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Analysis of the rabbit's electroretinogram following unilateral transection of the optic nerve
Exp. E,ye Res. (1972) 13, 227-235
Analysis of the Rabbit’s Electroretinogram Following Unilateral Transection of the Optic Nerve BARRY S. WINKLER*
Department of Physiobogy,Xchool of Medicines 8tote University of Weza York at Buffalo, Buffalo. NW York 14214, U.S.A. (Receivrd 29 Octobrr 1971, Boston) The effect of ganglion cell and centrifugal fiber activity on the electroretinogram of adult albino rabbits was investigated by measuring the electroretinogram 2-7 months after unilateral transection of the optic nerve. Histological examination of the denervated eyes showed that about 2Oqg of the ganglion cells still survived. The electroretinograms of the control and optic nerve-severed (denervated) eyes were compared in darkand lightadapted states, and before and after low doses of nembutal were administered. The electroretinograms from the two eyes (control and denervated) were the same in all cases. Ganglion cells and centrifugal fibers, if the latter are present, in the rabbit, do not influence the electroretinogram.
1. Introduction C’entrifugal fibers to the retina were first described by Cajal (1891), who found thin fibers ascending through the ganglion cell layer and ending in arborizations around the amacrine cell in the pigeon. Ventura and Mathieu (1959) found several types of centrifugal fibers in the ganglion cell and the inner plexiform layers in man, dog, cat, and rabbit. On the other hand, Brindley and Hamasaki (1961,1962b, 1966) concluded that the cat’s optic nerve does not contain centrifugal fibers, becausean intracranial lesion produced no degeneration of the optic nerve in the intraorbital region up to 9 days after the lesion. However, anucleation of the eye produced degeneration in the remaining nerve in 4 days. There appears to be good anatomical evidence and general agreement that the avian brain contains centrifugal fibers which arise in the caudal midbrain (isthmo-optic nucleus) and passvia the isthmo-optic tract and the contralateral optic nerve to the retina (Cowan, Adamson and Powell, 1961; Cowan and Powell, 1963). However, there is no agreement that mammals possessa similar pathway. Physiological evidence for the existence of centrifugal fibers comes from experiments where either sectioning the optic nerve or an electrical stimulus applied centrally in the visual pathway was found to alter the activity of the retina. Dodt (1956) found a delayed retinal spike, delayed relative to the antidromic spikes, following electrical stimulation of the contralateral optic tract in rabbits and suggestedthat the delayed spike was evidence for a centrifugal effect on the retina. Gills (1966) reported that after sectioning one optic nerve in humans, caused by removal of a pituitary adenoma, the electroretinograms (ERGS) were larger than those from the control, normal eye. Jacobson and Gestring (1958) found that the effects of barbiturates on the ERG of the cat were abolished by optic nerve transection. On the other hand, Brindley and Hamasaki (1962a) showed that the ERG of the consciouscat was the samewhether the optic nerve was intact or cut through. * Present U.S.A.
address:
Institute
of Biological
Sciences,
Oakland
University,
Rochester,
Michigan
48063,
228
BARRY
S. WII”;kI,Ef:
Experiment,s were undertaken to investigate quantitatively any influence of ccntrifugal fiber and ganglion cell activity on the ERG in the rabbit following unilateral sectioning of the optic nerve. The evidence presented suggests that centrifugal fibcr~ and ganglion cells play no role in the generation of t.he ERG potentials. 2. Materials
and Methods
Fifteen adult albino ra,bbit,s weighing ‘J- 3 kg each were anesthetized with pentobarbitone (30 mg/kg; intravenous), and were also given local infiltration of the orbit with xylocaine. An incision was made at the superior margin of the cartilaginous layer bordering the supra-orbital ridge, and Tenon‘s capsule was opened. The superior rectus was transected and the cartilaginous supraorbital ridge was removed to expose the optic nerve. The nerve was severed with a sharp Ziegler’s knife which was inserted into the center of the nerve and moved at right angles. Under direct visual observation, the cut nerve was observed to completely retract into the orbit indicating that it was totally severed. Care was taken to avoid damaging the long and short ciliary arteries, which are the major blood supply to the retina. The incision was closed with wound clips, which remained in place for about 1 week. The pupillary reflex of the operated eye usually disappeared