Name: the power of interstimulus interval for the assessment of temporal processing in rodents
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Description: Watch this Scientific Journal Video about The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents at JoVE.com

This work was supported in part by grants from NIH (National Institute on Drug Abuse, DA013137; National Institute of Child Health and Human Development HD043680; National Institute of Mental Health, MH106392; National Institute of Neurological Diseases and Stroke, NS100624) and the interdisciplinary research training program supported by the University of South Carolina Behavioral-Biomedical Interface Program. Dr. Landhing Moran is currently a Scientific Officer at the NIDA Center for Clinical Trials Network.

All animal protocols were reviewed and approved by the Animal Care and Use Committee at the University of South Carolina (federal assurance number: D16-00028).
1. Defining Parameters and Calibration of the Startle Apparatus
<ol>
	<li>Set up the startle response system (see Table of Materials) according to the manufacturer&#8217;s instructions.
	<ol>
		<li>Enclose the startle platform in a 10 cm-thick double-walled isolation cabinet.</li>
	</ol>
	</...

<ol>
	<li>Braff, D., Stone, C., Callaway, E., Geyer, M., Glick, I., Bali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prestimulus+effects+on+human+startle+reflex+in+normals+and+schizophrenics.">Prestimulus effects on human startle reflex in normals and schizophrenics.</a> Psychophysiology. 15 (4), 339-343 (1978).</li><li>Castellanos, F. X., Fine, E. J., Kaysen, D., Marsh, W. L., Rapoport, J. L., Hallett, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+gating+in+boys+with+Tourette's+Syndrome+and+ADHD:+Preliminary+results.">Sensorimotor gating in boys with Tourette's Syndrome and ADHD: Preliminary results.</a> Biological Psychiatry. 39 (1), 33-41 (1996).</li><li>Moran, L. M., Booze, R. M., Mactutus, C. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+to+light+in+a+case+of+hysterical+blindness+studied+by+reinforcement-inhibition+and+conditioning+methods.">Sensitivity to light in a case of hysterical blindness studied by reinforcement-inhibition and conditioning methods.</a> Yale Journal of Biology and Medicine. 6, 61-67 (1933).</li><li>Hoffman, H. S., Searle, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+variables+in+the+modification+of+startle+reaction+in+the+rat.">Acoustic variables in the modification of startle reaction in the rat.</a> Journal of Comparative and Physiological Psychology. 60, 53-58 (1965).</li><li>Hoffman, H. S., March, R. R., Stein, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+background+acoustic+stimulation+in+controlling+startle.">Persistence of background acoustic stimulation in controlling startle.</a> Journal of Comparative and Physiological Psychology. 68 (2), 280-283 (1969).</li><li>Ison, J. R., Hammond, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+startle+reflex+in+the+rat+by+changes+in+the+auditory+and+visual+environments.">Modification of the startle reflex in the rat by changes in the auditory and visual environments.</a> Journal of Comparative and Physiological Psychology. 75 (3), 435-452 (1971).</li><li>Moran, L. M., Hord, L. L., Booze, R. M., Harrod, S. B., Mactutus, C. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflex+modification+in+the+domain+of+startle:+I.+Some+empirical+findings+and+their+implications+for+how+the+nervous+system+processes+sensory+input.">Reflex modification in the domain of startle: I. Some empirical findings and their implications for how the nervous system processes sensory input.</a> Psychological Review. 87 (2), 175-189 (1980).</li><li>Ison, J. R., Agrawal, P., Pak, J., Vaughn, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+temporal+acuity+with+age+and+with+hearing+impairment+in+the+mouse:+A+study+of+the+acoustic+startle+reflex+and+its+inhibition+by+brief+decrements+in+noise+level.">Changes in temporal acuity with age and with hearing impairment in the mouse: A study of the acoustic startle reflex and its inhibition by brief decrements in noise level.</a> The Journal of the Acoustical Society of America. 104, 1696-1704 (1998).</li><li>. Startle response: Acoustic startle reflex response 101 Available from: <a target="_blank" href="https://mazeengineers.com/acoustic-startle-response/">https://mazeengineers.com/acoustic-startle-response/</a> (2014)</li><li>Curzon, P., Zhang, M., Radek, R. J., Fox, G. B., Buccafusco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behavioral+assessment+of+sensorimotor+processes+in+the+mouse:+Acoustic+startle,+sensory+gating,+locomotor+activity,+rotarod,+and+beam+walking.">The behavioral assessment of sensorimotor processes in the mouse: Acoustic startle, sensory gating, locomotor activity, rotarod, and beam walking.</a> Methods of behavior analysis in neuroscience. , (2009).</li><li>Geyer, M. A., Swerdlow, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+startle+response,+prepulse+inhibition,+and+habituation.">Measurement of startle response, prepulse inhibition, and habituation.</a> Current Protocols in Neuroscience. , (2001).</li><li>Parisi, T., Ison, J. 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W., Geisser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+methods+in+the+analysis+of+profile+data.">On methods in the analysis of profile data.</a> Psychometrika. 24, 95-112 (1959).</li><li>Fendt, M., Li, L., Yeomans, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+stem+circuits+mediating+prepulse+inhibition+of+the+startle+reflex.">Brain stem circuits mediating prepulse inhibition of the startle reflex.</a> Psychopharmacology (Berl). 156 (2-3), 216-224 (2001).</li><li>Koch, M., Schnitzler, H. 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The present protocol describes the power of varying ISI for the assessment of temporal processing for studies employing either cross-sectional or longitudinal experimental designs. Examining the effects of sensory modality, psychostimulant exposure, or age on the shape of the ISI function demonstrated its utility in revealing a differential sensitivity to the manipulation of ISI (i.e., shifts in the point of maximal inhibition) or a relative insensitivity to the manipulation of ISI (i.e., sharper inflec...

