Name: electroconvulsive seizures in rats and fractionation of their hippocampi to examine seizure-induced changes in postsynaptic density proteins
Uploaded: 2017-08-15T14:00:00
Description: Watch this Scientific Journal Video about Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins at JoVE.com

The authors thank Dr. Eric C. Bolton for allowing us to use his centrifuge for fractionation and Dr. Graham H. Diering in Dr. Richard L. Huganir's lab at John's Hopkins University for providing us with the small-scale protocol for the PSD fractionation.

All experimental procedures including animal subjects have been approved by the Institutional Animals Care and Use Committee at the University of Illinois at Urbana-Champaign.
1. Maintaining a Rat Colony
<ol>
	<li>Breed Sprague-Dawley rats (see the Table of Materials) and maintain them in standard conditions with a 12-h light-dark cycle and ad libitum access to food and water.</li>
	<li>Wean the rat pups at postnatal day (P) 28 and house them in groups of 2 - 4 male or femal...

Here, we describe an ECS induction method in rats that elicits the global stimulation of neuronal activity in their hippocampi. ECS is an animal model of electroconvulsive therapy, which is clinically used to treat drug refractory depressive disorders in humans1,2,3. Despite use of electroconvulsive therapy to treat severe depression, the precise underlying mechanism remains unclear. Because ECS induces anti-depressant-like beha...

Electroconvulsive therapy has been used to treat patients with major depressive disorders, including severe drug-resistant depression, bipolar depression, Parkinson&#39;s diseases, and schizophrenia1,2. In this therapy, seizure is generated by electrical stimulus delivered to the head of anesthetized patients via epicranial electrodes1,2,3. Repetitive administration of ECS has been clinically beneficial to drug-resistant depressive disorders1,2,3. However, the exact mechanism underlying the long-term efficacy of the antidepressant effect in humans has remained elusive. ECS is an animal model of electroconvulsive therapy and is widely used to investigate its therapeutic mechanism. In rodents, both acute ECS and chronic ECS treatment promote adult neurogenesis in the hippocampi and reorganize the neural network4,5, which is likely to contribute to improvements in cognitive flexibility. Furthermore, global elevation of brain activity by ECS alters the abundance of transcripts, such as a brain derived neurotropic factor6, and multiple proteins, including metabotropic glutamate receptor 17 and the N-methyl-D-aspartate (NMDA) type glutamate receptor subunits7. These changes are involved in mediating long-term modification of synapse number, structure, and strength in the hippocampus7,8,9.
In ECS models, electrical stimulation is delivered to rodents via stereotaxically implanted electrodes, corneal electrodes, or ear electrodes to evoke generalized tonic-clonic seizures10,11. Stereotaxic implantation of electrodes involves brain surgery and requires significant time to improve the experimenter&#39;s surgical skills to minimize injury. Less invasive corneal electrodes could cause corneal abrasion and dryness and require anesthesia. The use of ear-clip electrodes bypasses these limitations because they can be used on rodents without surgery or anesthesia and cause minimal injury. Indeed, we found that current delivered to awake rats via ear-clip electrodes reliably induces stage 4-5 tonic-clonic seizures and alters synaptic proteins in their hippocampi10.
To examine the ECS-induced abundance of synaptic proteins in the specific brain regions of the rodents, it is important to choose the experimental methods that are most suitable for their detection and quantification. Subcellular fractionation of the brain allows for the crude isolation of soluble cytosolic proteins; membrane proteins; organelle-bounds proteins; and even proteins in special subcellular structures, such as the PSD12,13,14. The PSD is a dense and well-organized subcellular domain in neurons in which synaptic proteins are highly concentrated at and near the postsynaptic membrane12,13,15. The isolation of the PSD is useful for the study of synaptic proteins enriched at the PSD, since dynamic changes in the abundance and function of postsynaptic glutamate receptors, scaffolding proteins, and signal transduction proteins in the PSD12,15,16,17 are correlated with synaptic plasticity and the synaptopathy observed in several neurological disorders17,18. A previous subcellular fractionation method used to purify the PSD involved the isolation of the detergent-insoluble fraction from the crude membrane fraction of the brain by the differential centrifugation of sucrose gradients14,19. The major challenge with this traditional method is that it requires large amounts of rodent brains14,19. Preparation of 10 - 20 rodents to isolate the PSD fraction per treatment requires extensive cost and time investment and is not practically feasible if there are many treatments.
To overcome this challenge, we have adapted a simpler method that directly isolates the PSD fraction, without sucrose gradient centrifugation20,21, and revised it to be applicable to PSD isolation from the hippocampi of a single rat brain.Our small-scale PSD fractionation method yields about 30 - 50 &#181;g of the PSD proteins from 2 hippocampi, sufficient for use in several biochemical assays, including immunoprecipitation and Western blotting. Western blotting demonstrates the success of our method for isolating the PSD by revealing the enrichment of postsynaptic density protein 95 (PSD-95) and the exclusion of presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin. Our ECS induction and small-scale PSD fractionation methods are easily adaptable to other rodent brain regions and provide a relatively simple and reliable way to evaluate the effects of ECS on the expression of PSD proteins.