immediately. as did the consensual reflex of the unoperated eye. ERGS were measured 2-7 months after transection of the nerve, using animals anesthetized with a 25% solution of urethane (0.8 mg/cm3) and topical atropine sulfate was applied to the eye. The animals were maintained on artificial respiration throughout the measurements and d-tubocurarine was injected int.ravenously to minimize movements of the animal. The eyelids were held open with sutures. The ERG was recorded from the cornea with a cotton wick electrode, with the indifferent electrode placed equidistant from the two eyes and as close to the nose as possible. A ground electrode was inserted in the tissue between the ears. The animal was allowed to darkadapt for at least 1 hr prior to recording. The electrodes were connected to it Grass P5 AC preamplifier with the -k amplitude frequencies usually set for a band pass of 0.1 to 2000 Hz. The output of the preamplifier was connected to a dual beam Tektronix 502 oscilloscope, so tha.t the ERGS of both eyes could be directly observed si~~~ultaneousl~. The traces were photographed by il camera in front of the oscilloscope. Two light sources were used in the experiments. One source was the Grass model PS-2 photostimulator which produced brief flashes of light of relative intensity. In the graphs, the 1,4, and 16 settings of the phot~)stimulntor refer to 0,06, and 1.2, respectively, in the logarithmic scale used for piotting ERG Hmplitude versus stimulus intensity. The other light source was a tungsten lamp which provided diffuse variable background &m~ination of the retinas. A fiber optic system supplied by the Bausch and Lomb Co.! Rochester, New York, was used to provide equal light stimulation of both eyes simultaneously. The range of light intensities used was increased by using neutral density filters. Both eyes were removed after ERG testing and fixed in Zenker’s or 10% formalill solutions for 24 hr; the eyes then were washed in water and dehydrated in alcohol prior to paraffin embedding. The eyes were c;ut through t,he vertical meridian starting at the nasal or temporal edge. Representative sections 10 and 15 pm in thickness were made at 0*5-mm intervals. Comparisonsbetween the nornlal and denervated eyes were made using the 15-pm section through the optic nerve; this sect,ionvxs typical of all the 16-1~msections observed with respect,l.o giulglion cell degeneration. ,411t,lte retinas were stained with hematoxylin and eosin. 3. Results Figure 1 shows typical ERG tracings from the normal and denervated eyes as a function of stimulus intensity in the dark-adapted state. The ERGS (a-wave, b-wave
ELECTRORETINOGRAM
AFTER
OPTIC
NERVE
SECTION
229
FIG. 1. Simultaneous recordings of ERGS in response to varying light intensities. Upper trace of each pair in all figures; control eye. Lower trace of each pair in all figures; denervated eye. Numbers be&de tracings indicate light intensities in relative logarithmic unite. Amplitude calibration, 100 pV; time mark, 10 maec. Note greater sensitivity and change in time scale for the bottom row of ERGS. In all pictures the flash occum at the beginning of the sweep and positive is upward.
and wavelets) are indistinguishable with respect to their form, amplitudes, latencies, and rates of rise. Figure 2 is a plot of the ERG amplitude versus stimulus intensity. At all intensities the amplitudes of the a- and b-waves are identical within experimental error. The average amplitude of the b-wave (ten experiments) measured from the trough of the a-wave was 604-+24 ,V and 579 f24 ~LV for the control and denervated 700
:
600 -
500
-
400
-
300
-
5
200 -
Log
Fm. 2. Dark-adapted a-wave and b-wave eye; 0, denervated eye; Q , superimposable Vertical lines represent S.D.
intensity
amplitudes responses.
as a function of stimulus intensity. 0, Control Each point is the average of ten experiments.
HAHi:Y
030
S. WISKLEI:
eyes, respect,ively, in response to the maximal light intensity. The maximal amp1itut-l~~ of the a-wave was 155&20 PV and 152+21 PY for the control and denerva,tccl eye>. respectively. Figure 3 showsERGS from both eyes in responseto varying rates of flicker stimulation (log 1 = 0.6). At low stimulus frequencies both the a- and b-waves are seen. while at rates greater than lO/sec only a positive potential is seen. The form of the ERGS from the two eyes is nearly identical with respect to the latencies and amplitudes of the potentials.
&&A I/C&C
II
ZO/sec
5/set
I O/set
4o/sec
60/set
FIG. 3. ERGs in response to repetitive light stimulation Amplitude calibration, 100 rV; time mark, 10 msec. Note
1
over the range of l/set to 60/set. Log Z = 0.6. change in time scale for bottom row of ERGS.
