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Ontogeny of flash-evoked potentials in unanesthetized rats
ht. 1. Devl. Neuroscience. Vol. 5. Nos 516.pp. 441454.1987. Printedin Great Britain.
ONTOGENY
0736-5748/87 $03.00+0.00 Pergamon Journals Ltd. @ 1987 ISDN
OF FLASH-EVOKED POTENTIALS UNANESTHETIZED RATS* GREG C. RIGDON
United
States Environmental
(Received
and ROBERT S. DYER
Protection Agency, Neurotoxicology Division/Neurophysiology Research Triangle Park, NC 27711, U.S.A.
15 May
1987; in revised form
IN
12 August
Branch, MD-74B,
1987; accepted 21 Augusr 1987)
Abstract-The effects of age and stimulation frequency (O.Usec, I .O/sec, Z.O/sec, or 4.O/sec) on flashevoked potentials (FEPs) were investigated in awake, unsedated, unrestrained rats. Animals were tested daily from postnatal day (PND) 8 to PND 20, and every 3 or 4 days thereafter until PND 41. On PND 9, a single negative wave (Nla) was observed following 0.2/set flash presentation. Animals tested on PND 10 exhibited a positive wave (P2) following the return of peak Nla to baseline. On PND 13 another negative wave (Nl) appeared on the leading shoulder of peak Nla. Peak Nl became the dominant negative wave on PND 14. Peak Nla merged into Nl and had disappeared by PND 19. Peak N3 was first observed as a negative shift following peak P2 on PND 15. Peaks N2 and P3 were not observed in the group average waveforms until PND 34. Peak latencies decreased through the fifth postnatal week. Peak amplitudes increased with age until after eye opening (PND 15). but were variable thereafter. No FEPs were observed following higher than O.Usec flash presentation until PND 13. Increasing stimulation frequency decreased Nl and P2 peak amplitudes, but had no effect on peak latencies. Key words: Ontogeny,
Flash-evoked
potential,
Vision,
Rat, Unanesthetized.
Flash-evoked potentials (FEPs) are light-flash-evoked alterations in the electrical potential of brain regions which receive visual input. The FEP reflects the functional integrity of the visual system, ’ t recorded from the rat visual cortex as a means of examining toxicant-induced damage to the visual system. Some neurotoxicants administered during the perinatal period may produce long-lasting changes in the visual system. For example, perinatal administration of methylmercury,’ trimethyltin,” triethyltin,“’ or carbon monoxide’ produces alterations in either peak latencies or peak amplitudes of FEPs recorded from adult rats. However, perinatal dysfunction need not be reflected in adult recordings in order to be important. Knowing the extent to which perinatal exposures produce short-term as well as long-term effects is also important. To make these determinations it is essential that we know the normal ontogeny of FEPs. The ontogeny of FEPs has been studied in anesthetized,‘3*‘n,26 paralysed-respired,‘Y and sedated’ rats. Malnutrition,3*4.22.2ythyroxine,% toluene,” and leadI have been shown to alter the development of the rat FJZP, recorded with these different preparations. However, to maximize understanding of the relationship between normal FEPs recorded from young and old rats, and to minimize concern that studies with toxicants might reflect interactions with anesthetics or sedatives, one must know the course of FEP ontogeny in awake, unrestrained, unsedated rats. In this paper we describe the normal ontogeny of FEPs in non-drugged rats. In addition, we describe a novel and simple method by which these recordings may be made. MATERIALS
AND METHODS
Timed pregnant Long-Evans rats were obtained on day 15 of gestation from Charles River Laboratories. Pups were randomly reassigned to dams on postnatal day (PND) 1 (birth = PND 0). Litters consisted of four male and four female pups. Weaning took place on PND 21 or 22. Recordings were obtained from awake, unanesthetized rats with previously implanted electrodes. To implant the electrodes animals were anesthetized with a combination of ketamine Address correspondence to: Dr Greg C. Rigdon, Department of Pharmacology, Cornwallis Road, Research Triangle Park, NC 27709, U.S.A. * Supported by a National Research Council Research Associateship Grant. 447
Burroughs Wellcome
Company,
3030
448
G. C. Rigdon and R. S. Dyer
(12.5-25.0 mg/kg, i.p.) and xylazine (3.0-12.5 mg/kg, i.p.). Animals remained surgically anesthetized for 15-20 min and body temperature was maintained with a heating pad. If an animal’s eyes were open at the time of surgery, they were kept moist with physiological saline solution. The recording electrodes used in pups on PND 8-20 were l-2 mm lengths of 0.01 in. non-insulated stainless steel wire inserted in gold-plated male Amphenol pins and bent perpendicular to the pin. Electrodes were inserted through the skull at three locations: over visual cortex (3.5 mm right of midline at lambda) with wire extending laterally; in the parietal bone (ground); and over frontal cortex (reference, 2 mm anterior and 2 mm lateral to bregma). The electrodes were inserted by exerting pressure upon the skull, which was extremely thin and easily punctured prior to PND 20. The electrodes were embedded in cranioplastic acrylic and the scalp sutured in place over the implant. With rats older than PND 20 the surgery was similar, but the electrodes used were stainless steel screws (00-90, l/16 in., Small Parts, Inc.) threaded into predrilled holes in the skull. Pups were returned to the dam within l-2 hr and would begin suckling within 3-4 hr after surgery. Most animals failed to gain weight the day following surgery, but proceeded to gain weight thereafter. Testing began 1 or 2 days following the surgery. Three separate groups of animals were tested daily, for 4 day periods; PND 9-12, PND 13-16, and PND 17-20. A small percentage of animals failed to receive all 4 tests, because head plugs became detached. Another group had been tested on PND 8. Other litters were tested on PND 23,24,28,29,33,34,40, or 41. Prior to PND 13 the recording chambers were warmed by placing isothermal pads (constant 37°C) under the floor. Rectal temperatures were obtained immediately after testing. Vigilance state may affect FEPs. While it was not possible to evaluate vigilance state systematically it was evident from behavioral observation that none of the pups were asleep during the test period. FEPs were obtained as averaged responses to 128 flashes from a click-muffled Grass strobe unit (10 ksec flash, 4.53 x lo-* lux-set). Testing took place in mirrored recording chambers (ambient lighting = 115 lux) that insured retinal stimulation regardless of the animals position. FEPs were obtained following stimulation frequencies of 0.2/set, l.O/sec, 2.0/set, or 4.0/set presented as blocks in random sequence. High- and low-frequency filters were set at 1 kHz and 0.8 Hz, respectively. The acquisition rate was 1000 Hz, and the recording epoch was 464 msec. Waveforms were averaged by a PDP 1l/70 computer and displayed on a Tekronix 4054 terminal as described previously.’ Peaks were initially labeled arbitrarily. Once the relationship of each peak to the adult pattern was evident, old waveforms were re-evaluated and relabeled as necessary. Peak amplitudes were measured from baseline, which was defined as the average voltage between 10 and 15 msec following the stimulus. The effects of age and stimulus frequency on peak latencies and peak amplitudes were analysed with multivariate analysis of variance (MANOVA, SAS Institute, Cary, NC) with appropriate Bonferroni correction procedures. RESULTS The techniques employed allowed recording of FEPs for 4-5 consecutive days in the rats younger than PND 20. After this the implants became loose due to rapid growth of the thin skull. With older animals, the implants would last much longer (2 weeks). Group average waveforms of FEPs recorded daily between PND 9 and 20 and on PND 23, 24, 28, 29, 33, 34, and 40 are presented in Fig. 1. The following descriptions of the FEP waveforms were taken from group averages of all individual waveforms recorded on a given day at a given frequency. Of the 8 pups tested on PND 8, none exhibited a response (O.Usec stimulation). Responses to O.Usec light flashes were first observed on PND 9 (5 of 20 animals). The response was a single negative wave (Nla) with a mean peak latency of 326.3 msec and a mean peak amplitude of 17.7 pV. The inflection point between baseline and the beginning of the wave was designated as Pl. No positive wave was observed to follow the return of wave Nla to baseline, although one may have fallen outside the analysis epoch. Because of the variability in their occurrence, mean values are not plotted for PND 9 rats. Fifteen of 17 animals tested on PND 10 exhibited a response to the light flash stimulation (0.2/set). All animals exhibited a response by PND 11. On PND 10 a positive wave, P2, with a mean peak latency of 361.4 msec and a mean peak amplitude
Ontogeny of flash-evoked potentials in PNDS N,a
N -20
unanesthetized
rats
449
PND 15 -501
N=14
N,
PND 23 N-16
I
PND 10 Nla
N:14
PND 33
PND 18 N=S
PND 19
P2 Nl
PND 14
PND 20
N = 14
N=8
N3
PND 40.41 N=9
A
Fig. 1. Group average waveforms of FEPs PND p-40, stimulation frequency = O.Usec. PND = postnatal day. N = number of animals. Number of animals is a subset of all animals tested. Some waveforms could not be included in group averages due to computer problems. Note variation in voltage scale for different waveforms. For PND 23, the +I00 (downward) scale marker is indicated, to fit more conveniently on the page. Sweep time is 464 msec. Last waveform includes animals PND 40 and 41.
of 10.7 )IV appeared following Nla. The prominant initial negative peak of the adult FEP waveform, Nl, appeared in the group average waveform on PND 13. Nl was first seen as a negative shift on the leading shoulder of Nla. By PND 14, Nl was the prominant negative wave and Nla had begun to merge into Nl. By PND 19 Nla could no longer be seen in the group average waveform. The rapid maturation of the FEP between PND 13 and 15 in an individual animal is shown in Fig. 2. PNOIJ
Nl
d-’ P, P2 PND 14 N3
(EYES OPEN,
Fig. 2. FEPs recorded from one animal on PND 13, 14 and 15, stimulation frequency =0.2&c. Each wave is average of 128 trials. Line indicates latency of peak Nl on PND 13. Sweep time is 464 msec.
G. C. Rigdon and R. S. Dyer
450
Fig. 3. Effect of age on peak latencies. Vertical lines represent standard error. Peak latencies are means of FEPs of all animals recorded on the given day at O.Usec stimulation frequency. See Fig. 4 for symbol identification.