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electroconvulsive seizures in rats and fractionation of their hippocampi to examine seizure-induced changes in postsynaptic density proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Using the detailed procedure presented here, one electrical shock (55 mA, 100 pulses/s for 0.5 s) delivered via ear-clip electrodes induced nonrecurring stage 4-5 tonic-clonic seizures in rats (Figure 1A-B). A total 8 of rats received acute ECS induction and displayed stage 4-5 tonic-clonic seizures. The seizures lasted about 10 s, and all rats recovered within 1 - 2 min of seizure cessation. Sham &#34;no seizure&#34; rats did not receive an ...

Watch this Scientific Journal Video about Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins at JoVE.com

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

The overall goal of the electroconvlusive seizure induction and subsequent smallscale fractionation of hippocampus is to induce global seizure activity in the rat brain, and to examine seizure activity induced alterations of proteins in the postsynaptic density of the hippocampus. This method can help answer key questions in the neural process of the film, such as which snaptic proteins are regulated in specific brain regions upon seizure, and whether such regulation can contribute to neuroplasticity. The main advantage of this electroconvulsive seizure induction protocol is that we can lively induce lo-bal allevation of law-dons brain activity without neuronal death.Demonstrating the procedure will be myself and Han Gil Jeong from Dr.Hee Jung Chung's laboratory. To begin this procedure, place a male rat in a clean, empty cage with a lid. Let the rat habituate for 30 minutes, then, set the pulse generator for ECS induction.Prepare the pulse generator by pushing the reset button and ensure that the ready button is lit. Make sure that the ear clips are not attached to the pulse generator. Next, press the shock button for a few seconds, then, plug the ear clips into the pulse generator.In this step, wet the ear clips with sterile saline and ensure that they are saturated. Next, wet the rat's ears with sterile saline by wrapping them in saline soaked gauze. Once they are wet, remove the gauze.Attach the clips to the ears at one clip per ear and position them beyond the main cartilage band. Press the shock button for a few seconds and observe the seizure. For the sham, no seizure control, treat the rat identically, but do not deliver the current.As clonus begins, disconnect the ear clips and record the seizure behavior according to a revised, Racine scale of one to five. This includes rearing with four limb clonus at stage four, and rearing and falling with four limb clonus at stage five. The seizure should last approximately 10 seconds.Record the seizure duration using a timer. Following seizure termination, return the rat to its home cage. Monitor the rat for another five minutes to make sure of its recovery from the seizures.Keep it singly housed in the cage, and return the cage to the recovery room. In this procedure, place two hippocampi from one rat onto a 30 milometer tissue culture dish and mince them into small pieces using scissors. After that, transfer the minced hippocampi to a manual glass homogenizer and add one milliliter of iced cold homogenization buffer.Next, insert a round pecil into a glass homogenizer. While the glass homogenizer is on ice, gently and steadily stroke up and down on the pecil, 10 to 15 times for one minute until small pieces of hippocampal