UFigure 4 showstypical ERGS from the normal and denervated eyes during various levels of background illumination from one experiment. No significant differences were found between the two eyes. The ERG undergoesmarked changeswith increasing background illumination. In responseto the highest intensity flash (log I = 1.2) at each background level, the rate of rise of the a-wave decreased(resulting in a less acute deflection from the baseline) as the background intensity was increased. The sharp upward rise of the b-wave was present at all background levels. However, in the Log intensity
FIG. 4. ERGs are shown in the dark and during various levels of background illumination. background of -4 is the lowest background intensity. Upper calibration refers to top row while calibration refers to all other rows. Amplitude calibration, 100 pV; time scale, 10 msec.
A log lower
140 -
I20 -
100 -
60-
-0,8
0
0.6
I.2
Log interwty
FIG. 5. Intensity-response curves of the amplitude of the a-wave in the dark- and light-adapted states. Test flashes are superimposed on a steady state of background illumination. Numbers on the right indicate background illumination in relative logarithmic units. Each point is the average of five experiments. 0, Control eye; 0, denervated eye; Q, superimposable responses.
Log
intensity
Fro. 6. Intensity-response curves of the amplitude of the b-wave in the dark- and light-adapted states. Test flashes are superimposed on a steady state of background illumination. Numbers on the right indicate background illumination in relative logarithmic units. Each point is the average of five experiments. Only the dark curve contains S.D. 0, Control eye; 0, denerrated eye; 0, superimposable responses.
232
BARRY
S. WINKLER
dark-adapted response the peak of the b-wave was round and smooth, while with low levels of background illumination the b-wave had several peaks. Further increases in background illumination resulted in a flat peak and, finally, at the highest background illumination there was a single sharp peak. The latencies of the a- and b-waves for a given stimulus intensity were not changed with increasing background illumination. The peak time of the b-wave, however, decreased markedly as the background intensity increased.
Nembutol
IOmg/kg
FIG. 7. ERGS in response to intravenous administration of Nembutal(l0 mg/kg). The tracings of the upper row were obtained shortly before injection of the drug. The indicated times (e.g., 45”) give the time in seconds after the start of the injection. Log I = 0. Amplitude calibration, 100 pV; time scale 10 meet.
The amplitude of the a- and b-waves decreased with increasing background illumination. Figures 5 and 6 show the amplitudes of the a- and b-waves, respectively, as a function of stimulus intensity in the dark- and light-adapted states. At all background levels the curves for both eyes are superimposable over the entire range of stimulus intensities. The a-wave shows little if any adaptation at low background intensities but then it adapts rapidly and fails to be evoked as the strength of the background illumination is further increased. In contrast to the a-wave, the b-wave appears to adapt at all background levels, although these particular curves are complex and not easily interpretable. Part of this complexity relates to the fact that the amplitude measurements are being made on different components or subunits of the b-wave as is suggested by the striking changes in the form and peak time of the b-wave in the light-adapted state. These adaptation curves are qualitatively identical to those reported by Dowling (1967) for the a- and b-waves of the rat ERG.
ELECTRORETINOGRARIAFTEROPTICNERVESECTION
233
The effect of Nembutal Following intravenous administration of Nembutal(l0 mg/kg), there was a sudden and marked increase in the amplitudes of the a- and b-waves (Fig. 7). The enhancement and prolongation of the a-wave resulted in an increase in the apparent latency of the b-wave, a finding in agreement with that of Noel1 (1958). In addition, as has been previously shown (Yonemura, Kawasaki and Tsuchida, 1966) the augmentation of the a- and b-waves was accompanied by an attenuation of the wavelets. These effects of Nembutal were identical in both the normal and denervated eyes.