Temporal processing deficits have been implicated as a potential underlying neural mechanism for alterations in higher-level cognitive processes commonly observed in neurocognitive disorders. Prepulse inhibition (PPI) of the auditory startle response (ASR) is a translational experimental paradigm commonly used to examine temporal processing deficits, revealing profound alterations in neurocognitive disorders such as schizophrenia1, attention deficit hyperactivity disorder2 and HIV-1 associated neurocognitive disorders3,4. Specifically, assessments of temporal processing in preclinical models of HIV-1 have revealed the generality, relative permanence, and suggested the diagnostic utility of PPI across the majority of the animals&#39; functional lifespan3,4,5,6.
Use of an approach varying interstimulus interval (ISI; i.e., the time between the prepulse and the startle stimulus) in the analysis of reflex modification dates back to Sechenov in 18637. The seminal studies of reflex modification, a measure of sensorimotor gating, employed an approach varying ISI to assess flexor response and audition in frogs7,8, as well as knee-jerk responses in humans9. The first clinical application of the reflex modification procedure assessed visual sensitivity in a man with hysterical blindness10. Over a century after the first reports of reflex modification, the approach of varying ISI was popularized across a series of seminal papers11,12,13. Despite the inherent differences in the seminal studies on reflex modification (i.e., species, experimental procedures, reflexes), they established a temporal relationship that was strikingly similar between species.
Assessment of prepulse inhibition using an approach varying ISI, as detailed in the present protocol, has multiple advantages over the popularized percent of control approach. First, the approach affords an opportunity to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve)3,15 in startle amplitude, as well as shifts in the peak point of response inhibition3,5. Additionally, when an approach varying ISI is employed, startle response is a relatively stable phenomenon1, suggesting the potential utility of the approach in longitudinal studies examining the progression of neurocognitive deficits5,15. Finally, PPI provides a critical opportunity to understand the underlying neural circuitry involved in neurocognitive disorders16.
In our study, we employed two experimental paradigms (Figure 1), including cross-modal PPI and gap prepulse inhibition (gap-PPI), to evaluate the utility of an approach varying ISI to delineate effects of sensory modality, psychostimulant exposure, and age. The cross-modal PPI experimental paradigm utilizes the presentation of an added stimulus (e.g., tone, light, air puff) as a discrete prestimulus prior to an acoustic startling stimulus. In sharp contrast, in the gap-PPI experimental paradigm, the absence of a background (e.g., removal of background noise, light, or air puff) serves as a discrete prestimulus. Here, we describe both experimental paradigms for the assessment of temporal processing, as well as statistical approaches for the analysis of PPI and gap-PPI. Within the discussion, we compared the conclusions one would draw from the variable ISI approach and the popularized percent of control approach.

