inducing ictal events. An optional modification for mice tissue is ...

Acute seizure models can successfully reproduce electrographic signatures reminiscent of ictal events observed in the electroencephalogram (EEG) of individuals experiencing a seizure. Researchers use these ictal-like events (herein referred to as &#39;ictal events&#39;) as surrogates for the seizure event1. Clinically, ictal events serve as a reliable proxy for seizure events since seizures are a neurological disorder which originates from the brain. In the epilepsy monitoring unit, neurologists rely upon the detection of ictal events to confirm the brain&#39;s epileptogenic region and isolate it for resection2. In the intensive care unit, physicians monitor ictal activity to assess if any seizure activity persists in sedated patients3. Controlling seizures remains to be a challenging issue for the medical community, as 30% of epilepsy patients are drug resistant to the available medication4,5, and 10% of medical cases involving drug-induced seizures are unresponsive to the standard treatment3. This presents a serious concern for the society, as 10% of the American population is prospected to experience one seizure event in their lifetime and 3% are expected to develop epilepsy6.
Studying seizures in chronic epilepsy models is expensive, laborious, and often take months to prepare7. It is also difficult to perform electrophysiological recordings in freely moving animals. Human clinical trials face similar issues, as well as additional complexities related to patient consent, variability in participants&#39; backgrounds, and the moral and ethical considerations involved8. Acute seizure models, on the other hand, are favorable because they are relatively convenient to prepare, cost-efficient, and capable of generating large volumes of ictal events for study9. Additionally, the tissue is fixed in a stable position, so the conditions are ideal for performing the electrophysiological recordings necessary to study seizure dynamics and the related underlying pathophysiology. Acute seizure models remain favorable over in silico (computer) models because they are based on biological material comprised of the brain&#39;s constituent neuronal network with all its inherent factors and synaptic connectivity, that may not be captured by even the most detailed computer models10. These features make acute seizure models poised to be efficient at screening for potential anti-seizure therapies and making preliminary findings before advancing them for further investigation in chronic epilepsy models and clinical trials.
Typically, acute seizure models are derived from the normal brain tissue that has been subjected to hyper-excitable conditions. To induce clinically relevant ictal events in healthy brain tissue, it is important to understand that the brain functions optimally in a critical state11 where excitation (E) and inhibition (I) are balanced12. A disruption of the E-I balance can lead to the hyper-excitable seizure state in which ictal events precipitate. Accordingly, within this conceptual framework, there are two major strategies to generate ictal events in brain slices (in vitro) or in whole-brain (in vivo) preparations: either decreased inhibition (&#34;disinhibition&#34;) or increased excitation (&#34;non-disinhibition&#34;). However, ictal events are highly ordered and synchronized events that require the influence of GABAergic interneurons to orchestrate the neural network activity13,14. For this reason, non-disinhibition models are the most effective for generating ictal events in isolated neural networks, such as in an in vitro brain slice15, whereas in vitro disinhibition models commonly lead to spiking activity reminiscent of interictal-like spiking. Furthermore, within this conceptual framework, a momentary synchronizing event can also reliably trigger an ictal event16. In fact, an ictal event can be triggered by any minor perturbation applied to the neural system17 when it is at a critical state transition (&#34;bifurcation&#34;) point18. Traditionally, these perturbations were induced by electrical stimulation. The recent development of optogenetics in neuroscience, however, now offers a more elegant strategy to induce critical state transitions16.
The methods described in this paper demonstrate how to generate ictal events on-demand in acute seizure models for both in vitro (step 1 of the Protocol) and in vivo studies (step 2 of the Protocol). They involve the choice of brain region, seizure induction method, study type, and species; however, the focus will be on the recommended choice of an acute 4-AP cortical seizure model because of its versatility in a wide variety of study types. The acute in vitro 4-AP seizure model is based on the standard protocol to prepare high-quality brain slices for electrophysiological recordings and imaging studies19. These protocols have already been used to make in vitro coronal brain slices from the somatosensory-motor cortex of mice16,20 and humans21. Modifications to generate ictal events in these types of brain slices have been previously demonstrated16 and the full details are described in the Protocol below. The acute in vivo 4-AP cortical seizure model is based on the standard protocol to prepare a craniotomy for imaging studies22. The modification is that no (glass slide) window is installed following the craniotomy. Instead, proconvulsant agents (4-AP) are topically applied to the exposed cortex to induce ictal events while the animal is under general anesthesia. To our knowledge, our group was the first to develop this acute in vivo cortical seizure model in mice16,23. The acute in vivo 4-AP cortical seizure model prepared from adult mice was developed to complement the in vitro slice model from juvenile tissue. The replication of findings in the adult in vivo seizure model helps to generalize the findings from slice models by addressing the inherent concerns regarding the non-physiological conditions of a 2D brain slice (versus a 3-D whole-brain structure) and the physiological differences between juvenile and adult tissue.
The method of on-demand ictal event initiation is demonstrated using either puffs of neurotransmitters with a picospritzer or optogenetic strategies. To the best of our knowledge, our group is the first to initiate ictal events in human tissue using neurotransmitters via a picospritzer16. For optogenetic strategies, the C57BL/6 mice strain is the conventional strain used for expressing transgenes. The expression of channelrhodopsin-2 (ChR2) in either GABAergic interneurons or glutamatergic pyramidal cells will provide the optional ability to generate ictal events on-demand with brief light pulses. Suitable optogenetic mice strains include the commercially available C57BL/6 variant that expresses ChR2 in either interneurons, using the mouse vesicular GABA transporter promotor (VGAT)24, or pyramidal cells, using the mouse thymus cell antigen 1 promotor (Thy1)25. These commercially available VGAT-ChR2 and Thy1-ChR2 mice offer the opportunity to activate GABAergic neurons or glutamatergic neurons, respectively, in the neocortex with blue (470 nm) light. The ability to generate ictal events on demand in acute seizure models can offer novel opportunities to study seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

