We thank the Harry Frank Guggenheim Foundation and the Natural Science and Engineering Research Council of Canada for grants to SMP that supported the research leading to the development of these methods.

1. Equipment and Room Set Up
<ol>
	<li>The play box apparatus is a clear, Plexiglas box measuring 50 cm X 50 cm X 50 cm with 1-2 cm of standard corncob bedding.</li>
	<li>All filming is conducted in the dark via a handheld camcorder under the night-shot option.</li>
	<li>The camcorder is placed at a 45&deg; angle facing downwards towards the play box on a tripod. All four corners must be visible in the shot to obtain a full view of the apparatus.</li>
</ol>
2. Methodology
<ol>
	<li>Animals are weaned between postnatal days 21-23. Depending on the experimental design, animals can be housed together in groups of 2-4 in the required male and female combinations.</li>
	<li>Play testing occurs during an animal's light cycle, although testing occurs in a dark room. The testing room must be dark prior to the entrance of the animals.</li>
	<li>Groups housed together are habituated for three, consecutive 30-min, sessions directly prior to testing following the same sequence as if being tested (see 2.5).</li>
	<li>Following habituation, pairs are individually isolated 24 hr prior to testing to maximize playful interactions (see 2.5).</li>
	<li>Animals are transported into a dark testing room and are then individually placed in the play apparatus and tested as pairs for 10 min. Subjects are then removed and placed together in their home cage.</li>
	<li>Following each play session, bedding is discarded and the play apparatus is thoroughly cleaned using the antiseptic Virkon (or its equivalent). This is a superior agent for cleaning the rat odors from the previous pair, as it does not leave the strong and persistent smell of alcohol wipes or sprays. Cover-spray may also be used to mask any scents given off by the subjects. New bedding is then added.</li>
	<li>Repeat steps (2.4-2.6) for additional trials.</li>
</ol>
3. Behavioral Analysis: Assessing the Content of Play
First, the distinctive targets competed over by rats in both serious fighting and play fighting make this species particularly valuable as a research subject for studying play, as escalation from playful to serious fighting can be unambiguously detected by a shift in body targets35. Second, the number of attacks launched against the nape of a partner can be measured. Third, given that defense is contingent on attack, the relative defensiveness of the recipient of attacks can be measured as the proportion of attacks defended. Fourth, when defending against a nape attack, the recipient can use one of several tactics to protect or extricate their nape from their partner's snout15, 25, 26. These tactics fall into two main categories.
<ol>
	<li>In evasion, the defender can walk, run, leap or dodge away (Figure 2a), and, by doing so, it not only moves its nape away from its attacker, but also ends up facing away from its attacker (Video of evasive defense).</li>
	<li>In facing defense, the defender turns to face its attacker, thus not only withdrawing its nape away, but also juxtaposing its teeth between its nape and the attacker's snout. Facing defense falls into two main categories: rotation around the longitudinal axis of the body and rotation, horizontally, around a vertical axis (usually between the rump and mid-body, although this can differ between the sexes27). Further, rotation around the longitudinal axis of the body can take one of two forms.</li>
</ol>
<ol class="lalpha">
	<li>Complete rotation involves the rat turning from a standing position to fully supine (Figure 2B). Partial rotation involves the rat turning its forequarters, but leaving one or both of its hind paws in contact with the ground (Figure 2C, Video to show difference between full rotation and partial rotation).</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/4288/4288fig2highres.jpg" src="/files/ftp_upload/4288/4288fig2.jpg" /> 
Figure 2. Three types of defense in response to a playful nape attack are shown in 61 days old male rats (A-C). A. Evasion. Following the attacker's lunge at the nape (a, b), the defender swerves away from its attacker (c). B. Complete rotation. A nape contact from behind (a) leads the defender to rotate (b, c) until it is lying supine and blocking the attacker with its outstretched paws (d). C. Partial rotation. An attacker's lunge to its partner's nape from the side (a, b) is followed by a rotation of the head, neck and shoulders by the defender, so withdrawing its nape from its attacker's snout, while maintaining support on the ground with its hind feet (c). From Pellis et al. 26; reprinted with permission of S. Karger AG, Basel.