Some of the animals’ eyes opened on PND 14 and all were completely open by PND 15. After eye opening the amplitude of the peaks increased and peak N3 was observed as a slow negative shift following P2. For example, eye opening occurred on PND 14 in the rat whose data are presented in Fig. 2. On PND 14, Pl became a distinct positive peak in the waveform preceding Nl. Peaks N2 and P3 of the adult FEP waveform were not apparent in the group average waveforms of developing animals until PND 34, although they could occasionally be observed in
individual waveforms prior to that time. Whether this late appearance reflected late development or masking by the N3 wave is unclear. As the animals aged, latencies decreased (Fig. 3). Peak P2 was picked as the most positive point following Nl. The small (not significant) increase in P2 latency between PND 16 and 19 probably reflected the merging of wave Nla into wave Nl (see Fig. 1). Statistical analysis (MANOVA) of the effects of age on peak latency was performed on the four age groups of animals (PND 9-12, PND 1316, PND 17-20, > PND 20). Within each age group there was a significant effect of age (P~O.025) on peak latencies. This was true for all peaks except for the PND 17-20 group when P2 and N3 latencies were not affected. There was no effect of age on peak amplitudes within the groups tested, but the amplitudes generally increased with age (between groups, Fig. 4). After its appearance as a positive wave on PND 14 the amplitude of peak Pl did not increase again until PND 23. Peak Nl increased with age until a maximum was reached on PND 29 (Fig. 4). Peaks P2 and N3 reached maximum values on days 23 and 33, respectively (Fig. 4).
10
12
14
16
(8
20
23 24 POSTNA,TI\L
28 29
33 34
41
DI\Y
Fig. 4. Effect of age on peak amplitudes. Vertical lines represent standard error. Peak amplitudes are means of FEPs of all animals recorded on the given day at O.Z/secstimulation frequency.
Ontogeny of flash-evoked potentials in unanesthetized ‘rats
. SWEEP = 464
Fig. 5. Group
average
451
l lll*ec
waveforms of FEPs recorded following 0.2hec. l.O/sec, Z.O/sec, or 4.Okec stimulation on PND 15. N= 14. Sweep time is 464 msec.
Before PND 13 there were no measurable FEPs if the flash was presented more rapidly than OZsec. On PND 13 there were responses to l.O/sec and 2.0/set stimulation, and by PND 14,9 of 14 animals exhibited responses to light flashes delivered at 4.0/set. Figure 5 shows the effects of increasing stimulus frequency on FEPs recorded on PND 15. Increasing frequency of stimulation significantly affected peak Nl, P2, and N3 amplitudes in each of the four age groups (the oldest group was studied through PND 28). Peak N3 amplitudes could not be observed following 4.0/set stimulation before PND 23 because the peak latency was longer than the inter-stimulus interval. Nl and P2 peak amplitudes were consistently decreased in all groups (significant P
G. C. Rigdon and R. S. Dyer
452
10
12
14
16
18
20
28
23 24
I’~lul~lltL 10 12
14
20
23 24
28
23’24
28
0-0.2/s
N3
0. 1.0/s
70 t
I
10
IS
POSTNATAL DAY
POSTNATAL DAY SO
16
12
14
16
18
20
/
23 24
POSTNATAL DAY
A- 2.0/s
28
10
12
14
16
18
20
POSTNATAL DAY
Fig. 6. Effect of stimulation frequency on amplitude of peaks Nl, Pl, P2, and N3. Values are means from all animals recorded on the day and frequency indicated. Vertical lines indicate standard errors. Sweep time is 464 msec.
of FEP ontogeny the experiments were concluded prior to or during the third postnatal week, when it was reported that nearly adult peak latency values were achieved.’ In this paper we show significant maturation of the FEP beyond the third postnatal week, for example, peak latencies continued to decrease through the fifth postnatal week. Also, peaks N2 and P3, which are prominent in the adult FEP waveform, were not seen in the group average waveform until PND 34. Two stages of rapid change in FEP ontogeny are: (1) PND 9-10 when the response to light flash is first observed, and (2) PND 13-14 when peak Nl appears and FEPs are observed following stimulation frequencies higher than 0.2/set. Some of the neuroanatomical events occurring during these periods are discussed below. The first observable cortical response evoked by light flash occurred on PND 9. The rat optic nerve is completely unmyelinated at birth and completely myelinated in adults.2.‘h The first myelinated axons are observed on PND 6-8.1h Geniculocortical afferents first appear in layer IV of area 17 between PND 6 and PND 8”’ and acetylcholinesterase activity associated with these axons is first detectable at this time as well.*’ This is also the period during which the first significant increase in number of dendritic spines on area 17 pyramidal cells occurs” (PND 6-9). The abrupt appearance of peak Nl and the appearance of FEPs following >0.2/sec stimulation on PND 13 coincides with a peak in acetylcholinesterase staining in layer IV of the rat visual cortexz and a significant increase in the number of dendritic spines on visual cortex pyramidal neurons*’ (PND 12-15). A ‘growth spurt’ also occurs at about this time in the lateral geniculate neurons which project to the visual cortex.24 Peak latencies continued to decrease into the fourth and fifth postnatal weeks in this study and peaks N2 and P3 were not observed in the group average waveform until PND 34. The decrease in peak latencies with age are probably related to myelination of the optic nerve which is 85%
Ontogeny
of flash-evoked
potentials
in unanesthetized rats
453
myelinated by PND 28 and completely myelinated after PND 40.1h Myelination alone, however, cannot account for all the latency changes, because conduction velocities in rat optic nerve decrease from PND 0 to PND 6 when the nerve is completely unmyelinated. I2 Changes in ionic conductance associated with development’ and increases in axon diameterI may play a role in increasing conduction velocities. The animal’s body temperatures increased with age and this was probably responsible for some of the decrease in peak latencies.’ The pattern of visual cortex pyramidal cell spine density continues to change between PND 15 and 60.14 Both findings indicate that the visual system is not mature until well after weaning (PND 21). Poststimulation neuronal recovery processes have been used to detect neurotoxicity in different sensory systems.h The ability of the visual system to respond to increasing stimulation frequencies developed with age, and only peak amplitudes were affected by increasing stimulation frequency in this study. Visual system recovery processes are polysynaptic,h therefore, simple maturational reductions in absolute and relative refractory periods in visual system neurons cannot explain the development of the recovery processes. The explanation is more likely to be related to maturation of inhibitory and excitatory neuronal circuits in the visual system. In summary, the ontogeny of the rat FEP proceeds in an orderly fashion beginning early in the second postnatal week and continuing into the fifth or sixth postnatal weeks. The development of the FEP can be studied in awake, unsedated animals and may provide a means of studying the effects of pharmacological and toxicological agents on the functional development of the visual system.
