Name: simultaneous detection of c-fos activation from mesolimbic and mesocortical dopamine reward sites following naive sugar and fat ingestion in rats
Uploaded: 2016-08-24T13:00:00
Description: Watch this Scientific Journal Video about Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats at JoVE.com

Thanks to Diana Icaza-Culaki, Cristal Sampson and Theologia Karagiorgis for their hard work on this project.

These experimental protocols have been approved by the Institutional Animal Care and Use Committee certifying that all subjects and procedures are in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.
1. Subjects
<ol>
	<li>Purchase and/or breed male Sprague-Dawley rats (260 - 300 g).</li>
	<li>House rats individually in wire mesh cages. Maintain them on a 12:12 hr light/dark cycle with rat chow and water available ad libitum.</li>
	<li>Assign appropriate samp...

The goal of the study was to determine if the source (VTA) and forebrain projection targets (NAC, AMY, mPFC) of DA reward-related neurons were simultaneously activated after novel ingestion of fat and sugar in rats using the cellular c-fos technique. The present study is a detailed description of the protocols of a study published previously69. It was hypothesized that the VTA, its major projection zones to the prelimbic and infralimbic mPFC, the core and shell of the NAC and the basolateral and central-cortic...

Brain dopamine (DA) has been implicated in central responses to intake of palatable sugars through proposed hedonic1,2, effort-related3 and habit-based4,5 mechanisms of action. The primary DA pathway implicated in these effects originates in the ventral tegmental area (VTA), and projects to the nucleus accumbens (NAC) core and shell, the basolateral and central-cortico-medial amygdala (AMY), and the prelimbic and infralimbic medial prefrontal cortex (mPFC) (see reviews6,7). The VTA has been implicated in sucrose intake8,9, and DA release is observed following sugar intake in the NAC10-15, AMY16,17 and mPFC18-20. Fat intake also stimulates DA NAC release21, and another DA-rich projection zone to the dorsal striatum (caudate-putamen) has been also associated with DA-mediated feeding22,23. Kelley24-27 proposed that these multiple projection zones of this DA-mediated system formed an integrated and interactive distributed brain network through extensive and intimate interconnections28-34.
In addition to the ability of DA D1 and D2 receptor antagonists to reduce intake of sugars35-37 and fats38-40, DA signaling has also been implicated in mediating the ability of sugars and fats to produce conditioned flavor preferences (CFP)41-46. Microinjections of a DA D1 receptor antagonist into the NAC, AMY or mPFC47-49 eliminate acquisition of CFP elicited by intragastric glucose. Whereas microinjections of either DA D1 or D2 receptor antagonists into the mPFC eliminates acquisition of fructose-CFP50, the acquisition and expression of fructose-CFP are differentially blocked by DA antagonists in the NAC and AMY51,52.
The c-fos technique53,54 has been employed to investigate neural activation induced by palatable intake and neural activation. The term &#34;c-fos activation&#34; will be used throughout the manuscript, and is operationally defined by increased transcription of c-Fos during neuronal depolarization. Sucrose intake increased fos-like immunoreactivity (FLI) in the central AMY nucleus, the VTA as well as the shell, but not core, of the NAC55-57. Whereas sucrose intake in sham-feeding rats significantly increased FLI in the AMY and the NAC, but not the VTA58, intragastric sucrose or glucose infusions significantly increased FLI in the NAC and central and basolateral nuclei of the AMY59,60. Repeated addition of sucrose to scheduled chow access increased FLI in the mPFC as well as the NAC shell and core61. A sucrose concentration downshift paradigm revealed that the greatest FLI increases occurred in the basolateral AMY and NAC, but not the VTA62. Following conditioning, extinction of sugar-related natural reward behaviors increased FLI in the basolateral AMY and the NAC63. Moreover, pairing sugar availability to a tone resulted in the tone subsequently increasing FLI levels in the basolateral AMY64. High-fat intake also increased FLI in NAC and mPFC sites65-67.
Most of the previously cited studies examined sugar and fat effects on c-fos activation in single sites that do not provide information about identification of reward-related distributed brain networks24-27. Further, many of the studies also did not delineate the relative contributions of sub-areas of the NAC (core and shell), AMY (basolateral and central-cortico-medial) and mPFC (prelimbic and infralimbic) that could potentially be examined by the advantage of excellent spatial, single-cell resolution in c-Fos mapping68. Our laboratory69 recently used c-fos activation and simultaneously measured alterations in the VTA DA pathway and its projection zones (NAC, AMY and mPFC) after novel ingestion of fats and sugars in rats. The present study describes the procedural and methodological steps to simultaneously analyze whether acute exposure to six different solutions (corn oil, glucose, fructose, saccharin, water and a fat emulsion control) would differentially activate FLI in sub-areas of the NAC, AMY, mPFC as well as the dorsal striatum. This simultaneous detection of differences allowed confirmation of significant effects on FLI in each site and determination as to whether changes in one particular site correlated with changes in related sites, thereby providing support for a distributed brain network24-27. These procedures tested whether the VTA, the prelimbic and infralimbic mPFC, the core and shell of the NAC, and the basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of glucose (8%), fructose (8%), corn oil (3.5%) and saccharin (0.2%) solutions.