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	<tbody>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through the Toronto Western Hospital&#39;s Suppliers</td>
		</tr>
		<tr>
			<td>1 mm syringe</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>25G 5/8&#8221; sterile needle</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Single edge razor blade (2x)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Instant adhesive glue</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Lens paper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Glass petri dish (2x)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Splinter forceps (2x)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>PVC handle micro spatula</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Micro spoon with flat end</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Detailing brush 5/0</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purcahsed from a boutique art store</td>
		</tr>
		<tr>
			<td>Wide bore transfer pipette</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Dental Tweezer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Thermometer (digital)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased on Amazon.ca</td>
		</tr>
		<tr>
			<td>Check carbogen tank (95%O2/5%CO2)&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through the Toronto Western Hospital&#39;s Suppliers</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>N/A</td>
			<td>Purchased through the Toronto Western Hospital&#39;s Suppliers</td>
		</tr>
		<tr>
			<td>brain slice incubation chamber (a.k.a. brain slice keeper)&#160;</td>
			<td>Scientific Systems Design Inc</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4 H2O)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate (HNaPO4&#183;H2O)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2&#183;2H2O)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
	</tbody>
</table>

generation and on-demand initiation of acute ictal activity in rodent and human tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

The application of 100 &#181;M 4-AP to good-quality (undamaged) 450 &#181;m-sized cortical brain slices from a juvenile VGAT-ChR2 mouse reliably induced recurrent ictal events (&#62; 5 s) within 15 min (Figure 1Ai). The application of 100 &#181;M 4-AP to slices of poor-quality resulted in bursting events or spiking activity (Figure 1Aii). On average, 40% of the slices from each dissected mouse brain successfully generated ictal e...

Watch this Scientific Journal Video about Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue at JoVE.com