An 80% reduction in the total number of ganglion cells was observed in the denervated retinas. This value was obtained by counting the number of ganglion cells in the 15-p”’ section through the optic nerve in 12 normal and denervated eyes. Comparisons were always made between the control and denervated eyes from the same animal. This average value of 80% includes retinas observed 2-7 months after the transection of the nerve. There were essentially no significant differences between the 2-month and the “r-month retinas with respect to the per cent degeneration of ganglion cells. Thus, degeneration was not dispersed in time during this period. The inner nuclear and outer nuclear layers appeared identical in the normal and denervated eyes. A variety of cell types were observed in the ganglion cell layer of the normal retina. They were classified as small, medium and large. Cell counts showed they represented about 60,25 and 15%, respectively of the total number of cells observed in the 15-pm section through the optic nerve. In the central region of the normal retina (Plate 1) the ganglion cells constituted an uninterrupted row in which the small ganglion cells were easily the most numerous. The ganglion cells decreased continually in number per unit area as the ora serrata was approached. All cell types were found in the periphery. The large cells were oval in shape and possessed a distinct nucleus. The small and medium cells were round and their nuclei were not easily seen. The denervated retinas lacked the close packing of ganglion cells in their central region (Plate 2). It was consistently observed that practically all the large and medium sized ganglion cells were missing in the denervated retinas, leaving the small cells as the only surviving cell type. Furthermore, the width of the inner plexiform layer in the denervated retinas was approximately 2576 smaller than its counterpart in t’he normal retjina. 4. Discussion The results of the present st’udy suggest that an intact optic nerve is not a prerequisite for the generation of the ERG of the rabbit. The ERGS of the control and denervat’ed eyes were observed under wide and diverse testing procedures that included : (1) responses to varying light intensities in the dark- and light-adapted states; (2) responses t,o repetitive stimulation; and (3) responses to low doses of Nemhutal. At no time in any experiment were the ERGS of t’he two eyes significantly different. Jacobson and Gestring (1958) found that Nembutal, a central nervous system depressant, evoked a larger ERG in the normal cat eye as compared to the optic
331
J
nerve-severed eye. They suggested that, there is a center in the braiu modulating thr* activity of the retina. However, it appears that the effects and conclusions reported by these authors regarding the importance of an intact’ optic nerve are not, supported l)y the findings of this or other studies. Arden, Granit and Ponte (1960) injected Nembutal in low doses to decerebrate cats and found an increased response of both single flashes and flickering wavelets. Since these experiments (Arden et al., 1960) were done on decerebrate cats. the investigators found no support for the conclusion of Jacobson and Gestring (1958) that increased b-waves after pentobarbitone administration required intact central connections through the optic nerve. In this study, injection of Nembutal produced an enhancement of the a- and bwaves in the normal and denervated eyes, controverting the findings of Jacobson and Gestring (1958). Although the actual presence of centrifugal fibers was not under investigation in this study of the rabbit ERG, it is concluded that activity from these fibers, if they are present, does not appear to influence the ERG. ERGS have been recorded previously from retinas possessing functionally and structurally impaired ganglion cells (Granit and Helme, 1939; Noell. 1953). The essential conclusion from these studies is that the ganglion cells do not generate any component of the ERG. Although Noel1 (1953) utilized the same procedure to isolate ganglion cell activity as in this study, he did not report the extent of ganglion cell degeneration or the precise conditions under which the ERG was observed. This investigation provided a more rigorous test for determining the influence of the ganglion cells on the ERG. Determination of this influence requires that the ganglion cells be destroyed and functionally inactivated. It was assumed that transection of the optic nerve and waiting for retrograde degeneration of the ganglion cells would fulfill this requirement. Histological examination of retinas 2-7 months after the transection proved, however, that 20% of the ganglion cells still survived. Uppermost in my mind was the reason for the survival of these cells. Incomplete transection was a possibility, but unlikely. The surgery was performed with the utmost care. The resistance to degeneration may be related to the age of the animal. Stone (1966) reported that sectioning one optic tract in 11-21-day-old kittens caused complete retrograde degeneration of the aflected ganglion cells within 24 days, while in the adult cat the ganglion cells showed no signs of degeneration after 10 weeks and required 2 years to degenerate completely. Therefore, it is likely that degeneration would have been more complete for the same time interval following transection of the nerve in younger rabbits. As a result of the incomplete destruction of the ganglion cells one could ask, “Do the surviving 20% in any way influence the ERG?” The experimental protocols contained a variety of tests in order that any influence of the ganglion cells, regardless of the subtlety of this influence, would be detected. In fact, no significant differences between the ERGS of the normal and denervated eyes were ever observed. Hence, in agreement with earlier studies, it is concluded that the ganglion cells do not appear to affect the generation of the a- and b-waves and wavelets of the ERG. ACKNOWLEDGMENTS This investigation was supported in part by a Training Grant (5 TO1 GM00341) from National Institute of General Medical Sciences, U.S. Public Health Service. I wish to thank Dr W. K. Noel1 for his encouragement and criticisms during the course of this work, and Mrs R. Albrecht for her assistance in preparing the histological sections.