tissue disappear. Then, transfer the homogenote to a 1.7 milometer microcentrifuge tube using a one milometer pipet and centrifuge the homogenote at 800 times G for 10 minutes at four degrees Celsius to separate the post nuclear supernatant from the pellet containing insoluble tissue and nuclei.Afterward, transfer 50 microliters and 950 microliters of the S1 fraction to two separate new 1.7 milliliter microcentrifuge tubes and store these tubes on ice. Also, save the P1 fraction pellet on ice. Subsequently, centrifuge the 950 microliter S1 fraction for 10 minutes at 13, 800 times G and four degrees Celsius to separate the supernatant enriched with cytosolic soluble proteins and the pellet enriched with membrane bound proteins, including synaptosomal proteins.Transfer the S2 fraction to a new 1.7 milliliter microcentrifuge and store it on ice. Following that, resuspend the P2 fraction pellet in 498 microliters of ice cold purified water. Add two microliters of one molar heeps to achieve a final concentration of four millimolar heeps.Incubate the suspension at four degrees Celsius with agitation for 30 minutes. After 30 minutes, store the resuspended P2 fraction on ice. In this step, centrifuge the P2 fraction for 20 minutes at 25, 000 times G and four degrees Celsius to separate the lysed supernatant and the lysed pellet.Transfer the LS2 fraction to a new 1.7 milliliter microcentrifuge tube and store it on ice. Then, resuspend the LP1 pellet in 250 microliters at 50 millimolar heeps, mixed with 250 microliters of one percent detergent, and 1 X PBS. Incubate the suspension at four degrees Celsius with gentle agitation for 15 minutes.Afterward, centrifuge the resuspended LP1 pellet for 3 hours at 25, 000 times G and four degrees Celsius to separate the non PSD fraction supernatant from the PSD fraction pellet. Remove the supernatant to a 1.7 milliliter microcentrifuge tube and resuspend the PSD pellet in 100 microliters of 50 millimolar heeps. In this figure, the representative western blots show the protein expression of NMDA receptive subunit GluN2B, ampa receptor, GluA2, and STEP 61 in the S2, P2, and PSD fractions from the hippocampi of sham, no seizure rats, and rats that received acute ECS.The cytoplasmic soluble protein alpha-Tubulin is enriched in the S2 fraction. Synaptophysin is a presynaptic vesicle protein and is enriched in the crude membrane P2 fraction but not in the PSD fraction, whereas PSD-95 is enriched in both the P2 and PSD fractions. Shown here, are the quantification of GluN2B and GluA2 in the P2 and PSD fractions at three and 24 hours after acute ECS.GluN2B and GluA2 expression in the P2 fraction was normalized to alpha-Tubulin in the S2 fraction. In GluN2B and GluA2 expression in the PSD fraction, was normalized to PSD 95 in the PSD fraction. Once mastered, this technique can be done in five to six hours if it is performed properly.Following this procedure, auto-masser like mass, spector-metry, based on protein mix can be performed in order to answer additional questions, what other synaptic proteins are altered by ECS. After its development, this technique paved the way for researchers in the field of neurological disease to explore the underlying mechanism of synaptic dysfunction in order for act of law-dons, trans-gen-ing motors of neurological diseases. After watching this video, you should have a good understanding of how to induce the ECS in law-dons, perform homogenization and PSD fractionation from the hippocampi and to examine alterations of synaptic proteins after ECS.