<ol class="lalpha" start="2">
	<li>Horizontal rotation around the vertical axis requires the defender to move its rump away from its attacker while moving its head toward its partner (Figure 3, Video of horizontal rotation).</li>
	<li>Following its immediate rotation around either its vertical axis or partially around its longitudinal axis, the defender can then rear and push against its partner, showing that the outcome, when using either of these two tactics, can be similar. Confounding tactics with outcomes can be even more striking when considering complete and partial rotations. When a defender executes a complete rotation, the movement is quick and fluid - if videotaped on a standard 30 fps recording, a rotation to fully supine takes 2-3 frames and the movement is continuous (Video).</li>
	<li>A partial rotation can also end in a fully supine position, but this typically involves the concerted actions of the attacker. In this case, the defender can partially rotate its forequarters and keep its body horizontal or even touching the ground, but the attacker can then press its body weight onto the defender. With persistent pressing and reaching for the nape by the attacker, the defender rotates more and more, until fully supine. Such a 'forced' supine occurs in a staccato manner, with incremental rotation of the body, and can take substantially more than 3 video frames (Video showing a partial rotation leading to the defender adopting a supine position).</li>
</ol>
Given that most studies are interested in the effects of particular manipulations on subjects, it is best to score the tactic attempted by the subject rather than the eventual outcome that may be forced by its partner's actions. This judgment can be made by examining the movements of the defender within the first 2-3 frames of the commencement of a defensive action (Video highlighting the first 2-3 frames, frame-by-frame).
<ol start="3">
	<li>Playful attacks can be measured when the snout of one rat either contacts or comes within 1-2 cm of its partner's nape (Video showing a playful attack compared to non-playful movements near partner rat).</li>
</ol>
<ol class="lalpha">
	<li>As noted above, the defensive tactic of the recipient can be categorized in the first few frames following an attack. A new sequence begins with a new attack (Video showing extended playful sessions compared to separate playful attacks).</li>
	<li>During an extended playful wrestle, such as is shown in Figure 1, rats can counterattack once they have successfully defended themselves, but these types of attacks are more difficult to score, especially in regard to the partner's defensive reaction. Because counterattacks are highly correlated with the frequency of initiating attacks16, these more complex phases of the interactions need not be scored unless there is good reason for doing so23. The scoring of initiating attacks and the tactics employed to defend against such attacks suffice to capture the relative differences in playfulness and defensive behavior of rats in different experimental conditions26, 29.</li>
</ol>
<img alt="Figure 3" fo:content-width="3.23in" fo:src="/files/ftp_upload/4288/4288fig3highres.jpg" src="/files/ftp_upload/4288/4288fig3.jpg" /> 
Figure 3. Facing defense involves a defender turning to face its attacker while pivoting on its hind legs in the horizontal plane around a vertical axis (a, b). From this position, the defender can block access to its nape (c) and launch a counterattack (d). From Pellis et al.25; adapted and reprinted with permission from APA.
Since most playful interactions start with a playful attack, the absolute number of such attacks can be measured within the time period of a test trial. The optimal size of the trial for our studies has been 10 min following 24 hr of social isolation15, 16. Brief periods of social isolation increases the motivation to engage in social play while not having a similar effect on other social behavior such as social investigation9. Although any amount of isolation has this effect, the impact of isolation on play reaches an asymptote around 24 hr27. While trials of 5 min can detect such isolation-induced changes, they are too short to detect subtle differences such as those between the sexes due to a ceiling effect - in this example, the greater propensity for males to play mostly emerges after the first 5 min. Even so, by 15 min, the animals appear fatigued by the vigorous play and consequently, trials lasting 10 min are a good compromise between economy of protocol and the ability to detect experimental effects15, 16, 27. Whether tested in the light or dark phase of the rats' daily cycle, our experience is that the amount of play increases if the rats are tested under red light instead of white light15, 16. With the advent of camcorders with night shot capability, filming in the dark is possible, and this also increases the amount of play performed in test trials29.