REFERENCES I. Albee R. R. and Mat&son J. L. (1983) Visual evoked response testing method for neonatal rats. Neurobehav. Toxicol. Terarol. 5, 497-501. studies during development of 2. Black J. A., Foster R. E. and Waxman S. G. (1982) Rat optic nerve: freeze-fracture myelination. Brain Res. 250, l-20. 3. Bronzino J. D., Morgane P. J., Forbes W. B., Stern W. C. and Resnick 0. (1975) Ontogeny of visual evoked responses in rats protein-malnourished during development. Biof. fsychiar. 10, 175-184. 4. Callison D. A. and Spencer J. W. (1968) Effect of chronic undernutrition and/or visual deprivation upon the visual evoked potential from the developing rat brain. Devf Psychobiol. 1, 19&204. 5. Connors B. W., Ransom B. R., Kunis D. M. and Gutnick M. J. (1982) Activity-dependent K+ accumulation in the developing rat optic nerve. Science 216, 1341-1343. challenges for the detection of toxicity. Fed. Proc. 42, 6. Dyer R. S. and Boyes W. K. (1983) Use of neurophysiological 3201-3206. and chloropent anesthesia differentially affect the flash evoked 7. Dyer R. S. and Boyes W. K. (1983) Hypothermia potentials of hooded rats. Brain Res. Bull. 10, 825-831. 8. Dyer R. S., Eccles C. U. and Annau Z. (1978) Evoked potential alterations following prenatal methylmercury. f’harmac. Biochem. Behav. 8, 137-141. H. S., Fechter L. D. and Annau Z. (1979) Prenatal carbon monoxide and 9. Dyer R. S., Eccles C. U., Swartzwelder adult evoked potentials in rats. 1. envir. H&h, Part C. Envir. Hfrh Sci. 13, 107-120. in rats perinatally exposed to 10. Dyer R. S., Howell W. E. and Reiter L. W. (1981) Visual evoked response alterations triethyltin. Neuroroxicology 2, 603-618. 11. Feinsod M., Hoyt W. F., Wilson W. B. and Spire J.-P. (1976) Visually evoked response use in neurological evaluation of posttraumatic subjective visual complaints. Archs Ophrhal. 94, 237-240. pharmacological and 12. Foster R. E., Connors B. W. and Waxman S. G. (1982) Rat optic nerve: electrophysiological, anatomical studies during development. Devl Brain Res. 3, 371-386. 13. Fox D. A., Lewkowski J. P. and Cooper G. P. (1977) Acute and chronic effects of neonatal lead exposure on development of the visual evoked response in rats.’ Tax.. app. Pharmac. 40, 449-%1. of pyramidal neurons after eye opening in the visual cortex of hooded rats: a 14. Juraska J. M. (1982) The development quantitative study. J. camp. Neural. 212, 208-213. of hypothermia to effects of chloral hydrate on flash evoked potenIS. Hetzler B. E. and Dyer R.-S. (1977) Contribution tials of hooded rats. Pharmac. Biochem. Behav. 21, 599-607. C. and Waxman S. G. (1984) Postnatal differentiation of rat optic nerve fibers: electron microscopic ob16. Hildebrand servations on the development of nodes of Ranvier and axoglial relations. J. camp. Neural. 224, 25-37. 17. Howell W. E., Miller D. and Dyer R. S. (1982) Impairment of visual system development following organotin exposure. Toxicologkr 2, 53.5. alterations due to diesel exhaust exposure during the 18. Laurie R. D. and Boyes W. K. (1981) Neurophysiological neonatal life of the rat. Envir. Int. 5, 36S368. M. and Salas M. (1985) Effects of neonatal toluene exposure on the development of evoked and 19. Lorenza-Jimenez spontaneous cortical activity in the rat. Neurobehav. Toxicol. Terarol. 7, 215-220. 20. Lund R. D. and Mustari M. J. (1977) Development of the geniculocortical pathway in rats. /. camp. Neural. 173, 289-306.
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21. Miller M. and Peters A. (1981) Maturation of rat visual cortex. II. A combined Golgi-electron microscope study of pyramidal neurons. J. camp. Neural. 203.555-573. 22. Mourek J., Himwich W. A., Myslivecek J. and Callison D. A. (t%7) The role of nutrition in the development of evoked cortical response in rat. Brain Res. 6, 241-251. 23. Myslivecek J. (1970) Electrophysiology of the developing brain--Central and Eastern European contributions. In Developmental Neurobiology (ed. Hinwich W. A.), pp. 475-527. Thomas, Springfield, IL. 24. Parnavelas J. Cl., Mounty E. J., Bradford R. and Leiberman A. R. (1977) The postnatal development of neurons in the dorsal lateral geniculate nucleus of the rat: a Golgi study. J. camp. Neurof. 171, 481-500. 25. Robertson R. T., Tijerina A. A. and Galtivan M. E. (1985) Transient patterns of acetytcholinesterase activity in visual cortex of the rat: normal development and the effects of neonatal monocular enucleation. Llevl Brain Rex 21, 203-214. 26. Rose G. H. (1968) The development of visualfy evoked etectrocorticat responses in the rat. Devl Psychobiot. 1,3540. 27. Rose G. H. and Ellingson R. J. (1970) Ontogenesis of evoked potentials. In Developmental Neurobiology (ed. Himwich W. A.), pp. 393-440. Thomas, Springfield, IL. 28. Salas M., Diax S. and Cintra L. (1976) Electrocortical effects of early postnatal thyroxine administration in the rat. Physiof. Be&w. 17, 239-244. 29. Sjostrom A., Conradi N. G. and Andersson S. A. (1984) Functional development of the visual system in normal and protein deprived rats. Acfa physiol. stand. fu), 585-594.