PLATE
of ganglion
PLATE
1. Photomicrograph through cells. Retina stained with
2. Photomicrograph
through
the central hematoxylin
region of a normal and cosin.
a comparable
but from a denervated retina. Close packing of ganglion absent due to degeneration of the ganglion cells. Retina
sectlon
of the
retina
illustrating
central
regon,
the close pa&in:!
as shown
m Hate
cells as seen in the normal retina is noticeably stained with hematoxglin and rosin.
I,
ELECTRORETINOGRAM
AFTER
OPTIC
NERVE
SECTION
235
REFERENCES &den, G., Granit, R. and Ponte, F. (1960). J. Neurophysiol. 23,305. Brindley, G. S. and Hamasaki, D. I. (1961). J. Physiol. (London) 159, 88P. Brindley, G. S. and Hamasaki, D. I. (1962s). J. Physiol. (London) 163, 558. Brindley, G. S. and Hamasaki, D. I. (1962b). J. Physiol. (London) 163,25P. Brindley, G. S. and Hamasaki, D. I. (1966). J. Physiol. (London) 184,444. Cajal, Ramon Y. (1891). Histologic du Systeme de Z’Homme et des Vertebres. vol. 2. Maloine, Paris. Cowan, W. M., Adamson, L. and Powell, T. (1961). J. Anat. 95, 545. Cowan, W. M. and Powell, T. (1963). Proc. Roy. Sot. Ser. R. 158,232. Dodt, E. (1956). J. Neurophysiol. 19, 301. Dowling, J. (1967). Science 155, 273. Gills, J. (1966). Amer. J. OphthaZmoZ. 62, 287. Granit, R. and Helme, T. (1939). J. Xeurophysiol. 2, 556. Jacobson. J. and Gestring, G. (1958). Ann. N. Y. Acud. Sci. 74, 362. Noell, W. K. (1953). USAF School of Aviation Medicine, Report No. 1, Randolph Field, Texas. Noel& W. K. (1958). A.2M.A. Arch. of Ophthalmol. 60, 702. Stone, J. (1966). .I. Comp. Neur. 126,585. Ventura. J. and Mathieu, M. (1959). Trans. Can. OphthaZmoZ. Sot. 22,184. Yonemura, D., Kawasaki, K. and Tsuchida, Y. (1966). Jap. J. OphthaZmoZ. 10, 155.
Analysis of the Rabbit’s Electroretinogram Following Unilateral Transection of the Optic Nerve BARRY S. WINKLER*
Department of Physiobogy,Xchool of Medicines 8tote University of Weza York at Buffalo, Buffalo. NW York 14214, U.S.A. (Receivrd 29 Octobrr 1971, Boston) The effect of ganglion cell and centrifugal fiber activity on the electroretinogram of adult albino rabbits was investigated by measuring the electroretinogram 2-7 months after unilateral transection of the optic nerve. Histological examination of the denervated eyes showed that about 2Oqg of the ganglion cells still survived. The electroretinograms of the control and optic nerve-severed (denervated) eyes were compared in darkand lightadapted states, and before and after low doses of nembutal were administered. The electroretinograms from the two eyes (control and denervated) were the same in all cases. Ganglion cells and centrifugal fibers, if the latter are present, in the rabbit, do not influence the electroretinogram.