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

This method can help answer key questions in the study of seizure mechanisms, such as, what neural subpopulations are responsible for electrographic seizure onset and termination. The main advantage of this technique, is that it can reproduce electrographic seizure events, on demand, and in vivo and in vitro models that are reminiscent of those observed clinically. This technique can also be used to search for potential anti-seizure therapies, by attempting to trigger electrographic seizure events during the application of different anti-seizure drug candidates.To prepare the cortical slices, glue the preclinical brain tissue onto the Vibratome stage, using instant adhesive glue. Gently place the specimin holder into the buffer tray. Insure that the dorsal portion of the brain is facing the Vibratome's blade.Slice the brain into 450 micrometer thick sections, in the dorsal to ventral direction. Make the first cut in the preclinical animal brain to remove the olfactory bulb. Then, make subsequent cuts until the somatosensory motor area is observed.Use a wide bore pipette. Transfer the targeted coronal slices to a Petrie dish containing cold dissection solution. For the coronal slices, make a transverse cut just below the neo cortical commissure, and then, cut at the midline to separate the hemispheres.Then, use a new razor blade to cut off any excess tissue from the slices. It's critical to perform the brain-slicing procedure efficiently. Every moment the brain tissue is not submerged in ACSF, is detrimental to its quality.Damaged, poor quality brain tissue is less likely to generate electrographic seizure events. Transfer the dorsal portion of the coronal slices, that contain the neo cortex, to a second Petrie dish filled with 35 degree Celsius ACSF, for a moment. Then, promptly transfer the slices to an incubation chamber, containing 35 degrees Celsius carbogenated ACSF.Leave the brain slices slightly submerged in the incubation chamber at 35 degrees Celsius for 30 minutes. Then, remove the incubation chamber from the water bath and allow it to return to room temperature. Wait one hour for the brain slices to recover, before performing electrophysiological recordings.In this procedure, cut out lens paper that is slightly larger than the brain slice. Use a wide-bore pipette or a detailing brush to transfer a brain slice onto the cut out lens paper that is held in place using a dental tweezer. Then, transfer the lens paper with a brain slice to the recording chamber, and secure it into position with a harp screen.Subsequently, profuse the brain slice in the recording chamber with carbogenated ACSF at 35 degrees Celsius at a rate of three milliliters per minute. Use a digital thermometer to ensure the recording chamber is 33 to 36 degrees Celsius. Then, backfill the glass electrodes with 10 microliters of ACSF, using a Hamilton syringe.Under the 20 times stereomicroscope, guide the recording glass electrode into the superficial cortical layer, two, three, using manual manipulators. View the electrical activity of the brain slice with standard software. To induce electrographic seizure-like activities, profuse the brain slice with ACSF containing 4-AP at 100 micromolar.View the electrical activity of the brain slice with standard software. To generate electrographic seizure-like events, using optogenetic strategy on the brain slices from optogenetic mice, use a manual manipulator to position a 1, 000 micron cord diameter optical fiber directly above the recording region. Apply a brief pulse of blue light to initiate an ictal event.A user-friendly MATLAB based program was specifically developed to detect and classify the various types of epileptiform events that occur in the in vitro and in vivo 4-AP seizure models. This detection program is available from the Valiante Labs GitHub repository. The application of 100 micromolar 4-AP to good quality 450 micron-sized cortical brain slices from a juvenile VGAT channelrhodopsin mouse, reliably induced recurrent ictal-like events within 15 minutes.The application of 100 micromolar 4-AP to slices of poor quality, resulted in bursting events or spiking activity. On average, 40%of the slices from each dissected preclinical brain, successfully generated ictal-like events. Moreover, 83%of the dissected mice resulted in at least one brain slice that successfully generated ictal-like events.In brain slices with spontaneously occurring ictal-like events, the application of a brief 30 millisecond light pulse on the brain slice, reliably triggered an ictal-like event that was identical in morphology. The same findings were made in brain slices from Thy one Channelrhodopsin two mice. Thus, regardless of which neuronal subpopulation was activated, any brief synchronizing event in the isolated cortical neural network, led to the onset of an ictal-like event.These ictal-like events were comprised of a sentinel spike, tonic-like firing, clonic-like firing, and burst-like activity toward the end. They were similar in nature to the electrographic signatures associated with clinical seizures. Following this procedure, other types of brain state transitions can be performed to address questions like, what are the neuro biological mechanisms underlying brain state transitions?And how can we regulate these transitions to prevent entry into various pathological brain states?

generation-on-demand-initiation-acute-ictal-activity-rodent-human

Research

JoVE Journal

Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

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 ...

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 ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury.
The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion.
In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

Severe spinal cord injuries often result in tissue defects. Two possibilities are described to successfully bridge such gaps to promote tissue adaptation, regenerative responses and functional improvement in rats via implantation of a mechanical microconnector system after acute injury and five weeks after complete spinal cord transection.

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...