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>
	</li>
	<li>Calibrate the response sensitivities using the startle calibration system.</li>
	<li>Attach the high-frequency loudspeaker 30 cm above the animal holder.
	<ol>
		<li>Measure and calibrate the loudspeaker using a sound level meter by placing the microphone inside the animal holder.</li>
	</ol>
	</li>
	<li>Affix a white LED light (22 lux) on the wall in front of the animal holder.
	<ol>
		<li>Measure the lux presented as a visual prepulse using a light meter.</li>
	</ol>
	</li>
	<li>Connect a semi-rigid plastic tube (0.64 mm diameter) to a compressed air tank via an airline regulator.
	<ol>
		<li>Set the air tank to 16 psi for the presentation of tactile prestimuli.</li>
		<li>Use a sound level meter to measure the amount of noise being emitted by the tactile stimulus inside the tube, 2.5 cm from the end of the animal holder. If using multiple chambers, ensure that all chambers are calibrated in the same manner. 
		Note: To prevent the tactile stimulus from being perceived as an acoustic stimulus, the sound of the air puff prepulse must be less than or equal to the white noise background. In the present set-up, the air puff prepulse emitted 70 db(A) inside the tube while the background white noise was also set to 70 db(A).</li>
	</ol>
	</li>
</ol>
2. Creation of Experimental Programs
<ol>
	<li>Open the Startle Response System software (see Table of Materials).</li>
	<li>Click Definitions and Select Define Trial.</li>
	<li>Define a pulse-only ASR trial. 
	Note: The pulse-only ASR trial is run during the habituation session, and 6 times at the beginning of every cross-modal PPI and gap-PPI session for habituation.
	<ol>
		<li>Type a Trial Name. Hit Enter.</li>
		<li>Record Data.</li>
		<li>Set the Analog Level to 720.</li>
		<li>Define the Wait Length as 20 ms.</li>
		<li>Introduce Background.</li>
		<li>End the Trial.</li>
		<li>Hit Accept to save the trial.</li>
	</ol>
	</li>
	<li>Click Definitions and Select Define Trial.&#160;</li>
	<li>Create six separate trial definitions for acoustic PPI, including one trial for each ISI (i.e., 0, 30, 50, 100, 200, 4000 ms).
	<ol>
		<li>Create a trial definition for the 0 ms ISI for acoustic PPI.
		<ol>
			<li>Type a Trial Name. Hit Enter.</li>
			<li>Record Data.</li>
			<li>Set the Analog Level to 720.</li>
			<li>Assign the Wait Length to 20 ms.</li>
			<li>Introduce Background.</li>
			<li>End the Trial.</li>
			<li>Hit Accept to save the trial.</li>
		</ol>
		</li>
		<li>Create remaining trial definitions for ISIs with both a prestimulus and a stimulus (i.e., 30, 50, 100, 200, 4000 ms).
		<ol>
			<li>Type a Trial Name. Hit Enter.</li>
			<li>Set the Analog Level to 600 at 0 ms to introduce the prestimulus.</li>
			<li>Assign the Wait Length to 20 ms to specify the length of the prestimulus.</li>
			<li>Set the Analog Level to 440 at 20 ms to remove the prestimulus.</li>
			<li>Define the Wait Length dependent upon ISI. 
			Note: Define the wait length as: 10 ms for the 30 ms ISI, 30 ms for the 50 ms ISI, 80 ms for the 100 ms ISI, 180 ms for the 200 ms ISI, and 3980 ms for the 4000 ms ISI. Only one wait length is included for each ISI.</li>
			<li>Record Data.</li>
			<li>Set the Analog Level to 720.</li>
			<li>Assign the Wait Length to 20 ms.</li>
			<li>Introduce Background.</li>
			<li>End the Trial.</li>
			<li>Hit Accept to save the trial.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Click Definitions and Select Define Trial.&#160;</li>
	<li>Create six separate trial definitions for visual or tactile PPI, including one trial for each ISI (i.e., 0, 30, 50, 100, 200, 4000 ms).