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Research

JoVE Journal

Neuroscience

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor learning. There exists a number of noninvasive electrophysiological methods investigating the excitability and plasticity of this pathway. However, most methods are based on quantification of compound potentials and neglect that the corticospinal pathway consists of many different connections that are more or less direct. Here, we present a method that allows testing excitability of different fractions of the corticospinal transmission. This so called H-reflex conditioning technique allows one to assess excitability of the fastest (monosynaptic) and also polysynaptic corticospinal pathways. Furthermore, by using two different stimulation sites, the motor cortex and the cervicomedullary junction, it allows not only differentiation between cortical and spinal effects but also assessment of transmission at the corticomotoneural synapse. In this manuscript, we describe how this method can be used to assess corticomotoneural transmission after low-frequency repetitive transcranial magnetic stimulation, a method that was previously shown to reduce excitability of cortical cells. Here we demonstrate that not only cortical cells are affected by this repetitive stimulation but also transmission at the corticomotoneuronal synapse at the spinal level. This finding is important for the understanding of basic mechanisms and sites of neuroplasticity. Besides investigation of basic mechanisms, the H-reflex conditioning technique may be applied to test changes in corticospinal transmission following behavioral (e.g., training) or therapeutic interventions, pathology or aging and therefore allows a better understanding of neural processes that underlie movement control and motor learning.

The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells or structures. These methods have been revolutionary for quantitative analysis of the mammalian brain, in which volumetric cell populations are too high to count manually, and stereology is now the technique of choice whenever estimates of three-dimensional quantities need to be extracted from measurements on two-dimensional sections. All stereological methods are in principle unbiased; however, they rely on proper knowledge about the structure of interest and the characteristics of the tissue. Stereology is based on Systematic Uniformly Random Sampling (SURS), with adjustment of sampling to the most efficient level in respect to precision, providing reliable, quantitative information about the whole structure of interest. Here we present the optical fractionator in conjunction with BrdU immunohistochemistry to estimate the production and survival of newly-formed neurons in the granule cell layer (including the sub-granular zone) of the rat hippocampus following electroconvulsive stimulation, which is among the most potent stimulators of neurogenesis. The optical fractionator technique is designed to provide estimates of the total number of cells from thick sections sampled from the full structure. Thick sections provide the opportunity to observe cells in their full 3-D extent and thus, allow for easy and robust cell classification based on morphological criteria. When correctly implemented, the sensitivity and efficiency of the optical fractionator provides accurate estimates with a fixed and predetermined precision.

Here, we present a stereological method, the optical fractionator, used to quantify the formation of new neurons, and their survival, in the rat hippocampus following electroconvulsive stimulation. When correctly implemented, the sensitivity and efficiency of stereological methods ensures accurate estimates with a fixed and predetermined precision.

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide limited information about muscle fiber membrane properties and underlying disease mechanisms. Muscle velocity recovery cycles (MVRCs) illustrate how the velocity of a muscle action potential depends on the time after a preceding action potential. MVRCs are closely related to changes in membrane potential that follow an action potential, thereby providing information about muscle fiber membrane properties. MVRCs may be recorded quickly and easily by direct stimulation and recording from multi-fiber bundles in vivo. MVRCs have been helpful in understanding disease mechanisms in several neuromuscular disorders. Studies in patients with channelopathies have demonstrated the different effects of specific ion channel mutations on muscle excitability. MVRCs have been previously tested in patients with neurogenic muscles. In this prior study, muscle relative refraction period (MRRP) was prolonged, and early supernormality (ESN) and late supernormality (LSN) were reduced in patients compared to healthy controls. Thereby, MVRCs can provide in vivo evidence of membrane depolarization in intact human muscle fibers that underlie their reduced excitability. The protocol presented here describes how to record MVRCs and analyze the recordings. MVRCs can serve as a fast, simple, and useful method for revealing disease mechanisms across a broad range of neuromuscular disorders.

Presented here is a protocol for the recording of muscle velocity recovery cycles (MVRCs), a new method of examining muscle membrane properties. MVRCs enable in vivo assessment of muscle membrane potential and alterations in muscle ion channel function in relation to pathology, and it enables the demonstration of muscle depolarization in neurogenic muscles.

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide ...