Physiology& Behavior,Vol. 49, pp. 355-365. Pergamon Press plc, 1991. Printed in the U.S.A.

0031-9384/91 $3.00 + .00

Rat Flash-Evoked Potential Peak N160 Amplitude: Modulation by Relative Flash Intensity 1'2 D A V I D W. H E R R , 3 W I L L I A M K. B O Y E S 4 A N D R O B E R T S. D Y E R

Health Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711 R e c e i v e d 26 July 1990

HERR, D. W., W. K. BOYES AND R. S. DYER. Rat flash-evoked potential peak N16o amplitude: Modulation by relativeflash intensity. PHYSIOL BEHAV 49(2) 355-365, 1991.--The flash-evoked potential (FEP) of rats has a large negative peak (N16o) approximately 160 ms following stimulation. This peak has been reported to be modulated by the subject's state of behavioral arousal and influenced by several test parameters. These experiments examined the influences of repeated testing, the number of stimuli/ session, interactions of ambient illumination and flash intensity, and the effect of pupillary dilation on the development and amplitude of peak N16o. The amplitude of peak N16o increased with dally testing and reached an asymptotic amplitude by about day 10. This amplitude was affected by the intensity of the flash stimulus relative to the ambient illumination (RFI) and appeared to reach a "ceiling" amplitude at greater than 50 dB RFI. The number of stimuli/session and dilation of the subject's pupils did not have a large influence on the growth or asymptotic level of peak N~6o amplitude. The data are consistent with the hypothesis that the growth of peak NI6o may represent a sensitization-like phenomenon. Flash-evoked potential

Peak N16o

Relative flash intensity

EVOKED potentials are often measured to assess neural function in sensory modalities. Flash-evoked potentials (FEPs) have been used to quantify changes in visual function produced by pharmaceutical compounds or toxicants (16, 32, 35-37, 45). The FEP consists of a series of positive and negative waves, which are often identified by their latency following stimulation. One such wave is a large negative peak which occurs at approximately 160 ms (peak N~6o) after flash presentation. This peak is the first of a series of waves known as the photic after discharge [PhAD; (1)]. It is hypothesized that both the PhAD and peak N16o are generated by a reverberating thalamo-cortical circuit which is activated by the photic stimulus [see (1,43) for reviews]. A similar circuit has also been proposed to be involved in the generation of cortical electroencephalogram (EEG) spindles [see (44) for review]. Because numerous substances have been found to alter the amplitude of peak N~6o (1, 3, 6, 16), it is important to improve our understanding of the functional significance of peak Nl6o and how it may be modulated by the test parametrics. Peak N~6o amplitude has been proposed to be influenced by the behavioral state of the test subject. Several investigators have proposed that a relaxed or habituated state is optimal for maximal

Sensitization

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elicitation of the PhAD, and that a state of behavioral arousal will suppress its expression (2, 4, 5, 18, 26, 34). These researchers suggested that a reduction in arousal allowed the expression of the PhAD. Since a habituated response decreases in amplitude, while a sensitized response increases in magnitude with repeated stimulus presentations (21,51), a reduction/habituation of arousal hypothesis was required to explain the observed increases in the PhAD. Recently, Dyer (17) has proposed that maximal expression of FEP peak N~6o may involve a sensitization-like process. This conclusion was based on a series of parametric manipulations of the testing procedures, and comparing the results with those predicted by theories of habituation and sensitization (21,51). The results showed that FEP peak N16o amplitude increased over 8 daily test sessions, and that the amplitude was larger at greater stimulus intensities. However, peak N16o amplitude may not have reached maximal levels over this test interval. Additionally, only four stimulus intensities with a constant ambient illumination were used. When examining the effect of flash intensity on FEPs, most previous investigators have varied the strobe intensity under constant ambient illumination, or have examined one or two flash intensities in a lighted or darkened apparatus (8, 13, 14, 28, 37,

IPortions of this manuscript were presented as a poster at the 19th Annual Meeting of the Society for Neuroscience, October 29-November 3, 1989, Phoenix, AZ [Soc. Neurosci. Abstr. 15(1):118; 1989]. 2The research descibed in this article has been reviewed by the Health Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. 3D.W.H. was supported by a NRC Research Associateship. 4Requests for reprints should be addressed to William K. Boyes, Neurotoxicology Division, MD-74B, HERL/NTD, US Environmental Protection Agency, Research Triangle Park, NC 27711. 355