	<ol>
		<li>Create a trial definition for the 0 ms ISI for visual or tactile PPI.
		<ol>
			<li>Type a Trial Name. Hit Enter.</li>
			<li>Record Data.</li>
			<li>Turn the Tactile on.</li>
			<li>Set the Analog Level to 720 and the Wait Length to 20 ms.</li>
			<li>Turn the Tactile off.</li>
			<li>Introduce Background.</li>
			<li>End the Trial.</li>
			<li>Hit Accept to save the trial.</li>
		</ol>
		</li>
		<li>Create remaining trial definitions for ISIs with both a prestimulus and a stimulus (i.e., 30, 50, 100, 200, 4000 ms). 
		Note: Visual and tactile can not be run concurrently because of software and hardware limitations. The modality presented is dependent upon the input into the hardware (i.e., whether the light is connected or the air puff is connected).
		<ol>
			<li>Type a Trial Name. Hit Enter.</li>
			<li>Turn the Tactile on to introduce the prestimulus. 
			Note: In this instance, tactile refers to the modality (i.e., either visual or air puff) that is connected to the hardware.</li>
			<li>Set the Wait Length to 20 ms.</li>
			<li>Turn the Tactile off to remove the prestimulus.</li>
			<li>Set the Analog Level to 440 at 20 ms.</li>
			<li>Define the Wait Length dependent upon ISI. 
			Note: Define the wait length as: 10 ms for the 30 ms ISI, 30 ms for the 50 ms ISI, 80 ms for the 100 ms ISI, 180 ms for the 200 ms ISI, and 3980 ms for the 4000 ms ISI.</li>
			<li>Record Data.</li>
			<li>Set the Analog Level to 720.</li>
			<li>Assign the Wait Length to 20 ms.</li>
			<li>Introduce Background.</li>
			<li>End the Trial.</li>
			<li>Hit Accept to save the trial.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Click Definitions and Select Define Trial.</li>
	<li>Create six separate trial definitions for acoustic gap-PPI, including one trial for each ISI (i.e., 0, 30, 50, 100, 200, 4000 ms).
	<ol>
		<li>Create a trial definition for the 0 ms ISI for acoustic gap-PPI.
		<ol>
			<li>Type a Trial Name. Hit Enter.</li>
			<li>Record Data.</li>
			<li>Set the Analog Level to 720 and the Wait Length to 20 ms.</li>
			<li>Introduce Background.</li>
			<li>End the Trial.</li>
			<li>Hit Accept to save the trial.</li>
		</ol>
		</li>
		<li>Create remaining trial definitions for ISIs with both a prestimulus and a stimulus (i.e., 30, 50, 100, 200, 4000 ms).
		<ol>
			<li>Type a Trial Name. Hit Enter.</li>
			<li>Set the Analog Level to 0 at 0 ms to introduce the prestimulus.</li>
			<li>Assign the Wait Length to 20 ms to specify the length of the prestimulus.</li>
			<li>Set the Analog Level to 440 at 20 ms to remove the prestimulus.</li>
			<li>Define the Wait Length dependent upon ISI. 
			Note: Define the wait length as: 10 ms for the 30 ms ISI, 30 ms for the 50 ms ISI, 80 ms for the 100 ms ISI, 180 ms for the 200 ms ISI, and 3980 ms for the 4000 ms ISI.</li>
			<li>Record Data.</li>
			<li>Set the Analog Level to 720.</li>
			<li>Assign the Wait Length to 20 ms.</li>
			<li>Introduce Background.</li>
			<li>End the Trial.</li>
			<li>Hit accept to save the trial.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Click Definitions and Select Define Trial.&#160;</li>
	<li>Create six separate trial definitions for the visual or tactile gap-PPI, including one trial for each ISI (i.e., 0, 30, 50, 100, 200, 4000 ms).
	<ol>
		<li>Create a trial definition for the 0 ms ISI for visual or tactile gap-PPI.
		<ol>
			<li>Type a Trial Name. Hit Enter.</li>
			<li>Turn the Tactile on.</li>
			<li>Record Data.</li>
			<li>Set the Analog Level to 720 and the Wait Length to 20 ms.</li>
			<li>Introduce Background.</li>
			<li>End the Trial.</li>
			<li>Hit Accept to save the trial.</li>
		</ol>
		</li>
		<li>Create remaining trial definitions for ISIs with both a prestimulus and a stimulus (i.e., 30, 50, 100, 200, 4000 ms).&#160;
		<ol>
			<li>Type a Trial Name. Hit Enter.</li>
			<li>Turn the Tactile on.</li>
			<li>Set the Analog Level to 0 ms.</li>
			<li>Turn the Tactile off.</li>
			<li>Set the Wait Length to 20 ms.</li>
			<li>Turn the Tactile on.</li>
			<li>Set the Analog Level to 440.</li>
			<li>Define the Wait Length dependent upon ISI. 
			Note: Define the wait length as: 10 ms for the 30 ms ISI, 30 ms for the 50 ms ISI, 80 ms for the 100 ms ISI, 180 ms for the 200 ms ISI, and 3980 ms for the 4000 ms ISI.</li>
			<li>Record Data.</li>
			<li>Set the Analog Level to 720.</li>
			<li>Assign the Wait Length to 20 ms.</li>
			<li>Introduce Background.</li>
			<li>End the Trial.</li>
			<li>Hit accept to save the trial.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Select Definitions and Define Session.
	<ol>
		<li>Create a habituation session.
		<ol>
			<li>Set the Background Analog Level to 440, the Number of Record Samples to 200 the Samples per Second to 2000, the Acclimation Period to 5 min, and the Sequence Repetitions to 36.</li>
			<li>Type 10 into the intertrial interval (ITI) list box.</li>
			<li>Click Add and select the pulse-only ASR trial.</li>
			<li>Click Save to save the habituation session.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Select Definitions and Define Session.</li>
	<li>Define the session for Cross-Modal PPI.
	<ol>
		<li>Set the Background Analog Level to 440, the Number of Record Samples to 200 the Samples per Second to 2000, the Acclimation Period to 5 min, and the Sequence Repetitions to 1.</li>
		<li>Define the intertrial interval (ITI) list.
		<ol>
			<li>Type 10 into the first 5 ITI list boxes.</li>
			<li>Type a variable ITI (15-25 s) into the next 72 ITI list boxes, representing trials with a prestimulus.</li>
		</ol>
		</li>
		<li>Click Add.
		<ol>
			<li>Select the pulse-only ASR trial and load it 6 times for Trials 1-6.</li>
			<li>Create 6-trial blocks for each prestimulus modality using a Latin Square design (Table 1).</li>
			<li>Load the 6-trial blocks in an ABBA counterbalanced order of presentation (e.g., acoustic, visual, visual, acoustic, acoustic, etc.) for cross-modal PPI. 
			Note: Each trial must be loaded individually. 
			Note: Each cross-modal PPI session includes a total of 78 trials.</li>
		</ol>
		</li>
		<li>Click Save to save the session.</li>
	</ol>
	</li>
	<li>Select Definitions and Define Session.
	<ol>
		<li>Define the session for Gap-PPI.
		<ol>
			<li>Set the Background Analog Level to 440, the Number of Record Samples to 200 the Samples per Second to 2000, the Acclimation Period to 5 min, and the Sequence Repetitions to 1.</li>
			<li>Define the intertrial interval (ITI) list.
			<ol>
				<li>Type 10 into the first 5 ITI list boxes.</li>
				<li>Type a variable ITI (15-25 s) into the next 36 ITI list boxes, representing trials with a prestimulus.</li>
			</ol>
			</li>
			<li>Click Load to load the trials.
			<ol>
				<li>Select the pulse-only ASR trial and load it 6 times for Trials 1-6.</li>
				<li>Create 6-trial blocks for each prestimulus modality using a Latin Square design (Table 1).</li>
			</ol>
			</li>
			<li>Click Save to save the session. 
			Note: Each gap-PPI session includes a total of 42 trials. Each session assesses one sensory modality.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Protocol Structure
<ol>
	<li>Use the F344/N rat strain, the most common inbred rat strain, for assessments. 
	Note: Cross-modal PPI and gap-PPI can be conducted in animals at a variety of ages, of both sexes, and regardless of hormonal status (i.e., ovariectomized, castrated, intact). Details regarding the animals used in the representative data are presented in the representative results.</li>
	<li>Handle the animals to allow for acclimation across a series of days prior to beginning experimentation.</li>
	<li>Randomize the order of animals for experimentation dependent upon the between-subjects&#8217; factors of interest (e.g., biological sex, treatment).</li>
	<li>Open the startle response system software. Click Run. Select the session of interest. 
	Note: Only one session is conducted per day and sessions need to be conducted in a sequential order (i.e., Habituation, Cross-Modal PPI, Gap-PPI)</li>
	<li>Input an Output File Name and click OK.</li>
	<li>Enter Subject, Group, and ID information and click Continue.</li>
	<li>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.</li>
	<li>Export data for analysis.
	<ol>
		<li>Click Reports | Concatenate Data. Load the data file and click Add. Click ASCII to save the data output.</li>
	</ol>
	</li>
</ol>
4. Data Analysis
<ol>
	<li>Calculate an adjusted V. Max for each trial by subtracting the V. Max from the Start value. 
	Note: The adjusted V. Max creates a measure of mean peak ASR amplitude.</li>
	<li>Graphically visualize results for the habituation session.
	<ol>
		<li>Plot group means and standard errors of the mean for each trial. Regression analyses can be conducted and fit with 95% confidence intervals.</li>
	</ol>
	</li>
	<li>Graphically visualize results for cross-sectional cross-modal PPI and gap-PPI.
	<ol>
		<li>Calculate mean values for each ISI by averaging across the 6 trials individually for each animal.</li>
		<li>Calculate and graph group means and standard errors of the mean for each ISI and sensory modality.</li>
	</ol>
	</li>
	<li>Statistically analyze cross-modal PPI and gap-PPI (Optional). 
	Note: Although the precise statistical approach will be dependent upon the experimental design and research question of interest, a mixed-design repeated measures ANOVA provides one appropriate approach.</li>
</ol>

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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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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transformation+of+percentage+for+use+in+the+analysis+of+variance.">The transformation of percentage for use in the analysis of variance.</a> Ohio Journal of Science. 38, 9-12 (1938).</li><li>Bartlett, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+transformations.">The use of transformations.</a> Biometrics. 3, 39-52 (1947).</li><li>Cochran, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+variance+when+experimental+errors+follow+the+poisson+or+bimodal+laws.">The analysis of variance when experimental errors follow the poisson or bimodal laws.</a> Annals of Mathematical Sciences. 11, 335-347 (1940).</li><li>Greenhouse, S. 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. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acoustic+startle+response+in+rats:+Circuits+mediating+evocation,+inhibition+and+potentiation.">The acoustic startle response in rats: Circuits mediating evocation, inhibition and potentiation.</a> Behavioural Brain Research. 89 (1-2), 35-49 (1997).</li></ol>

None of the authors have conflicts of interest to declare.

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 inflections of the ISI curve, flattening of the ISI curve). Use of two experimental paradigms, including cross-modal PPI and gap-PPI, demonstrates that the utility of ISI is independent of the addition (i.e., cross-modal PPI) or removal (i.e., gap-PPI) of a discrete prestimulus.
Critical experimental design considerations for the completion of cross-modal PPI and gap-PPI are included within the protocol. First, a Latin-Square experimental design is implemented for the presentation of ISIs within 6-trial blocks, controlling for variation due to the order of ISI presentation. Second, the use of two control trials, including both the 0 and 4000 ms ISIs, provides reference control trials within the test session. The use of the 4000 ms ISI is particularly critical, as it most appropriately resembles the other (i.e., 30, 50, 100, 200) prepulse + pulse trials, but without the expectation of significant inhibition. Third, a counterbalanced (i.e., ABBA) experimental design is employed within cross-modal PPI to account for the repeated measurement of sensory modalities within a test session. Finally, inclusion of a variable ITI during the prepulse + pulse trials prevents an animal from expecting, and thus preparing for, the start of a trial. Thus, the implementation of a comprehensive number of ISIs in accordance with an appropriate experimental design allows for the determination of relatively precise and defined response functions; functions which provide a critical opportunity to assess the construct of temporal processing.
Methodology described within the present protocol contrasts other contemporary protocols for the analysis of PPI, which have popularized an approach that commonly employs a single ISI19,20. The popularized approach is commonly analyzed using percent inhibition, calculated as follows: 100 x {[(startle response amplitude during control trials)-(startle response amplitude during prepulse + pulse trials)] / {startle response amplitude during control trials)}. Two major caveats of the contemporary protocols, including the preclusion of the assessment of temporal processing and inappropriate statistical analyses, are discussed in turn below.
Percent inhibition was calculated for the 100 ms ISI within the representative data to demonstrate the limitations of the popularized approach (Table 2).&#160;For example, results for the assessment of acoustic gap-PPI and tactile gap-PPI suggest that animals fail to display any significant inhibition. Examination of Figure 2, utilizing an approach varying ISI, however, reveals that the animals did not fail to inhibit, but displayed a significant shift in the point of maximal inhibition (i.e., 50 ms in acoustic gap-PPI, 30 ms in tactile gap-PPI). Most notably, however, use of percent inhibition precludes the use of longitudinal experimental designs for assessing the development of temporal processing as a function of age, a well-recognized phenomena14,21. Thus, like any percent of control measure, percent inhibition fails to disambiguate changes in PPI from changes in baseline startle response precluding the assessment of temporal processing.
Despite the apparent simplicity of the popularized approach, inferences drawn from statistical analysis must be made with extreme caution. Assumptions of the analysis of variance (i.e., normality of sampling distribution of means, homogeneity of error variance, independence of errors, absence of outliers22) may not be met with subjectively determined percentage data23. Specifically, the error variance for percentage data are not normally distributed24, but instead are more appropriately described by a Poisson or bimodal distribution25. In sharp contrast, a repeated-measures ANOVA provides one valid and reliable method for the statistical analysis of the ISI function. However, it is vital to account for the potential violation of sphericity, an assumption only present in models involving repeated measures, either using planned orthogonal contrasts or the post hoc Greenhouse-Geisser df correction factor26 (pGG).
Utilization of an approach varying ISI, however, is not without limitation. First, hardware and software limitations allow only two prestimulus modalities to be assessed at once. Notably, a differential sensitivity to the manipulation of context (i.e., concurrent visual or tactile stimulus in acoustic PPI) was previously reported in Long-Evans rats14 and HIV-1 Tg animals6. Second, relative to the popularized approach, there is a greater experimental time for the ISI approach (i.e., ~30 minutes for cross-modal PPI; ~20 minutes for gap-PPI).
Thus, an approach varying ISI provides an experimental method for the assessment of temporal processing. In addition to the aforementioned strengths of the approach, the serial neural circuit mediating PPI has been well-established27,28, allowing for the assessment of neural circuitry alterations in neurocognitive disorders. Additionally, cross-modal PPI and gap-PPI may serve as a diagnostic screening tool for neurocognitive disorders (e.g., HAND4,5,6). Utilization of the variable ISI approach, therefore, may potentially have translational clinical utility for neurocognitive disorders.

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 stimuli. However, no significant decrease in ASR is observed at the 4000 ms ISI because of the large time interval between the prepulse and startling stimulus. Either the addition (i.e., cross-modal PPI) or removal (i.e., gap-PPI) of a discrete prestimulus produced robust inhibition at the 30, 50, 100, and 200 ms ISIs; inhibition that was dependent upon sensory modality, psychostimulant exposure or age. The power of the ISI approach is revealed by examining these effects on changes in the ISI function (i.e., sharper inflections of the ISI curve, flattening of the ISI curve, and shifts in the point of maximal inhibition).
The utility of an approach varying the ISI to delineate effects of sensory modality in cross-modal PPI are illustrated in Figure 2A (F344/N controls between 8 and 10 months of age, n=20). Due to hardware and software limitations, only two prestimulus modalities can be assessed at once. Following habituation, concurrent acoustic and visual prepulse stimuli were used to examine PPI. Next, concurrent acoustic and tactile prepulse stimuli were used to assess PPI. Data for acoustic PPI is presented from the experimental paradigm including the concurrent presentation of acoustic and visual prestimuli (i.e., visual context). 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 ms ISI following the presentation of a discrete acoustic prestimulus, at the 50 ms ISI following the presentation of a discrete visual prestimulus, and at the 200 ms ISI following the presentation of a discrete tactile prestimulus. Additionally, a flatter ISI function, indicative of a relative&#160;insensitivity to the manipulation of ISI, is observed following the presentation of an acoustic prestimulus relative to a visual or tactile prestimulus. A repeated measures ANOVA was conducted to statistically analyze the data, confirming our observations and revealing a significant prestimulus modality x ISI interaction [F(10,190)=22.8, pGG&#8804;0.001, &#951;p2=0.546] with a prominent linear-linear component [F(1,19)=36.1, p&#8804;0.001, &#951;p2=0.655]. Notably, the interaction&#160;accounted for a large proportion of variance within the model, evidenced via measures of &#951;p2.
Following an animal&#39;s experience with each prestimulus in cross-modal PPI, the generalizability of sensory modality effects was assessed in gap-PPI. Acoustic gap-PPI, visual gap-PPI and tactile gap-PPI were each conducted separately. Figure 2B 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 (i.e., 30 ms) relative to acoustic gap-PPI and visual gap-PPI (i.e., 50 ms). Additionally, a relative insensitivity to the manipulation of ISI, evidenced by a relatively flatter ISI function was observed in tactile gap-PPI and visual gap-PPI relative to acoustic gap-PPI. As in cross-modal PPI, a significant prestimulus modality x ISI interaction [F(10,190)=17.6, pGG&#8804;0.001, &#951;p2=0.481] with a prominent linear-quadratic component [F(1,19)=58.5, p&#8804;0.001, &#951;p2=0.755] was revealed; an effect which again, accounts for a significant proportion of the variance.
After the completion of cross-modal PPI and gap-PPI, animals repeatedly orally self-administered methylphenidate (MPH). A post-test assessment of cross-modal PPI with concurrent acoustic and visual prestimuli and acoustic gap-PPI were conducted at approximately 14 months of age following 22-27 days of MPH exposure. The pre-test and post-test ISI functions for acoustic PPI are illustrated in Figure 3A. Most notably, at the post-test assessment, 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 revealed, with inhibition at the 30 ms ISI during the pre-test assessment and the 100 ms ISI at the post-test assessment, suggesting a differential sensitivity to the manipulation of ISI. A repeated-measures ANOVA confirmed these observations, revealing a significant test session x ISI interaction [F(5,95)=7.4, pGG&#8804;0.003, &#951;p2=0.280] with a prominent linear-quadratic component [F(1,19)=10.6, p&#8804;0.004, &#951;p2=0.358].
Following the post-test cross-modal PPI assessment, acoustic gap-PPI was conducted to assess the generalizability of the effects of psychostimulant exposure on temporal processing. Figure 3B illustrates the generalizability of varying the ISI to delineate effects of psychostimulant exposure. The point of maximal inhibition was at the 50 ms ISI during both the pre-test and post-test assessment. However, a significantly flatter ISI function was observed following MPH exposure. A repeated-measures ANOVA confirmed these observations, revealing a significant test session x ISI interaction [F(5, 95)=3.6, pGG&#8804;0.013, &#951;p2=0.159] with a prominent linear-cubic component [F(1,19)=9.1, p&#8804;0.007, &#951;p2=0.325].