<ol start="4">
	<li>Given that defense is contingent on an attack occurring, defensiveness should be scored as a proportion of attacks leading to defense. Similarly, given that any given type of defense is contingent on a defense occurring, scoring types of defense should also be in terms of proportions (e.g., of the defenses that occurred, X% were complete rotations). Scoring defense as an absolute measure can be misleading. For example, a low frequency of complete rotations could arise because of low rates of attack or because the subjects in that particular experimental condition used the complete rotation infrequently even if they had been attacked just as frequently as the animals in the control condition.</li>
</ol>
<ol class="lalpha">
	<li>Thus, when scoring play, there is a sequential analysis: how many attacks are launched by the subjects (number/unit time), how many attacks are defended against (i.e., probability of defense), and when defending, what is the likelihood of using the different tactics of defense (%tactics).</li>
</ol>
4. Behavioral Analysis: Using the Content of Play to Assess Play Partner Relationships
With the onset of puberty, there is an overall decline in the amount of play fighting8, 40. This is because they decrease the frequency of their launching of nape attacks, but maintain the same, high probability of defense17. Among male rats, the pattern of play is modified by the development of dominance relationships that coalesce around puberty37. This is reflected in the types of defensive tactics used.
<ol>
	<li>In males, with the onset of puberty, there is a relative decline in their use of the complete rotation tactic (Different Video of Full Rotation as shown in above parts) and an increase in their use of the partial rotation tactic (Different Video of Partial Rotation as show in above parts), with the other tactics remaining at about the same frequency at all ages16.</li>
	<li>However, in subordinate males, it is dependent on their partner as to which of these tactics they will use. When playing with another subordinate male or a female, a subordinate male rat is most likely to use the partial rotation tactic when attacked, but when playing with the dominant male in the colony, it is more likely to use the complete rotation tactic when attacked18, 19, 24, 36. Thus, with the onset of dominance relationships, the relative use of tactics is a good marker to monitor changes in an experimental subordinate rat's social relationships12.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+the+absence+of+the+pubertal+shift+in+the+playful+defense+of+female+rats.">Mechanisms underlying the absence of the pubertal shift in the playful defense of female rats.</a> Dev. Psychobiol. 33, 147-156 (1998).</li><li>Takahashi, L. K., Lore, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Play+fighting+and+the+development+of+agonistic+behavior+in+male+and+female+rats.">Play fighting and the development of agonistic behavior in male and female rats.</a> Aggress. Behav. 9, 217-227 (1983).</li><li>Taylor, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fighting+in+juvenile+rats+and+the+ontogeny+of+agonistic+behavior.">Fighting in juvenile rats and the ontogeny of agonistic behavior.</a> J. Comp. Physiol. Psych. 94, 953-961 (1980).</li><li>Thor, D. H., Holloway, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Play+solicitation+behavior+in+juvenile+male+and+female+rats.">Play solicitation behavior in juvenile male and female rats.</a> Anim. Learn. Behav. 11, 173-178 (1983).</li><li>Thor, D. H., Holloway, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+analysis+of+social+play+behavior+in+juvenile+rats.">Developmental analysis of social play behavior in juvenile rats.</a> Bull. Psychon. Soc. 22, 587-590 (1984).</li><li>Vanderschuren, L. J. M. J., Niesink, R. J. M., van Ree, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+play+behavior+in+rats.">The neurobiology of play behavior in rats.</a> Neurosci. Biobehav. Rev. 21, 309-326 (1997).</li><li>Whishaw, I. Q., Kolb, B., Tees, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+decorticate+rat.">The decorticate rat.</a> Cerebral cortex of the rat. , 239-268 (1990).</li></ol>

