This work was funded by the American Diabetes Association (ADA), Grant JF1-10-14 (MDiF). We would like to thank the personnel of the Laboratory of Animal Resources at WSU. Authors gratefully acknowledge assistance with the statistical analysis of data from Neil Paton, Ph.D.

TNTs of mice and rats are determined by using the incremental hot-plate analgesia meter [iHPAM, IITC Inc. Life Science (Woodland Hills, CA)]. The equipment consists of several components: an aluminum plate (10 x 20 cm) with the heating system underneath and a Plexiglas observation chamber above; a heat controlling unit, software for data acquisition (IITC Part # Soft Series 8), a PC (personal computer) and a footswitch for remote start, stop or reset control of the unit. The heating system also allows starting/stopping/resetting of the heating process manually through a keypad in front of the equipment. The iHPAM can be set to various heating rates, standby/cutoff temperatures.
1. Equipment Set-up
<ol>
	<li>Before testing animals, visually inspect the unit to corroborate the chosen heating rates (e.g., 6 &deg;C/min), initial standby and final cut-off temperatures (e.g., 28 &deg;C and 55 &deg;C, respectively).</li>
	<li>Verify all connections. To keep electronic copy of the results, the unit must be connected to a computer running Software Series 8. Alternatively, hard copies of the results can be printed out via the serial port of unit.</li>
	<li>Test the equipment. Press the footswitch once to make sure the Software Series 8 records the data: initial standby, final cutoff temperatures and time in seconds required to reach the cut-off temperature.</li>
	<li>As a further control test, the investigators involved in the experiments may need to experience the device on themselves. Withdrawing the hand from the metal plate at the moment of pain perception (46-48 &deg;C) must neither cause harm to the skin nor produce any later discomfort.</li>
	<li>Once the unit has been set and the results from steps 1.3 and 1.4 recorded they can be compared. This is helpful to detect potential problems that could invalidate the use of the unit. For instance, discrepancies between initial standby/final cut-off temperatures and the actual reading of the unit, and/or differences between temperatures displayed by the unit and the actual plate temperature required for hand withdrawal.</li>
</ol>
2. Testing Normal Animals
Either young or adult Wistar rats [post-natal (p) age 21-25 days], or mice (p21-25) of any genetic background or gender can be used. The mice used in our experiments are of the strain C57BL/6J.
<ol>
	<li>The night before the experiments to be performed, acclimate the animals by placing their cages on the bench where the iHPAM is placed. Two hours before performing the tests, turn on the iHPAM to familiarize the animals with the background noise of the unit. At this point, the iHPAM can be set up and tested to verify proper function (see Steps 1). Food and water is provided ad libitum throughout the test, except when the animal is in the observation chamber (see Step 2.1). 
	Note: It is important not to assume that the laboratory conditions in behavioral experiments are controlled by default. Special attention should be paid to genetic and environmental factors that may influence the outcome of the behavioral studies (reviewed in 7).</li>
	<li>Gently place a normal mouse/rat in the Plexiglass observation chamber on top of the heating plate of the unit. Allow the animal to acclimate to the warmed surface (set at 28 &deg;C) until the animal shows a comfortable behavior. 
	Note: Under this conditions normal adult rats show an initial exploratory behavior, occasionally standing up towards the lid of the observation chamber while sniffing. After a few minutes, rats may show casual bouts of grooming and a relaxed behavior. Sometimes, rats lay with a relaxed body posture (prone extension) while casually sniffing the surroundings. Normal mice are usually more active than rats. However, mice show a relaxed exploratory behavior after few minutes in the observation chamber. It is exceptionally rare to observe either escaping behaviors (e.g., jumping, trying to escape from the observation chamber) or defensive attitudes (e.g., squealing). It is also important to note that mice can communicate pain from one mouse to another8. Therefore, it may be important to test mice placed in separate cages.</li>
	<li>Place mirrors in the back of the observation chamber, to permit observations at all angles. A tripod holding a digital video camera may be of help to determine the exact moment at which nocifensive (pain avoiding) behavior had occurred.</li>
	<li>Once the animal is comfortable in the observation chamber heat up the plate by pressing the footswitch. When the plate temperature heats up at a rate of 6 &deg;C/minute, normal animals exhibit nocifensive behavior involving either hind-paw* usually temperatures raging from 46 to 48 &deg;C3,5,9,10. Thus, the animal is typical removed form the observation chamber after approximately 3 minutes. 
	Note: Observing the typical nocifensive response of a rat or mouse i.e., hind-paw licking, while shaking and/or lifting, is enough to terminate the thermal stimulus. This is achieved by pressing the footswitch. At this moment, the software records the plate's temperature and the plate immediately cools down until standby temperature (28 &deg;C) is reached (~0.5 min). This temperature i.e., the one that evokes any nocifensive reaction is regarded as the noxious heat threshold (TNT) of the normal (control) animal. 
	*Sometimes, particularly in mice, nocifensive behavior to noxious temperature may first be observed in one or both forepaws (e.g., licking while sitting), few seconds before hind-paws. However, fore-paw licking is a normal component of the grooming behavior. Hence, only hind-paw reactions are assessed. In occasions mice may groom their hind-paws as well. However, normal grooming do not last more than a few seconds, does not involve intensive licking and usually ends after cleaning its claws.</li>
	<li>Gently remove the animal from the plate and place it in its respective cage. The heat threshold measurement is then repeated for other control (normal) animal. 
	Note: TNTs can be determined in a single animal at intervals of 1-2 minutes without removing the animal from the observation chamber. TNT determinations in rats or mice can be repeated several times during several days. This is particularly useful to determine the mean TNT of an individual animal as well as its variation with age.</li>
	<li>The TNT of control animals is expressed as the mean of three or more thresholds &plusmn; SEM.</li>
	<li>Statistical analysis of data obtained is performed to compare different treatment groups of mice or rats. Parametric procedures including Analysis of Variance may be appropriately used where data are normally distributed and where treatment groups are homoskedastic; when these conditions have been satisfied the following approaches can be applied. Analysis of Variance followed by the Newman-Keuls test to detect differences in multiple measurements i.e., repeated individual TNTs with/without treatment or at different time points (e.g., data in Figure 3). The paired Student's t-test may be used to compare two data groups i.e., TNTs before vs. after drug treatment; the unpaired t-test may be used to determine differences in TNT changes in drug-treated vs. vehicle-treated animals (e.g., data in Figure 4).</li>
</ol>
3. Testing Diabetic Animals
The streptozotocin (STZ)-induced diabetic rat is the most commonly used animal model to study mechanisms of PDN and to assess analgesic drugs and therapies11,12. Rats made diabetic by a single intraperitoneal dose of STZ (60 mg/kg) can be tested for thermal hyperalgesia during the 3rd week after onset of diabetes11. It is important to note that not all diabetic rats develop PDN. However, animals that do develop hyperalgesia (~50%) can be easily identified. Indeed, their TNTs are lower than normal*.
*To determine TNTs of STZ-diabetic rats, a starting plate temperature of 15 &deg;C instead of 28 &deg;C is used. The reason for these change is that most diabetic animals with PDN are expected to be hyperalgesic2,11. Therefore, they will have lower than normal noxious heat thresholds (e.g., &lt;45 &deg;C).
<ol>
	<li>A diabetic rat is placed in the observation chamber on top of the testing apparatus. The animal is allowed to acclimate to the plate surface until the animal shows a comfortable behavior. 
	Note: Typically, a diabetic rat does not show much of the normal behavior. Diabetic rats are usually hypoactive, lethargic; often showing restricted movements and slow grooming. It is rare to observe standings towards the lid of the observation chamber. After few minutes, diabetic rats may calmly stay in a corner of the chamber with casual sniffing of the surroundings.</li>
	<li>Once the diabetic animal is comfortable in the observation chamber, the plate is heated up as in Step 2.3 the diabetic rat may exhibit nocifensive behavior involving either hind/fore-paw. This usually happens at non-noxious plate temperatures (e.g., &lt;45 &deg;C), or after no more than 5 minutes in the chamber with the 6 &deg;C/min temperature increase. 
	Note: The nocifensive response of a young adult diabetic rat does not differ from that of a normal rat. However, the lowest temperature evoking such behavior is significantly lowered.</li>
	<li>See Step 2.4</li>
	<li>The mean of more than three thresholds in the day of the experiment expressed as &deg;C &plusmn; SEM is considered the noxious heat threshold of the diabetic animal. 
	Note: A similar paradigm to the one explained for normal or diabetic rats can be followed to determine the thermal anti-nocifensive effect of analgesics on heat thresholds.</li>
</ol>
4. Testing Analgesia