The shape of the ISI function also affords an opportunity to assess the development of temporal processing across age. In a longitudinal study (F344/N controls, male: n=20, female: n=17), cross-modal PPI with concurrent acoustic and visual prestimuli was conducted every sixty days from postnatal day (PD) 30 to PD 150. The development of temporal processing in visual PPI is illustrated in Figure 4A. Within visual PPI, the point of maximal inhibition at all ages is at the 50 ms ISI. However, a sharper inflection of the ISI function is observed across age, suggesting a perceptual sharpening which occurs with development. A repeated-measures ANOVA, with sex as the between-subjects factor and age, ISI, and trial as within-subjects factors, confirmed these observations revealing a significant age x ISI interaction [F(10,350)=12.6, pGG&#8804;0.001, &#951;p2=0.265] with a prominent linear-quadratic component [F(1,35)=32.6, p&#8804;0.001, &#951;p2=0.482] and a significant ISI x sex interaction [F(5,175)=4.0, pGG&#8804;0.014, &#951;p2=0.104] with a prominent quadratic component[F(1,35)=5.2, p&#8804;0.028, &#951;p2=0.130].
At every age, acoustic gap-PPI was conducted following cross-modal PPI. The experiences an animal has had have a direct impact on its responses, necessitating the use of a sequential experimental design (i.e., always conducting cross-modal PPI prior to gap-PPI). Figure 4B illustrates the development of temporal processing, assessed using acoustic gap-PPI. At PD 30, a relative&#160;insensitivity to the manipulation of ISI was observed, evidenced by a flatter ISI function, relative to PD 90 or PD 150. Observations of the sharpest ISI function at PD 150 suggest a perceptual sharpening that occurs across development. Additionally, a prominent shift in the point of maximal inhibition is revealed, with maximal inhibition occurring at the 30 ms ISI at PD 30 and the 50 ms ISI at PD 90 and PD 150, suggesting a differential sensitivity to the manipulation of ISI. Statistically, the observation of a significant age x ISI interaction [F(10,350)=10.4, pGG&#8804;0.001, &#951;p2=0.230] with a prominent linear-quadratic component [F(1,35)=70.5, p&#8804;0.001, &#951;p2=0.668] and an ISI x sex interaction [F(5,175)=3.8, pGG&#8804;0.010, &#951;p2=0.097] with a prominent quadratic component [F(1,35)=11.0, p&#8804;0.002, &#951;p2=0.184240] confirms our observations.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58659/58659fig1.jpg" /> 
Figure 1: Prepulse Inhibition Experimental Paradigms. A) Animals exhibit a baseline auditory startle response when an acoustic startle stimulus is presented. B) During cross-modal prepulse inhibition (PPI), the presentation of a discrete prestimulus (i.e., acoustic tone, light, air puff) 30 to 500 ms16 prior to an acoustic startle stimulus, produces robust inhibition. C) During gap prepulse inhibition (gap-PPI), the removal of a discrete prestimulus (gap in background noise, light, or air puff) 30 to 200 ms17 prior to an acoustic startle stimulus produces robust inhibition. The image is adapted from Maze Engineers18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58659/58659fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58659/58659fig2.jpg" /> 
Figure 2: Cross-Sectional Assessment of Temporal Processing: Sensory Modality. A) Representative analysis of the effect of sensory modality on the interstimulus interval (ISI) function in cross-modal prepulse inhibition (PPI). B) Representative analysis of the effect of sensory modality on ISI in gap prepulse inhibition (gap-PPI). Control results from McLaurin et al.6 are reanalyzed in a novel manner to assess the effect of sensory modality. Data are presented as mean &#177; standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58659/58659fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58659/58659fig3.jpg" /> 