No conflicts of interest declared.

Play fighting is a complex, dynamic interaction in which the participants continually influence each other's behavior. As a consequence, a scoring scheme that is sensitive to those dynamics and the contingent nature of the actions performed is needed. The scheme outlined in this paper has proven successful in comparing across sexes, individual differences, through development, strain differences and for experimental treatments4, 14, 16, 20, 24, 29, 30, 31, 32, 32, 36. Moreover, the scheme presented can...

Rats have been the most intensively used animals in the laboratory with which to study play10, 21, 41, and several attempts have been made to develop scoring schemes to measure the effects of experimental treatments on play fighting8, 9, 37, 38, 39. However, while these schemes have had some success in monitoring the frequency of play, they are less suitable as ways to monitor changes in the content of play due to their lack of sensitivity to the dynamics of the interaction13. The main problem is that, during fighting, the use of tactics by one partner is often contingent on the performance of particular tactics by the other2, 15. We have developed a scoring scheme that takes the dynamics of the interaction into account and so is better suited to detect and characterize the subtle changes that occur during play with experimental manipulation16, 20, 26. In this paper, we focus on the measurement of the defensive tactics performed by the recipient of a playful attack.
During serious fighting, animals attack and defend particular body targets, using species-typical weapon systems5. In rats, the primary weapons are the teeth, which are used to bite the flanks and lower dorsum2. Such serious fighting is readily exhibited in a resident-intruder context, in which an unfamiliar adult male is introduced into the cage of a resident adult male2, 15. As in serious fighting, in play fighting, animals also compete to gain some advantage over one another1. Rats, for example, attack and defend the nape of the neck15, which if contacted is gently nuzzled with the snout11, 32. Therefore, it is possible to discern playful attacks from aggressive attacks based not only on the manner of physical interactions (nuzzle vs. bite), but also on the body targets attacked (nape vs. flanks and lower dorsum). In addition, such playful nuzzling should not be confused with social investigation or allogrooming. Social investigation involves sniffing the partner, most frequently on the face and anogenital area. Because these behaviors appear to coincide with playful behaviors, and therefore may be considered aspects of playful interactions, it has been shown that these social interactions precede play behavior. Furthermore, changes in the motivation to engage in social investigation are independent of comparable changes in play9. There are several features that distinguish allogrooming from play20. First, allogrooming usually occurs when the rats huddle together following a protracted period of playing. Second, while playful attack is centered on the nape, allogrooming can involve contact with many parts of the partner's body, although it sometimes is focused on the nape. Third, this grooming involves gentle nibbling and pulling of the partner's fur with the teeth. Fourth, unlike allogrooming, most playful attacks to the nape are defended by the recipient, which leads to complex sequences of wrestling.
The main focus of this paper is to show what may be fruitfully measured during those complex sequences of play that can include wrestling. As shown below, the various tactics used by the defender to protect its nape lead to different possible outcomes. These outcomes, such as wrestling on the ground, boxing or chasing, have in some studies been measured as independent behavior patterns, but we have found that, in the majority of cases, these outcomes depend on the defensive tactic adopted by the recipient of a nape attack15. For example, evasive tactics often lead to chases and rolling over onto the back leads to wrestling. Thus, outcome based measurements, such as boxing and wrestling, are actually composites arising from the actions of both participants. Using a dynamic approach, our framework provides a means of scoring moments in the interaction when the actions measured are attributable to only one of the participants28, an approach that will be more evident to the reader once the examples of how the measuring framework is used are presented.
As shown in Figure 1, the rat on the left is seen approaching another from the rear (a) and then pouncing toward the nape of its neck (b). However, before contact is made, the defender rotates around the longitudinal axis of its body (c) to face its attacker (d). As the attacker moves forward, the defender is pushed onto its side (e), but then rolls over onto its back as the attacker continues to reach for its nape (f-h). From the supine position, the defender launches an attack toward its partner's nape (i), but is blocked by its partner's hind foot (j, k). Following another attempt to gain access to its partner's nape, the rat on top is pushed off (l) by the supine animal's hind feet (m), which thus enables the original defender to regain its footing (n) and lunge to attack its partner's nape (o).
<img src="/files/ftp_upload/4288/4288fig1.jpg" alt="Figure 1"/> 
Figure 1. A pair of juvenile male rats is shown attacking and defending the nape during a play fight. From Pellis &amp; Pellis15; reprinted with permission of John Wiley &amp; Sons.