The anti-nociceptive properties of drugs (e.g., analgesics) as well as the determination of pharmacologic parameters such as the minimum effective dose can be easily obtained by using the steps explained above. It is predicted that proper doses of an analgesic administered to rats/mice would significantly increase their TNTs. This is particularly sensitive in animals with thermal hyperalgesia3. However, to avoid bias in the interpretation of the results, the investigators involved in the behavioral observation of an animal group must not know or be aware if the animals had been treated or not. Further, drugs to be injected by the observer may be prepared by another investigator and labeled A, B, C, etc. Dosage is provided in a printout, listing individual doses per identified animal.
Depending on the drug being tested, different doses of a potential analgesic may be administered to normal or diabetic animals 5-15 min prior behavioral tests. Proper controls must be included (i.e., age-matched animals injected with vehicle alone and/or diabetic rats without PDN) and tested using identical conditions (e.g., same starting temperature of the plate). Chronic effects of drugs can be tested as well.
5. Representative Results
The lowest plate temperature evoking nocifensive reactions in either hind-paw of young adult normal non-treated rats or mice were determined following steps 2-1 to 2-6. As shown in Figure 1, TNT of normal young adult rats and mice were 47.2 &plusmn; 0.2 &deg;C and 47.5 &plusmn; 0.5 &deg;C, respectively.
We have observed neither significant daily variation of individual's TNTs (Figure 2A) nor significant changes of TNTs among individuals of the same age (Figure 2B). TNT differences due to gender were not observed (not shown).
TNTs of young adult STZ-diabetic rats were determined in a similar fashion as for normal rats (Steps 3.1 to 3.3). However, the starting plate's temperature was set to 15 &deg;C instead of 28 &deg;C. The nocifensive behavior of STZ-diabetic rats was assessed at least three times in an individual and in a daily fashion starting eleven days post-STZ injection. As shown in Figure 3, a significant drop (p&lt;0.01) in the TNT of STZ-diabetic rats becomes evident two weeks post-STZ injection. The mean TNT of STZ-diabetic rats was 45.6 &plusmn; 0.1 &deg;C (n=16, pooled values from days 14-23).
Diabetic rats exhibiting significantly lower TNT than normal were considered to have PDN. These rats were used to test the acute anti-nociceptive properties of an analgesic (compound A). TNTs were determined in normal and diabetic rats injected with either a single dose of the compound or vehicle alone five minutes before the test. As shown in Figure 4, a single intra-peritoneal dose of the compound significantly increased TNTs of diabetic rats when compared to rats treated with vehicle alone (45.6 &deg;C vs 47.6 &deg;C, respectively). The anti-nocifensive effect of compound A was also observed in normal rats; although significant (p&lt;0.01), it was less pronounced (47.4 &deg;C vs 48.0 &deg;C, respectively).
<img alt="Figure 1" src="/files/ftp_upload/3785/3785fig1.jpg" /> 
Figure 1. Determination of TNTs of normal young adult rats or mice: The TNTs of young adult rats (p21-24) or mice (p20-21) were tested in the iHPAM following the steps outlined above. A total of 12 rats and 25 mice were tested. Measurements were performed during several days and the results were pooled. Results are expressed as mean &plusmn; SEM. The minimum and maximum TNT values recorded in these rats were 46.2 &deg;C and 48.6 &deg;C respectively. In the case of mice, the maximum and minimum TNT values recorded were 48.8 &deg;C and 44.9 &deg;C, respectively.
<img alt="Figure 2" src="/files/ftp_upload/3785/3785fig2.jpg" /> 
Figure 2. Age-dependency of TNTs in rats and mice: A) Plotted are the TNTs of four normal rats (n=4) of 21 days of age determined at least three times daily during 5 days. B) TNTs of mice littermates determined once at indicated ages. The numbers on top of each determination point represents the number of littermates used.
<img alt="Figure 3" src="/files/ftp_upload/3785/3785fig3.jpg" /> 
Figure 3. Diabetic rats become hyperalgesic 3 weeks after the onset of diabetes: TNTs of six (n=6) age matched young adult STZ-diabetic rats were determined every day starting 11 days post-induction of diabetes. Shown are daily TNT averages (mean &plusmn; SEM). Diabetic TNTs determined between 11-13 days post-STZ injection were within the normal range (red line). Two weeks after STZ injection TNTs decreased significantly to 45.1 &plusmn; 0.4 &deg;C, (p&lt;0.05).
<img alt="Figure 4" src="/files/ftp_upload/3785/3785fig4.jpg" /> 
Figure 4. Acute anti-nocifensive effects of compound B in STZ-diabetic rats. Young adult normal rats (blue group n=12) and age matched STZ-diabetic hyperalgesic rats (green group n=6) were tested in the iHPAM after intraperitoneal injection of vehicle (dark bars) or compound A (light bars). The mean TNT &plusmn; SEM for each treatment (vehicle control or compound A-treated) are shown. Asterisks denote statistical differences (p&lt;0.05).

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We have decided not to disclose the identity of the analgesic used in these experiments. Part of the results shown here has been presented in the 71st Scientific Sessions of the American Diabetes Association (San Diego CA) and the 47th Annual Meeting of the European Association for the Study of Diabetes (Lisbon, Portugal).

Similarly to the classic hot plate test to quantify thermal hyperalgesia4,13, the assay of nociception described here permits a fast and reliable way to quantify nocifensive behavior in rats and mice. However, contrary to classic test, the incremental hot-plate method is non-invasive and virtually stress-free. Although some restrain is necessary to perform the test (i.e., the animal must be in the observation chamber), rats or mice are accustomed to similar areas (e.g., housing cages).
<...

<table border="1">
	<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Incremental Hot-Plate Analgesia Meter </td>
 <td>IITC Inc. Life Science </td>
 <td>Part #PE34 </td>
 </tr>
 <tr>
 <td>Soft Series 8</td>
 <td>IITC Inc. Life Science</td>
 <td>Part # Series8</td>
 </tr>
 <tr>
 <td>Streptozotocin</td>
 <td>Calbiochem</td>
 <td>572201</td>
 </tr>
 </tbody>
 </table>

rapid determination of the thermal nociceptive threshold in diabetic rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

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Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

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27.4K Views.  Wright State University. Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Video: Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

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 ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Measurement &amp; Analysis of the Temporal Discrimination Threshold Applied to Cervical Dystonia

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being asynchronous (typically 30-50 ms). It has been shown to be abnormal (prolonged) in neurological disorders, including cervical dystonia, a phenotype of adult onset idiopathic isolated focal dystonia. The TDT is a quantitative measure of the ability to perceive rapid changes in the environment and is considered indicative of the behavior of the visual neurons in the superior colliculus, a key node in covert attentional orienting. This article sets out methods for measuring the TDT (including two hardware options and two modes of stimuli presentation). We also explore two approaches of data analysis and TDT calculation. The application of the assessment of temporal discrimination to the understanding of the pathogenesis of cervical dystonia and adult onset idiopathic isolated focal dystonia is also discussed.

Methods for the measurement and analysis of the temporal discrimination threshold are presented, and its application to the study of the pathogenesis of cervical dystonia are discussed.

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...