Figure 3: Cross-Sectional Assessment of Temporal Processing: Psychostimulant Exposure. A) Representative analysis of the effect of psychostimulant exposure (pretest vs. posttest) on the interstimulus interval (ISI) function in acoustic prepulse inhibition (PPI). B) Representative analysis of the effect of psychostimulant exposure on ISI in acoustic gap prepulse inhibition (gap-PPI). Control results from McLaurin et al.6 are reanalyzed in a novel manner as the pretest component for psychostimulant exposure. Data are presented as mean &#177; standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58659/58659fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58659/58659fig4.jpg" /> 
Figure 4: Longitudinal Assessment of Temporal Processing. A) Representative analysis of the effect of age on the interstimulus interval (ISI) function in visual prepulse inhibition (PPI). B) Representative analysis of the effect of age on the ISI function in acoustic gap prepulse inhibition (gap-PPI). Data are presented as mean &#177; standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58659/58659fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Trial Block</td>
			<td colspan="6">Interstimulus Interval&#160;</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0</td>
			<td>30</td>
			<td>50</td>
			<td>100</td>
			<td>200</td>
			<td>4000</td>
		</tr>
		<tr>
			<td>2</td>
			<td>30</td>
			<td>50</td>
			<td>100</td>
			<td>200</td>
			<td>4000</td>
			<td>0</td>
		</tr>
		<tr>
			<td>3</td>
			<td>50</td>
			<td>100</td>
			<td>200</td>
			<td>4000</td>
			<td>0</td>
			<td>30</td>
		</tr>
		<tr>
			<td>4</td>
			<td>100</td>
			<td>200</td>
			<td>4000</td>
			<td>0</td>
			<td>30</td>
			<td>50</td>
		</tr>
		<tr>
			<td>5</td>
			<td>200</td>
			<td>4000</td>
			<td>0</td>
			<td>30</td>
			<td>50</td>
			<td>100</td>
		</tr>
		<tr>
			<td>6</td>
			<td>4000</td>
			<td>0</td>
			<td>30</td>
			<td>50</td>
			<td>100</td>
			<td>200</td>
		</tr>
	</tbody>
</table>
Table 1: Latin Square Experimental Design
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sensory Modality</td>
			<td>Cross-Modal PPI</td>
			<td>Gap-PPI</td>
		</tr>
		<tr>
			<td>Auditory</td>
			<td>85.7 (2.0)</td>
			<td>25.0 (4.3)</td>
		</tr>
		<tr>
			<td>Visual</td>
			<td>72.6 (2.7)</td>
			<td>52.8 (5.3)</td>
		</tr>
		<tr>
			<td>Tactile</td>
			<td>73.2 (3.0)</td>
			<td>-3.6 (8.5)</td>
		</tr>
		<tr>
			<td>Psychostimulant Exposure</td>
			<td>Cross-Modal PPI</td>
			<td>Gap-PPI</td>
		</tr>
		<tr>
			<td>Pretest Assessment</td>
			<td>85.7 (2.0)</td>
			<td>25.0 (4.3)</td>
		</tr>
		<tr>
			<td>Posttest Assessment</td>
			<td>90.5 (1.3)</td>
			<td>52.6 (4.5)</td>
		</tr>
		<tr>
			<td>Age</td>
			<td>Cross-Modal PPI</td>
			<td>Gap-PPI</td>
		</tr>
		<tr>
			<td>PD 30</td>
			<td>51.3 (3.7)</td>
			<td>29.7 (4.4)</td>
		</tr>
		<tr>
			<td>PD 90</td>
			<td>73.8 (2.2)</td>
			<td>39.6 (5.7)</td>
		</tr>
		<tr>
			<td>PD 150</td>
			<td>66.3 (2.9)</td>
			<td>45.0 (3.9)</td>
		</tr>
	</tbody>
</table>
Table 2: Percent of Control

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.9K Views.  University of South Carolina. 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.

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

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 ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

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.

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

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 ...