<table border="1">
 <tbody>
 <tr>
 <td>Equipment</td>
 <td>Comments (optional)</td>
 </tr>
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 <td>Sony DCR-DVD103 Camcorder</td>
 <td>Optical Zoom- 20x, Night shot 
 *See Below</td>
 </tr>
 <tr>
 <td>Play Apparatus</td>
 <td>50 X 50 X 50 cm Plexiglas</td>
 </tr>
 <tr>
 	<td></td>
 <td>* Any camcorder will work for recording, although an optical zoom of 20x or above is desired for optimal zoom.</td>
 </tr>
 </tbody>
</table>

peering into the dynamics of social interactions: measuring play fighting in rats

Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements of one animal are countered by the actions of its partner, play fighting is a complex, dynamic interaction. This dynamic complexity raises methodological problems about what to score for experimental studies. We present a scoring schema that is sensitive to the correlated nature of the actions performed. The frequency of play fighting can be measured by counting the number of playful nape attacks occurring per unit time. However, playful defense, as it can only occur in response to attack, is necessarily a contingent measure that is best measured as a percentage (#attacks defended/total # attacks X 100%). How a particular attack is defended against can involve one of several tactics, and these are contingent on defense having taken place; consequently, the type of defense is also best expressed contingently as a percentage. Two experiments illustrate how these measurements can be used to detect the effect of brain damage on play fighting even when there is no effect on overall playfulness. That is, the schema presented here is designed to detect and evaluate changes in the content of play following an experimental treatment.

Two studies illustrate how the multi-faceted scoring scheme that we have developed can be used to diagnose the effects of experimental manipulations on the play fighting of rats. The first shows that the method can be used to pinpoint the specific component of play changed by an experimental manipulation and the second shows how the method can be used to measure changes in the relationships following an experimental manipulation.
(1) The effects of neonatal decortication
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Watch this Scientific Journal Video about Peering into the Dynamics of Social Interactions: Measuring Play Fighting in Rats at JoVE.com

Peering into the Dynamics of Social Interactions: Measuring Play Fighting in Rats

Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements of one animal are countered by the actions of its partner, play fighting is a complex, dynamic interaction. This dynamic complexity raises methodological problems about what to score for experimental studies. We present a scoring schema that is sensitive to the correlated nature of the actions performed. Two experiments illustrate how these measurements can be used to detect the effect of brain damage on play fighting even when there is no effect on overall playfulness. That is, the schema presented here is designed to detect and evaluate changes in the content of play following an experimental treatment.

Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements ...

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15.2K Views.  University of Lethbridge. Играть в борьбе с крысой включает в себя атаку и защиту затылка, который при контакте, мягко ткнулся носом в морду. Потому что движение одного животного противостоит действиям своего партнера, игры боевых является сложным, динамичным взаимодействием. Эта дина...

Video: Peering into the Dynamics of Social Interactions: Measuring Play Fighting in Rats

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language affect our decisions in personal and professional life alike 1. These "friend or foe" judgements are often based on first impressions, which in turn may affect our decisions to "approach or avoid". Previous studies investigating the neural correlates of social cognition tended to use static facial stimuli 2. Here, we illustrate an experimental design in which whole-body animated characters were used in conjunction with functional magnetic resonance imaging (fMRI) recordings. Fifteen participants were presented with short movie-clips of guest-host interactions in a business setting, while fMRI data were recorded; at the end of each movie, participants also provided ratings of the host behaviour. This design mimics more closely real-life situations, and hence may contribute to better understanding of the neural mechanisms of social interactions in healthy behaviour, and to gaining insight into possible causes of deficits in social behaviour in such clinical conditions as social anxiety and autism 3.

This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions.

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency low-energetic stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility. The goal of the research was the development of a miniature, endoscopically implantable device to a submucosal pocket. The implantable device is a fully customized electronic package which was specifically designed for the purpose of experiments in the submucosa. The device is equipped with a lithium-ion battery which can be recharged wirelessly by receiving an incident magnetic field from the charging/transmitting coil. The uplink communication is achieved in a MedRadio band at 432 MHz. The device was endoscopically inserted into the submucosal pocket of a live domestic pig used as an in vivo model, specifically in the stomach antrum. The experiment confirmed that the designed device can be implanted into the submucosa and is capable of bidirectional communication. The device can perform bipolar stimulation of muscle tissue.

The application of high-frequency low-energetic stimulation can alleviate the symptoms of gastric dysmotility. In this research, a miniature, endoscopically implantable and wirelessly rechargeable device which is implanted into a submucosal pocket is presented. Successful both-way communication and stimulation control were achieved during an experiment on live pig.

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency ...

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) ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...