<table>
	<tbody>
		<tr>
			<td>SR-Lab Startle Response System</td>
			<td>San Diego Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolation Cabinet</td>
			<td>Industrial Acoustic Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SR-Lab Startle Calibration System</td>
			<td>San Diego Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-Frequency Loudspeaker</td>
			<td>Radio Shack</td>
			<td>model #40-1278B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sound Level Meter</td>
			<td>Bruel &#38; Kjaer</td>
			<td>model #2203</td>
			<td></td>
		</tr>
		<tr>
			<td>Perspex Cylinder</td>
			<td>San Diego Instruments</td>
			<td></td>
			<td>Included with the SR-Lab Startle Response System</td>
		</tr>
		<tr>
			<td>SR-Lab Startle Response System Software</td>
			<td>San Diego Instruments</td>
			<td></td>
			<td>Included with the SR-Lab Startle Response System</td>
		</tr>
		<tr>
			<td>Light Meter</td>
			<td>Sper Scientific, Ltd.</td>
			<td>model #840006</td>
			<td></td>
		</tr>
		<tr>
			<td>Airline Regulator</td>
			<td>Craftsman</td>
			<td>model #16023</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS Statistics 24</td>
			<td>IBM</td>
			<td></td>
			<td>Used for Statistical Analyses (Optional)</td>
		</tr>
	</tbody>
</table>

the power of interstimulus interval for the assessment of temporal processing in rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

A prominent non-monotonic ISI function was observed in cross-modal PPI (Figures 2A, 3A, 4A) and gap-PPI (Figures 2B, 3B, 4B). Baseline startle responses were observed at the 0 and 4000 ms ISIs, included as reference trials within a test session. The importance of the 4000 ms ISI cannot be understated, as it most closely resembles the PPI test trials (i.e., 30, 50, 100, 200 ms ISIs) in that the subject receives both the prepulse and startling stim...

Watch this Scientific Journal Video about The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents at JoVE.com