ONTOGENY
0736-5748/87 $03.00+0.00 Pergamon Journals Ltd. @ 1987 ISDN
OF FLASH-EVOKED POTENTIALS UNANESTHETIZED RATS* GREG C. RIGDON
United
States Environmental
(Received
and ROBERT S. DYER
Protection Agency, Neurotoxicology Division/Neurophysiology Research Triangle Park, NC 27711, U.S.A.
15 May
1987; in revised form
IN
12 August
Branch, MD-74B,
1987; accepted 21 Augusr 1987)
Abstract-The effects of age and stimulation frequency (O.Usec, I .O/sec, Z.O/sec, or 4.O/sec) on flashevoked potentials (FEPs) were investigated in awake, unsedated, unrestrained rats. Animals were tested daily from postnatal day (PND) 8 to PND 20, and every 3 or 4 days thereafter until PND 41. On PND 9, a single negative wave (Nla) was observed following 0.2/set flash presentation. Animals tested on PND 10 exhibited a positive wave (P2) following the return of peak Nla to baseline. On PND 13 another negative wave (Nl) appeared on the leading shoulder of peak Nla. Peak Nl became the dominant negative wave on PND 14. Peak Nla merged into Nl and had disappeared by PND 19. Peak N3 was first observed as a negative shift following peak P2 on PND 15. Peaks N2 and P3 were not observed in the group average waveforms until PND 34. Peak latencies decreased through the fifth postnatal week. Peak amplitudes increased with age until after eye opening (PND 15). but were variable thereafter. No FEPs were observed following higher than O.Usec flash presentation until PND 13. Increasing stimulation frequency decreased Nl and P2 peak amplitudes, but had no effect on peak latencies. Key words: Ontogeny,
Flash-evoked
potential,
Vision,
Rat, Unanesthetized.
Flash-evoked potentials (FEPs) are light-flash-evoked alterations in the electrical potential of brain regions which receive visual input. The FEP reflects the functional integrity of the visual system, ’ t recorded from the rat visual cortex as a means of examining toxicant-induced damage to the visual system. Some neurotoxicants administered during the perinatal period may produce long-lasting changes in the visual system. For example, perinatal administration of methylmercury,’ trimethyltin,” triethyltin,“’ or carbon monoxide’ produces alterations in either peak latencies or peak amplitudes of FEPs recorded from adult rats. However, perinatal dysfunction need not be reflected in adult recordings in order to be important. Knowing the extent to which perinatal exposures produce short-term as well as long-term effects is also important. To make these determinations it is essential that we know the normal ontogeny of FEPs. The ontogeny of FEPs has been studied in anesthetized,‘3*‘n,26 paralysed-respired,‘Y and sedated’ rats. Malnutrition,3*4.22.2ythyroxine,% toluene,” and leadI have been shown to alter the development of the rat FJZP, recorded with these different preparations. However, to maximize understanding of the relationship between normal FEPs recorded from young and old rats, and to minimize concern that studies with toxicants might reflect interactions with anesthetics or sedatives, one must know the course of FEP ontogeny in awake, unrestrained, unsedated rats. In this paper we describe the normal ontogeny of FEPs in non-drugged rats. In addition, we describe a novel and simple method by which these recordings may be made. MATERIALS
AND METHODS
Timed pregnant Long-Evans rats were obtained on day 15 of gestation from Charles River Laboratories. Pups were randomly reassigned to dams on postnatal day (PND) 1 (birth = PND 0). Litters consisted of four male and four female pups. Weaning took place on PND 21 or 22. Recordings were obtained from awake, unanesthetized rats with previously implanted electrodes. To implant the electrodes animals were anesthetized with a combination of ketamine Address correspondence to: Dr Greg C. Rigdon, Department of Pharmacology, Cornwallis Road, Research Triangle Park, NC 27709, U.S.A. * Supported by a National Research Council Research Associateship Grant. 447
Burroughs Wellcome
Company,
3030
448
G. C. Rigdon and R. S. Dyer
(12.5-25.0 mg/kg, i.p.) and xylazine (3.0-12.5 mg/kg, i.p.). Animals remained surgically anesthetized for 15-20 min and body temperature was maintained with a heating pad. If an animal’s eyes were open at the time of surgery, they were kept moist with physiological saline solution. The recording electrodes used in pups on PND 8-20 were l-2 mm lengths of 0.01 in. non-insulated stainless steel wire inserted in gold-plated male Amphenol pins and bent perpendicular to the pin. Electrodes were inserted through the skull at three locations: over visual cortex (3.5 mm right of midline at lambda) with wire extending laterally; in the parietal bone (ground); and over frontal cortex (reference, 2 mm anterior and 2 mm lateral to bregma). The electrodes were inserted by exerting pressure upon the skull, which was extremely thin and easily punctured prior to PND 20. The electrodes were embedded in cranioplastic acrylic and the scalp sutured in place over the implant. With rats older than PND 20 the surgery was similar, but the electrodes used were stainless steel screws (00-90, l/16 in., Small Parts, Inc.) threaded into predrilled holes in the skull. Pups were returned to the dam within l-2 hr and would begin suckling within 3-4 hr after surgery. Most animals failed to gain weight the day following surgery, but proceeded to gain weight thereafter. Testing began 1 or 2 days following the surgery. Three separate groups of animals were tested daily, for 4 day periods; PND 9-12, PND 13-16, and PND 17-20. A small percentage of animals failed to receive all 4 tests, because head plugs became detached. Another group had been tested on PND 8. Other litters were tested on PND 23,24,28,29,33,34,40, or 41. Prior to PND 13 the recording chambers were warmed by placing isothermal pads (constant 37°C) under the floor. Rectal temperatures were obtained immediately after testing. Vigilance state may affect FEPs. While it was not possible to evaluate vigilance state systematically it was evident from behavioral observation that none of the pups were asleep during the test period. FEPs were obtained as averaged responses to 128 flashes from a click-muffled Grass strobe unit (10 ksec flash, 4.53 x lo-* lux-set). Testing took place in mirrored recording chambers (ambient lighting = 115 lux) that insured retinal stimulation regardless of the animals position. FEPs were obtained following stimulation frequencies of 0.2/set, l.O/sec, 2.0/set, or 4.0/set presented as blocks in random sequence. High- and low-frequency filters were set at 1 kHz and 0.8 Hz, respectively. The acquisition rate was 1000 Hz, and the recording epoch was 464 msec. Waveforms were averaged by a PDP 1l/70 computer and displayed on a Tekronix 4054 terminal as described previously.’ Peaks were initially labeled arbitrarily. Once the relationship of each peak to the adult pattern was evident, old waveforms were re-evaluated and relabeled as necessary. Peak amplitudes were measured from baseline, which was defined as the average voltage between 10 and 15 msec following the stimulus. The effects of age and stimulus frequency on peak latencies and peak amplitudes were analysed with multivariate analysis of variance (MANOVA, SAS Institute, Cary, NC) with appropriate Bonferroni correction procedures. RESULTS The techniques employed allowed recording of FEPs for 4-5 consecutive days in the rats younger than PND 20. After this the implants became loose due to rapid growth of the thin skull. With older animals, the implants would last much longer (2 weeks). Group average waveforms of FEPs recorded daily between PND 9 and 20 and on PND 23, 24, 28, 29, 33, 34, and 40 are presented in Fig. 1. The following descriptions of the FEP waveforms were taken from group averages of all individual waveforms recorded on a given day at a given frequency. Of the 8 pups tested on PND 8, none exhibited a response (O.Usec stimulation). Responses to O.Usec light flashes were first observed on PND 9 (5 of 20 animals). The response was a single negative wave (Nla) with a mean peak latency of 326.3 msec and a mean peak amplitude of 17.7 pV. The inflection point between baseline and the beginning of the wave was designated as Pl. No positive wave was observed to follow the return of wave Nla to baseline, although one may have fallen outside the analysis epoch. Because of the variability in their occurrence, mean values are not plotted for PND 9 rats. Fifteen of 17 animals tested on PND 10 exhibited a response to the light flash stimulation (0.2/set). All animals exhibited a response by PND 11. On PND 10 a positive wave, P2, with a mean peak latency of 361.4 msec and a mean peak amplitude
Ontogeny of flash-evoked potentials in PNDS N,a
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Fig. 1. Group average waveforms of FEPs PND p-40, stimulation frequency = O.Usec. PND = postnatal day. N = number of animals. Number of animals is a subset of all animals tested. Some waveforms could not be included in group averages due to computer problems. Note variation in voltage scale for different waveforms. For PND 23, the +I00 (downward) scale marker is indicated, to fit more conveniently on the page. Sweep time is 464 msec. Last waveform includes animals PND 40 and 41.
of 10.7 )IV appeared following Nla. The prominant initial negative peak of the adult FEP waveform, Nl, appeared in the group average waveform on PND 13. Nl was first seen as a negative shift on the leading shoulder of Nla. By PND 14, Nl was the prominant negative wave and Nla had begun to merge into Nl. By PND 19 Nla could no longer be seen in the group average waveform. The rapid maturation of the FEP between PND 13 and 15 in an individual animal is shown in Fig. 2. PNOIJ
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Fig. 2. FEPs recorded from one animal on PND 13, 14 and 15, stimulation frequency =0.2&c. Each wave is average of 128 trials. Line indicates latency of peak Nl on PND 13. Sweep time is 464 msec.
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Fig. 3. Effect of age on peak latencies. Vertical lines represent standard error. Peak latencies are means of FEPs of all animals recorded on the given day at O.Usec stimulation frequency. See Fig. 4 for symbol identification.
Some of the animals’ eyes opened on PND 14 and all were completely open by PND 15. After eye opening the amplitude of the peaks increased and peak N3 was observed as a slow negative shift following P2. For example, eye opening occurred on PND 14 in the rat whose data are presented in Fig. 2. On PND 14, Pl became a distinct positive peak in the waveform preceding Nl. Peaks N2 and P3 of the adult FEP waveform were not apparent in the group average waveforms of developing animals until PND 34, although they could occasionally be observed in
individual waveforms prior to that time. Whether this late appearance reflected late development or masking by the N3 wave is unclear. As the animals aged, latencies decreased (Fig. 3). Peak P2 was picked as the most positive point following Nl. The small (not significant) increase in P2 latency between PND 16 and 19 probably reflected the merging of wave Nla into wave Nl (see Fig. 1). Statistical analysis (MANOVA) of the effects of age on peak latency was performed on the four age groups of animals (PND 9-12, PND 1316, PND 17-20, > PND 20). Within each age group there was a significant effect of age (P~O.025) on peak latencies. This was true for all peaks except for the PND 17-20 group when P2 and N3 latencies were not affected. There was no effect of age on peak amplitudes within the groups tested, but the amplitudes generally increased with age (between groups, Fig. 4). After its appearance as a positive wave on PND 14 the amplitude of peak Pl did not increase again until PND 23. Peak Nl increased with age until a maximum was reached on PND 29 (Fig. 4). Peaks P2 and N3 reached maximum values on days 23 and 33, respectively (Fig. 4).
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Fig. 4. Effect of age on peak amplitudes. Vertical lines represent standard error. Peak amplitudes are means of FEPs of all animals recorded on the given day at O.Z/secstimulation frequency.
Ontogeny of flash-evoked potentials in unanesthetized ‘rats
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Fig. 5. Group
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Before PND 13 there were no measurable FEPs if the flash was presented more rapidly than OZsec. On PND 13 there were responses to l.O/sec and 2.0/set stimulation, and by PND 14,9 of 14 animals exhibited responses to light flashes delivered at 4.0/set. Figure 5 shows the effects of increasing stimulus frequency on FEPs recorded on PND 15. Increasing frequency of stimulation significantly affected peak Nl, P2, and N3 amplitudes in each of the four age groups (the oldest group was studied through PND 28). Peak N3 amplitudes could not be observed following 4.0/set stimulation before PND 23 because the peak latency was longer than the inter-stimulus interval. Nl and P2 peak amplitudes were consistently decreased in all groups (significant P
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Fig. 6. Effect of stimulation frequency on amplitude of peaks Nl, Pl, P2, and N3. Values are means from all animals recorded on the day and frequency indicated. Vertical lines indicate standard errors. Sweep time is 464 msec.
of FEP ontogeny the experiments were concluded prior to or during the third postnatal week, when it was reported that nearly adult peak latency values were achieved.’ In this paper we show significant maturation of the FEP beyond the third postnatal week, for example, peak latencies continued to decrease through the fifth postnatal week. Also, peaks N2 and P3, which are prominent in the adult FEP waveform, were not seen in the group average waveform until PND 34. Two stages of rapid change in FEP ontogeny are: (1) PND 9-10 when the response to light flash is first observed, and (2) PND 13-14 when peak Nl appears and FEPs are observed following stimulation frequencies higher than 0.2/set. Some of the neuroanatomical events occurring during these periods are discussed below. The first observable cortical response evoked by light flash occurred on PND 9. The rat optic nerve is completely unmyelinated at birth and completely myelinated in adults.2.‘h The first myelinated axons are observed on PND 6-8.1h Geniculocortical afferents first appear in layer IV of area 17 between PND 6 and PND 8”’ and acetylcholinesterase activity associated with these axons is first detectable at this time as well.*’ This is also the period during which the first significant increase in number of dendritic spines on area 17 pyramidal cells occurs” (PND 6-9). The abrupt appearance of peak Nl and the appearance of FEPs following >0.2/sec stimulation on PND 13 coincides with a peak in acetylcholinesterase staining in layer IV of the rat visual cortexz and a significant increase in the number of dendritic spines on visual cortex pyramidal neurons*’ (PND 12-15). A ‘growth spurt’ also occurs at about this time in the lateral geniculate neurons which project to the visual cortex.24 Peak latencies continued to decrease into the fourth and fifth postnatal weeks in this study and peaks N2 and P3 were not observed in the group average waveform until PND 34. The decrease in peak latencies with age are probably related to myelination of the optic nerve which is 85%
Ontogeny
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myelinated by PND 28 and completely myelinated after PND 40.1h Myelination alone, however, cannot account for all the latency changes, because conduction velocities in rat optic nerve decrease from PND 0 to PND 6 when the nerve is completely unmyelinated. I2 Changes in ionic conductance associated with development’ and increases in axon diameterI may play a role in increasing conduction velocities. The animal’s body temperatures increased with age and this was probably responsible for some of the decrease in peak latencies.’ The pattern of visual cortex pyramidal cell spine density continues to change between PND 15 and 60.14 Both findings indicate that the visual system is not mature until well after weaning (PND 21). Poststimulation neuronal recovery processes have been used to detect neurotoxicity in different sensory systems.h The ability of the visual system to respond to increasing stimulation frequencies developed with age, and only peak amplitudes were affected by increasing stimulation frequency in this study. Visual system recovery processes are polysynaptic,h therefore, simple maturational reductions in absolute and relative refractory periods in visual system neurons cannot explain the development of the recovery processes. The explanation is more likely to be related to maturation of inhibitory and excitatory neuronal circuits in the visual system. In summary, the ontogeny of the rat FEP proceeds in an orderly fashion beginning early in the second postnatal week and continuing into the fifth or sixth postnatal weeks. The development of the FEP can be studied in awake, unsedated animals and may provide a means of studying the effects of pharmacological and toxicological agents on the functional development of the visual system.