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40, 46). Therefore, previous studies did not permit a quantitative assessment of interactions between the flash intensity and ambient illumination in modulating the FEP. It was hypothesized that the relative flash intensity (RFI; flash intensity relative to the ambient illumination) would be crucial in determining the stimulus intensity. Habituation to sensory input is typically greater with less intense eliciting stimuli. Conversely, sensitization is usually greater at more intense stimulus levels (21,51). Ambient illumination and flash intensity have been shown to influence both retinal cell responses (52) and detection thresholds in humans (23). Thus, these factors are important variables which affect the response of the visual system to flash stimuli. If sensitization or habituation to the test stimulus affects peak N ~ amplitude, then the RFI should be an important variable in determining the modulating influences of these constructs. A series of four experiments was designed to study the effects of parametric manipulations of the test procedures on the development and amplitude of FEP peak N~6o. This portion of the FEP was selected for detailed study due to the hypothesized involvement of behavioral constructs in the expression of peak N16o (1, 2, 4, 17) and due to the need for an understanding of how these constructs may influence changes in peak N I ~ amplitude produced by xenobiotics (16). In the first study, the time course of development of peak N16o was determined using a variable number of stimuli per session. Secondly, we examined the effect of a wide range of relative stimulus intensities on the development and final amplitude of peak N~6o. This was accomplished by varying both the intensity of the stimulus and the ambient illumination of the test apparatus. The relationship between peak N~6o amplitude and the RFI was determined using a nonlinear regression model. In the third investigation, the predictive validity of the model was tested using an RFI not included in deriving the relationship. Because some of the testing conditions involved stimulation in a dark chamber, while an illuminated environment was used in others, the degree of pupil dilation was expected to vary between conditions. Additionally, some investigators dilate the subject's pupils in toxicological experiments to prevent xenobiotic-induced pupillary constriction. Thus, in the last experiment, we examined the impact of dilating the test subject's pupils on the development of FEP peak Nl6 o. METHOD All studies used naive adult male Long-Evans rats (60-90 days old) purchased from Charles River (Raleigh, NC). Animals were housed singly and allowed to acclimate to the animal colony for at least one week (12-h light-dark cycle, lights on 7:00; 2 2 _ 2°C; 40 -+ 20% relative humidity) prior to surgery. Subjects were anesthetized with sodium pentobarbital (50 mg/kg, IP) and concurrently atropinized (2 mg/kg, SC) to decrease respiratory distress. Rats were implanted with epidural stainless steel screw electrodes (00-90 x 1/16"), which were presoldered to nichrome wire. The surface area of the electrodes in contact with the dura was approximately 0.8 mm 2. Visual cortex electrodes were implanted l mm anterior to lambda and 4 mm left and right of midline. Ground and reference electrodes were located 2 mm anterior to bregma and 2 mm left and right of midline, respectively. The sharp distribution gradients for all of the FEP peaks 0 5 ) indicate that the potentials originate in cortical tissue around the active electrode and that the reference electrode is relatively indifferent to retinal and diffuse cortical potentials induced by the flash stimulus. The wires were attached to gold pins and connected to a female plastic Amphenol connector, which was in turn fastened to the skull with cranioplastic acrylic (Plastics One, Roanoke, VA). The wound was closed, painted with 0.2% nitrofurazone ointment (Thames Pharmacal Co., Inc., Ronkonkoma, NY), and the rat was admin-

istered a prophylactic IM injection of 67,500 units of penicillin G. Subjects were allowed 3-7 days of recovery prior to beginning the experiment. Testing occurred in a mirrored rectangular box (8 cm wide, 20 cm long, 38 cm high), which allowed free movement, yet assured uniform stimulus intensity regardless of the exact location of the subject. The mirror on the rear wall extended to 11.5 cm above the floor where it was interrupted by a Grass PS22 Photic Stimulator (Grass Instrument Co., Quincy, MA) enclosed in a soundattenuating box mounted exterior to the chamber. The test chamber was enclosed in an insulated faraday cage to reduce extraneous noise and electrical signals. This system has previously been used to record FEPs (16, 17, 37). EEG signals were passed to differential preamplifiers (100 x gain), further amplified 100 x (total gain of 10,000 x ), bandpass filtered between 0.8 and 1,000 Hz (rolloff = 6 dB/octave), digitized at a minimum of 2,000 Hz, and sampled for at least 256 ms following stimulation. Signals were averaged using either a PDP 11/70 computer or a Nicolet Pathfinder. The systems' amplitude and latency response factors were calibrated using a Grass Square Wave Generator (Model SWC 1C) prior to beginning each experiment. Peak amplitudes and latencies were measured from each animal's waveform. Amplitudes (in p.V) were measured from baseline, which was defined as the average voltage between 5-10 ms after the flash. Latency from stimulus presentation was calculated in ms. Component peaks of the FEP were identified by their approximate latency in group averages, as well as their polarity. Flash intensity and overhead illumination were quantified using a photometer (Model 450 with Model 550-3 Pulse Integration Module; EG&G Inc., Salem, MA). In each experiment, photic stimulation was a 10 ~ts flash presented at 0.3 Hz. Unless stated otherwise, 64 trials/test session were collected. Subjects were randomly assigned to treatments, after counterbalancing days of recovery from surgery across groups. Animals were allowed a minimum of 15 min to adjust to transfer to the laboratory prior to beginning each test session. Each animal was acclimated to handling and the test chamber one day prior to beginning the actual sessions. This consisted of connecting the rat to the recording cable and placing the subject in the chamber, with the appropriate ambient illumination, for 15 min. No flash stimuli were presented during this session. On each subsequent test day, a 10-min acclimation period with the appropriate ambient illumination occurred prior to beginning stimulation. Data were analyzed using a repeated measures analysis of variance [ANOVA; (38)]. Degrees of freedom for within-subject factors and their interactions were adjusted using a GeisserGreenhouse correction factor (19, 20, 27). Significant overall effects were further examined using step down ANOVAs, which used Bonferroni corrections to maintain a familywise p :=L I,LI

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TEST DAY FIG. 1. Effect of number of photic stimuli/session on the development of mean FEP peak N~6o amplitude (Experiment 1; n = 8-12 rats/group). Subjects received 32, 64, 128, or 256 photic stimuli/ session for 15 days. Peak N~6o amplitude increased over days for each group and appeared to reach a common asymptotic amplitude by day 10-11. Although a significant interaction between the number of flash stimuli/session and day of testing was found, no significant effects of number of stimuli/ session were indicated on any given day. Overlapping error bars are not shown.

stimuli/session on the rate of peak N160 growth. Subjects were assigned to one of four independent groups, each of which received a different number of photic stimuli per test day [32 flashes (n= 10), 64 flashes ( n = 8 ) , 128 flashes (n= 10), or 256 flashes (n = 12)]. Each group was tested once/day for 15 consecutive days. The flash intensity was 409 lux-s and the ambient illumination was 115 lux. These conditions have been used in our laboratory in several previous investigations (16, 17, 37). The number of photic stimuli/session was a between-subject factor, and test day was a within-subject factor.

Experiment 2-1nfluence of Relative Flash Intensity on Peak N16o Development Because the previous experiment indicated that peak N16o asymptotic amplitude was reached by day 13, all further investigations involved only 13 consecutive test days. This study involved 12 independent groups, each of which was tested under a different combination of ambient illumination and flash intensity. Ambient illumination was either 0, 115, or 250 lux, and flash intensity was either 409, 81, 24, or 5 lux-s. These flash intensities correspond to settings of 16, 4, 1, and 1 plus a 0.6 neutral density filter on the Grass PS22 Photic Stimulator, respectively. Thus, this experiment was a factorial design with both flash intensity and ambient illumination serving as between-subject factors, while test day was a within-subject variable. The photometric energy of each flash stimulus was converted to power by multiplying the lux-s value by 105. The RFI (in dB of photometric power) was calculated according to the following equation:

(flash power (lux)) RFI = 10 x log (ambient illumination (lux)) • For groups tested with an ambient illumination of 0 lux, the RFI was calculated setting the ambient illumination equal to 1 lux. These combinations of ambient illumination and flash intensity produced the following RFIg: 76 (n = 8), 69 (n= 6), 64 (n = 6), 57 (n = 8), 56 (n = 8), 52 (n = 8), 48 (n = 8), 45 (n = 7), 43 (n = 8), 40 (n = 8), 36 (n = 7), and 33 (n = 7). Weighted nonlinear regression (38) was used to model several relationships in these experiments, using the group mean response and weighting it inversely proportionally to its variance. The development of peak Nl6o over time was fit to the general logistic response function: N16o Amplitude = (Asymptotic Nt6o Amplitude)" × (Day) '~ (Day) ~ + (Ts0) ~ A similar function has been used to model the response of the visual system to light stimuli successfully in several other experimental paradigms (23, 31, 52, 54) and allowed the calculation of the number of test days required to reach half plateau amplitude for peak N16o (T~o), and a "fitting" exponent (ct). Based on the data, the asymptotic peak N16o amplitude was set equal to the mean response over days 10-13 (see the Results section). The relationship between the asymptotic NI6o amplitude and the RFI was fit using quadratic and cubic polynomials, as well as a segmented-line regression (24, 33, 57). Improvement of fit was assessed using an F-test based on the "extra sum of squares principle"

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