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

This method can help answer key questions in the field of neuroscience by characterizing neurocognitive disorders and their progression across the lifespan via cross-sectional and longitudinal assessments. The main advantage of this technique is that it utilizes the systematic manipulation of ISI to afford a critical opportunity for the evaluation of a temporal processing. The implications of this technique extend for the assessment of neurocircuitry alterations in neurocognitive disorders, because the serial neural circuit mediating PPI has been well-established.To begin this procedure, open the Startle Response system software, define a pulse-only ASR trial by selecting Definitions, then Define Trial. Type a trial name, hit enter, and Record Data. Set the Analog Level to 720.Define the Wait Length as 20 milliseconds, and introduce Background. End the trial and hit Accept to save it. To create a 30-milliseconds trial definition for acoustic PPI, select Definitions, then Define Trial.Type a trial name and hit enter. Set the Analog Level to 600 at zero millisecond to introduce the pre-stimulus. Assign the Wait Length to 20 milliseconds to specify the length of the pre-stimulus.Then, set the Analog Level to 440 at 20 milliseconds to remove the pre-stimulus. Define the Wait Length dependent upon ISI, and specify Record Data. Set the Analog Level to 720 and assign the Wait Length to 20 milliseconds.Next, introduce Background, then end the trial and hit Accept to save the trial. Afterward, create a 30-milliseconds definition for acoustic gap PPI. Selection Definitions, one, Define Trial.Type a trial name and hit enter. Set the Analog Level to zero at zero millisecond to introduce the pre-stimulus. Assign the Wait Length to 20 milliseconds to specify the length of the pre-stimulus.Then, set the Analog Level to 440 at 20 milliseconds to remove the pre-stimulus. Subsequently, define the Wait Length dependent upon ISI, then specify Record Data. Set the Analog Level to 720.Assign the Wait Length to 20 milliseconds, and introduce Background. End the trial, hit Accept to save the trial. To create a habituation session, select Definitions and Define Session.Set the number of Record Samples to 200, the Samples per Second to 2, 000, the Background analog level to 440, the Acclimation Period to 5 minutes, and the sequence Repetitions to 36. Type 10 into the ITI List box. Next, click Add, and select the Pulse Only ASR trial.Click Save to save the Habituation session. Subsequently, define the session for cross-modal PPI by selecting Definitions and Define Session. Set the number of Record Samples to 200, the Samples per Second to 2, 000, the Acclimation Period to five minutes, the Background analog level to 440, and the sequence Repetitions to one.Then, define the ITI list by typing 10 into the first five ITI List boxes. Type the ITI values into the next 72 ITI list boxes, representing trials with a pre-stimulus. Afterward, click Add, select the Pulse Only ASR trial, and load it six times for trials one to six.Load the six-trial blocks in an A-B-B-A counterbalanced order of presentation for a cross-modal PPI. Then, click Save to save the session. Next, define the session for gap PPI by selecting Definitions and Define Session.Set the number of Record Samples to 200, the Samples per Second to 2, 000, the Acclimation Period to five minutes, the Background analog level to 440, and the sequence Repetitions to one. To define the ITI list, type 10 into the first five ITI List boxes. Type the ITI values into the next 72 ITI List boxes, representing trials with a pre-stimulus.Then, select the Pulse Only ASR trial and add it six times for trials one to six. Create six trial blocks for each pre-stimulus modality using a Latin square design and save the session. In this procedure, handle the animals to allow for acclimation across a series of days prior to beginning experimentation.Open the Startle Response system software. Click Run and select the session of interest. Then, input an Output File Name and click OK.Enter Subject, Group, and ID information and click Continue.Place the animal into the startle apparatus, using an animal enclosure that is most appropriate for the size of the animal. Click OK to begin the session. When it is done, export data for analysis by clicking Reports, then, Concatenate Data.Load the data file and click Add. Then, click ASCII to save the data output. The utility of an approach varying the ISI to delineate effects of sensory modality and cross-modal PPI are illustrated here.A prominent shift in the point of maximal inhibition is dependent upon sensory modality, suggesting a differential sensitivity to the manipulation of ISI. Specifically, maximal inhibition is observed at the 30-milliseconds ISI following the presentation of a discrete acoustic pre-stimulus at the 50-milliseconds ISI following the presentation of a discrete visual pre-stimulus and at the 200-milliseconds ISI following the presentation of a discrete tactile pre-stimulus. Following an animal's experience with each pre-stimulus in cross-modal PPI, the generalizability of sensory modality effects was assessed in gap PPI.This figure demonstrates the generalizability of varying the ISI to delineate effects of sensory modality. A prominent shift in the point of maximal inhibition suggesting a differential sensitivity to the manipulation of ISI was observed in tactile gap PPI relative to acoustic gap PPI and visual gap PPI. The utility of an approach varying the ISI to delineate effects of psychostimulant in cross-modal PPI are illustrated here.At the post-test assessment, most notably, a relative flattening of the ISI function is observed, suggesting a relative insensitivity to the manipulation of ISI relative to the pre-test assessment. Additionally, a prominent shift in the point of maximal inhibition is also revealed, supporting a differential sensitivity to the manipulation of ISI. Specifically, maximal inhibition is observed at the 30-millisecond ISI during the pre-test assessment and at the 100-millisecond ISI during the post-test assessment.Subsequently, the generalizability of the varying ISI approach to delineate effects of psychostimulant exposure was assessed in auditory gap PPI. A significantly flatter ISI function was observed at the post-test assessment relative to the pre-test assessment, suggesting a relative insensitivity to the manipulation of ISI. The development of temporal processing can be assessed using a longitudinal experimental design.The development of temporal processing in visual PPI is illustrated here. The point of maximal inhibition at all ages is at the 50-millisecond ISI, however, a sharper inflection of the ISI function is observed across age, suggesting a perceptual sharpening which occurs with development. Subsequently, the generalizability of the varying ISI approach to examine the development of temporal processing was assessed in auditory gap PPI.At post-natal day 30, an insensitivity to the manipulation of ISI was observed, evidenced by a flatter ISI function relative to PD90 or PD150. While attempting this procedure, it's important to implement critical experimental design considerations, including a Latin square experimental design for the presentation of ISIs, two control trials including the zero and 4, 000-millisecond ISI and a variable ITI. Following this procedure, other neurocognitive assessments including a signal-detection operant task and neuroanatomical assessments can be performed to further assess temporal processing deficits and their underlying neural mechanisms.

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A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

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

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

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

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

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

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 ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their consequences. This process is integral to the normal human behavior and has been shown to be disrupted by neurological and psychiatric disorders such as addiction, schizophrenia, and depression. Rodents have long been used to uncover the neurobiology of human cognition. To this end, several behavioral tasks have been developed; however, most are non-automated and are labor-intensive. The recent development of the open-source microcontroller has enabled researchers to automate operant-based tasks for assessing a variety of cognitive tasks, standardizing the stimulus presentation, improving the data recording and consequently, improving the research output. Here, we describe an automated delay-based reinforcement-guided decision-making task, using an operant T-maze controlled by custom-written software programs. Using these decision-making tasks, we show the changes in the local field potential activities in the anterior cingulate cortex of a rat whilst it performs a delay-based cost-and-benefit decision-making task.

A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them.

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their ...