Methodology

The methodologies available for the study of rhythms can be subdivided into those methods available for animal use (primarily rodents) and those methods available for use with human subjects. In general, the same methods used to study other rhythmic processes can be used to study the rhythmic nature of seizures. Conversely, those mechanisms used to investigate seizures can be examined for a rhythmic component. For example, the seizure inductive methodologies described elsewhere in this book could be examined for rhythmic components by producing the seizures at different times of the day and then examining for rhythmicity in seizure severity.

To further characterize the rhythm, an investigator could place the subjects in constant conditions and determine if the rhythm still exists and whether it continues with a periodicity of its own (Figure 12.3), i.e., whether the rhythm is circadian or diurnal.

FIGURE 12.3

Artistic depiction of a standard "double plotted" circadian rhythm with the peak of the rhythm occurring in the dark and the nadir occurring in the light. This type of figure is generated by plotting the 24-h rhythm of interest on a linear scale and then duplicating the result to the right of the first plot. The next day's measurement of the rhythm is plotted beneath the first day with the remaining days of the experiment following below. This allows the figure to be "read" like a book, from left to right and top to bottom. Experimental treatments are usually placed to the right of the double plotted figure at the time they are begun. Under an external light:dark cycle (days 1 to 5), the rhythm is fixed to the onset and offset of darkness. When the subject is placed in constant conditions (days 6 to 15), the rhythm begins to "free run" following its own endogenous period (tau) which in this case is slightly longer than 24 h. When the subject is placed back into a light:dark cycle (days 16 to 20), the rhythm is entrained to the new photoperiod.

FIGURE 12.3

Artistic depiction of a standard "double plotted" circadian rhythm with the peak of the rhythm occurring in the dark and the nadir occurring in the light. This type of figure is generated by plotting the 24-h rhythm of interest on a linear scale and then duplicating the result to the right of the first plot. The next day's measurement of the rhythm is plotted beneath the first day with the remaining days of the experiment following below. This allows the figure to be "read" like a book, from left to right and top to bottom. Experimental treatments are usually placed to the right of the double plotted figure at the time they are begun. Under an external light:dark cycle (days 1 to 5), the rhythm is fixed to the onset and offset of darkness. When the subject is placed in constant conditions (days 6 to 15), the rhythm begins to "free run" following its own endogenous period (tau) which in this case is slightly longer than 24 h. When the subject is placed back into a light:dark cycle (days 16 to 20), the rhythm is entrained to the new photoperiod.

In general, if an investigator wants to determine if a specific process is rhythmic, at least six time points throughout the cycle of interest are necessary. This number of time points allows for the statistical interpretation of the rhythm and the generation of a curve depicting the rhythm. Specific mathematical treatments available for characterizing and interpreting rhythms include cosinor analysis, power spectrum analysis (fast Fourier transformations), and periodograms.37 These mathematical formulations are available in specific software programs developed by companies specializing in rhythms research (Table 12.1). Each of these methods has advantages

TABLE 12.1

Companies Providing Hardware or Software that Collect or Analyze Rhythmic Processes

Company

name and address

Products available

Ambulatory Monitoring, Inc.

Actillume®, Mini-Motionlogger Actigraph®, and other

731 Saw Mill River Road

monitoring devices for measurement of rhythms in

P.O. Box 609

human subjects

Ardsley, NY 10502-0609

Circadian Technologies, Inc.

Techniques available for treatment of shift work, jet lag

125 Cambridge Park Drive

and other circadian disorders in human subjects

Cambridge, MA 02140

Data Sciences International

Temperature and activity monitoring: implantable

4211 Lexington Avenue North

transmitters for animal research

St. Paul, MN 55126-6164

Harvard Apparatus

Feeding/drinking monitors; activity monitoring in

22 Pleasant Street

animals

South Natick, MA 01760

Mini-Mitter Company

Implantable transmitters; temperature and activity

P.O. Box 3386

monitoring in animals; Tau® software for analyzing

Sunriver, OR 97707

rhythms

Nalgene Company

Wheel running apparatus for rodents

P.O. Box 20365

Rochester, NY 14602

Stanford Software Systems

Collect® software for analyzing rhythms

574 Sims Road

Santa Cruz, CA 95060

Stoelting Company

Activity monitoring apparatus for animals

620 Wheat Lane

Wood Dale, IL 60191

Note: The majority of these companies have Internet sites that can be accessed using available search engines.

and disadvantages. Cosinor analysis seeks to impose a cosine rhythm on the data set. This assumes that the actual data set is rhythmic and that it is biologically expressed as a cosine waveform. Power spectrum analysis also imposes rhythms on the data set, but uses more sophisticated approaches to rhythm interpretation (both sine and cosine values). An advantage of the previous two methods is that the "power" or strength of the rhythm can be determined (i.e., how well the data set fits the hypothetical curve generated). Periodograms, on the other hand, do not assume any inherent rhythmic nature to the data set and, therefore, may provide a more realistic description of the rhythm. However, the power of the rhythm generated by a periodogram cannot be determined because periodograms do not have an inherent rhythm to be compared against. For a more complete description of these methods, Enright provides a detailed discussion.37

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