REFERENCES I. Albee R. R. and Mat&son J. L. (1983) Visual evoked response testing method for neonatal rats. Neurobehav. Toxicol. Terarol. 5, 497-501. studies during development of 2. Black J. A., Foster R. E. and Waxman S. G. (1982) Rat optic nerve: freeze-fracture myelination. Brain Res. 250, l-20. 3. Bronzino J. D., Morgane P. J., Forbes W. B., Stern W. C. and Resnick 0. (1975) Ontogeny of visual evoked responses in rats protein-malnourished during development. Biof. fsychiar. 10, 175-184. 4. Callison D. A. and Spencer J. W. (1968) Effect of chronic undernutrition and/or visual deprivation upon the visual evoked potential from the developing rat brain. Devf Psychobiol. 1, 19&204. 5. Connors B. W., Ransom B. R., Kunis D. M. and Gutnick M. J. (1982) Activity-dependent K+ accumulation in the developing rat optic nerve. Science 216, 1341-1343. challenges for the detection of toxicity. Fed. Proc. 42, 6. Dyer R. S. and Boyes W. K. (1983) Use of neurophysiological 3201-3206. and chloropent anesthesia differentially affect the flash evoked 7. Dyer R. S. and Boyes W. K. (1983) Hypothermia potentials of hooded rats. Brain Res. Bull. 10, 825-831. 8. Dyer R. S., Eccles C. U. and Annau Z. (1978) Evoked potential alterations following prenatal methylmercury. f’harmac. Biochem. Behav. 8, 137-141. H. S., Fechter L. D. and Annau Z. (1979) Prenatal carbon monoxide and 9. Dyer R. S., Eccles C. U., Swartzwelder adult evoked potentials in rats. 1. envir. H&h, Part C. Envir. Hfrh Sci. 13, 107-120. in rats perinatally exposed to 10. Dyer R. S., Howell W. E. and Reiter L. W. (1981) Visual evoked response alterations triethyltin. Neuroroxicology 2, 603-618. 11. Feinsod M., Hoyt W. F., Wilson W. B. and Spire J.-P. (1976) Visually evoked response use in neurological evaluation of posttraumatic subjective visual complaints. Archs Ophrhal. 94, 237-240. pharmacological and 12. Foster R. E., Connors B. W. and Waxman S. G. (1982) Rat optic nerve: electrophysiological, anatomical studies during development. Devl Brain Res. 3, 371-386. 13. Fox D. A., Lewkowski J. P. and Cooper G. P. (1977) Acute and chronic effects of neonatal lead exposure on development of the visual evoked response in rats.’ Tax.. app. Pharmac. 40, 449-%1. of pyramidal neurons after eye opening in the visual cortex of hooded rats: a 14. Juraska J. M. (1982) The development quantitative study. J. camp. Neural. 212, 208-213. of hypothermia to effects of chloral hydrate on flash evoked potenIS. Hetzler B. E. and Dyer R.-S. (1977) Contribution tials of hooded rats. Pharmac. Biochem. Behav. 21, 599-607. C. and Waxman S. G. (1984) Postnatal differentiation of rat optic nerve fibers: electron microscopic ob16. Hildebrand servations on the development of nodes of Ranvier and axoglial relations. J. camp. Neural. 224, 25-37. 17. Howell W. E., Miller D. and Dyer R. S. (1982) Impairment of visual system development following organotin exposure. Toxicologkr 2, 53.5. alterations due to diesel exhaust exposure during the 18. Laurie R. D. and Boyes W. K. (1981) Neurophysiological neonatal life of the rat. Envir. Int. 5, 36S368. M. and Salas M. (1985) Effects of neonatal toluene exposure on the development of evoked and 19. Lorenza-Jimenez spontaneous cortical activity in the rat. Neurobehav. Toxicol. Terarol. 7, 215-220. 20. Lund R. D. and Mustari M. J. (1977) Development of the geniculocortical pathway in rats. /. camp. Neural. 173, 289-306.
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21. Miller M. and Peters A. (1981) Maturation of rat visual cortex. II. A combined Golgi-electron microscope study of pyramidal neurons. J. camp. Neural. 203.555-573. 22. Mourek J., Himwich W. A., Myslivecek J. and Callison D. A. (t%7) The role of nutrition in the development of evoked cortical response in rat. Brain Res. 6, 241-251. 23. Myslivecek J. (1970) Electrophysiology of the developing brain--Central and Eastern European contributions. In Developmental Neurobiology (ed. Hinwich W. A.), pp. 475-527. Thomas, Springfield, IL. 24. Parnavelas J. Cl., Mounty E. J., Bradford R. and Leiberman A. R. (1977) The postnatal development of neurons in the dorsal lateral geniculate nucleus of the rat: a Golgi study. J. camp. Neurof. 171, 481-500. 25. Robertson R. T., Tijerina A. A. and Galtivan M. E. (1985) Transient patterns of acetytcholinesterase activity in visual cortex of the rat: normal development and the effects of neonatal monocular enucleation. Llevl Brain Rex 21, 203-214. 26. Rose G. H. (1968) The development of visualfy evoked etectrocorticat responses in the rat. Devl Psychobiot. 1,3540. 27. Rose G. H. and Ellingson R. J. (1970) Ontogenesis of evoked potentials. In Developmental Neurobiology (ed. Himwich W. A.), pp. 393-440. Thomas, Springfield, IL. 28. Salas M., Diax S. and Cintra L. (1976) Electrocortical effects of early postnatal thyroxine administration in the rat. Physiof. Be&w. 17, 239-244. 29. Sjostrom A., Conradi N. G. and Andersson S. A. (1984) Functional development of the visual system in normal and protein deprived rats. Acfa physiol. stand. fu), 585-594.