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simultaneous detection of c-fos activation from mesolimbic and mesocortical dopamine reward sites following naive sugar and fat ingestion in rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

All representative results described below have been published previously69, and are re-presented here to support &#34;proof of concept&#34; in indicating the effectiveness of the technique.
Solution Intakes 
Significant differences in baseline saccharin intakes were observed over the first four days for all animals (F(3,108) = 57.27, p &#60; 0.001) with intakes (Day 1: 1.3 (&#177; 0.2) ml; Day 2...

Watch this Scientific Journal Video about Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats at JoVE.com

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

The overall goal of this experimental protocol is to determine whether the VTA, and it's major mesotelencephatic DA projection zones would display coordinate and simultaneous c-Fos activation after novel intake of corn oil, glucose, fructose, or saccharine solutions. This method can help answer key questions in the neurobiology field, such as motivation, and identify whether multiple brain sites are activated in a coordinated fashion in response to palatable stimuli. The main advantage of this technique is that it combines the established behavioral assays, cellular activation techniques, and the statistical analyses.To set up the apparatus, use a calibrated centrifuge tube with a robber stopper, and a 45-degree angle metal sipper tube to provide an accurate measurement of the solution presented to the rat. Secure it to the home cage by a taut metal spring, and to allow visibility of the calibration. Three to five days before the training, restrict food rations to the rat to reduce its body weight to 85%of its original weight, in order to increase motivation to consume the solution.In the meantime, provide 10 milliliters of pre-training solution, with a 0.2%saccharine, to the animal for four days, over a one-hour session, to maximize the probability that the rat will sample the subsequent test solution with short latency. Weigh the tube before and after each session to obtain the intake measurement. Then, perform an intake test on the fifth day on subgroups receiving one of the six solutions.Ensure that the rats sample solution with short latency, and exclude the subjects from the study if this requirement is not met. To prepare the tissue, after sacrificing the animal, remove the brain from the skull quickly. Use the rongeurs to carefully crack and remove the bone from the brain, starting from rear to front.Work in the area below the cerebellum, and ensure the rongeurs are between the bone and the meningeal pia mater. Once the top and the sides of the skull are removed, use a small spatula to lift the brain from its base, and snip the cranial nerves with small scissors. Then, fix the brain in 4%paraformaldehyde solution overnight at four degrees Celsius.The next day, place the brain in the sucrose solution at room temperature, until it settles at the bottom of the container. After that, remove the olfactory bulb. Subsequently, remove the cerebellum and the pons.Then, mount the brain coronally, with the caudal part fixed to the stage of the microtome, and cut coronal sections of 40 mirometers. In this procedure, treat the sections with five milliliters of 5%normal goat serum, and 0.2%Triton x-100 in PBS, for one hour. Next, incubate the treated sections with primary antibody at four degrees Celsius for 36 hours, in the wells containing one milliliter of PBS.After 36 hours, rinse the sections three times with five milliliters of PBS for 10 minutes each time. Then, incubate the sections with secondary antibody at room temperature for two hours, in the wells containing one milliliter of PBS. Afterward, rinse the sections three times in five milliliters of PBS for 10 minutes each time.Next, incubate the rinsed sections for two hours in a commercially-available Avidin horseradish peroxidase mixture. After two hours, rinse the sections three times in five milliliters of PBS, for ten minutes each time. Subsequently, react the sections with DAB in the presence of hydrogen peroxide, for five to ten minutes.After that, double label the VTA sections by incubating them with a tyrosine hydroxylase antibody, in five millilters of PBS, overnight at four degrees Celsius. The next day, rinse the sections three times in five milliliters of PBS, for ten minutes each time. Following that, incubate the sections with secondary antibody in five milliliters of PBS, at room temperature, for two hours.After two hours, rinse the sections three times in five milliliters of PBS, for ten minutes each time. To visualize the antibody using a secondary antibody peroxidase complex, react it with a combination of DAB, and a 0.3%nickel sulfate solution, for five to ten minutes. Count the cells by double-clicking the software icon.Go to the menu bar, click on Acquisition, and then Live Image. After that, bring the region of interest into focus, and click the screen to establish a reference point. Next, go to the grid toolbar, and click Display Grid, and Use Grid Labels.Outline the region of interest with a predetermined trace. Then, count all the cells in each region of interest, and select Plus in the left-hand sidebar to register counts of the c-Fos cells. Consider a cell positive for c-Fos when a defined dark-red circle is observed.Repeat this process for each site, and ensure the inter-rater reliability of the two uninformed raters for each section in each region of interest, always exceeds 0.8. Afterward, count the c-Fos positive neurons in the regions of interest. Delineate whether c-Fos immunoreactivity is present in the TH-positive and TH-negative cells in the VTA.This image shows that the fat or sugar intake differentially increases c-Fos activation in the entire amygdala, as well as in the basolateral, and central-cortico-medial amygdala subareas. In this image, actual amygdala c-Fos activation was observed in animals exposed to intakes of corn oil, glucose, as well as fructose. C-Fos activation in the VTA was also observed in animals exposed to corn oil and water.Black arrows indicate the representative, double-labeled, TH c-Fos positive cells, while gray arrows indicate the representative c-Fos only cells. Once mastered, this technique can be done in three to four weeks, if performed properly. While attempting this procedure, it's important to remember to follow precise methodology, storage and counting.Thanks for watching, and good luck with your experiments.

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JoVE Journal

Neuroscience

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

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 ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...