1. Introduction C’entrifugal fibers to the retina were first described by Cajal (1891), who found thin fibers ascending through the ganglion cell layer and ending in arborizations around the amacrine cell in the pigeon. Ventura and Mathieu (1959) found several types of centrifugal fibers in the ganglion cell and the inner plexiform layers in man, dog, cat, and rabbit. On the other hand, Brindley and Hamasaki (1961,1962b, 1966) concluded that the cat’s optic nerve does not contain centrifugal fibers, becausean intracranial lesion produced no degeneration of the optic nerve in the intraorbital region up to 9 days after the lesion. However, anucleation of the eye produced degeneration in the remaining nerve in 4 days. There appears to be good anatomical evidence and general agreement that the avian brain contains centrifugal fibers which arise in the caudal midbrain (isthmo-optic nucleus) and passvia the isthmo-optic tract and the contralateral optic nerve to the retina (Cowan, Adamson and Powell, 1961; Cowan and Powell, 1963). However, there is no agreement that mammals possessa similar pathway. Physiological evidence for the existence of centrifugal fibers comes from experiments where either sectioning the optic nerve or an electrical stimulus applied centrally in the visual pathway was found to alter the activity of the retina. Dodt (1956) found a delayed retinal spike, delayed relative to the antidromic spikes, following electrical stimulation of the contralateral optic tract in rabbits and suggestedthat the delayed spike was evidence for a centrifugal effect on the retina. Gills (1966) reported that after sectioning one optic nerve in humans, caused by removal of a pituitary adenoma, the electroretinograms (ERGS) were larger than those from the control, normal eye. Jacobson and Gestring (1958) found that the effects of barbiturates on the ERG of the cat were abolished by optic nerve transection. On the other hand, Brindley and Hamasaki (1962a) showed that the ERG of the consciouscat was the samewhether the optic nerve was intact or cut through. * Present U.S.A.
address:
Institute
of Biological
Sciences,
Oakland
University,
Rochester,
Michigan
48063,
228
BARRY
S. WII”;kI,Ef:
Experiment,s were undertaken to investigate quantitatively any influence of ccntrifugal fiber and ganglion cell activity on the ERG in the rabbit following unilateral sectioning of the optic nerve. The evidence presented suggests that centrifugal fibcr~ and ganglion cells play no role in the generation of t.he ERG potentials. 2. Materials
and Methods
Fifteen adult albino ra,bbit,s weighing ‘J- 3 kg each were anesthetized with pentobarbitone (30 mg/kg; intravenous), and were also given local infiltration of the orbit with xylocaine. An incision was made at the superior margin of the cartilaginous layer bordering the supra-orbital ridge, and Tenon‘s capsule was opened. The superior rectus was transected and the cartilaginous supraorbital ridge was removed to expose the optic nerve. The nerve was severed with a sharp Ziegler’s knife which was inserted into the center of the nerve and moved at right angles. Under direct visual observation, the cut nerve was observed to completely retract into the orbit indicating that it was totally severed. Care was taken to avoid damaging the long and short ciliary arteries, which are the major blood supply to the retina. The incision was closed with wound clips, which remained in place for about 1 week. The pupillary reflex of the operated eye usually disappeared immediately. as did the consensual reflex of the unoperated eye. ERGS were measured 2-7 months after transection of the nerve, using animals anesthetized with a 25% solution of urethane (0.8 mg/cm3) and topical atropine sulfate was applied to the eye. The animals were maintained on artificial respiration throughout the measurements and d-tubocurarine was injected int.ravenously to minimize movements of the animal. The eyelids were held open with sutures. The ERG was recorded from the cornea with a cotton wick electrode, with the indifferent electrode placed equidistant from the two eyes and as close to the nose as possible. A ground electrode was inserted in the tissue between the ears. The animal was allowed to darkadapt for at least 1 hr prior to recording. The electrodes were connected to it Grass P5 AC preamplifier with the -k amplitude frequencies usually set for a band pass of 0.1 to 2000 Hz. The output of the preamplifier was connected to a dual beam Tektronix 502 oscilloscope, so tha.t the ERGS of both eyes could be directly observed si~~~ultaneousl~. The traces were photographed by il camera in front of the oscilloscope. Two light sources were used in the experiments. One source was the Grass model PS-2 photostimulator which produced brief flashes of light of relative intensity. In the graphs, the 1,4, and 16 settings of the phot~)stimulntor refer to 0,06, and 1.2, respectively, in the logarithmic scale used for piotting ERG Hmplitude versus stimulus intensity. The other light source was a tungsten lamp which provided diffuse variable background &m~ination of the retinas. A fiber optic system supplied by the Bausch and Lomb Co.! Rochester, New York, was used to provide equal light stimulation of both eyes simultaneously. The range of light intensities used was increased by using neutral density filters. Both eyes were removed after ERG testing and fixed in Zenker’s or 10% formalill solutions for 24 hr; the eyes then were washed in water and dehydrated in alcohol prior to paraffin embedding. The eyes were c;ut through t,he vertical meridian starting at the nasal or temporal edge. Representative sections 10 and 15 pm in thickness were made at 0*5-mm intervals. Comparisonsbetween the nornlal and denervated eyes were made using the 15-pm section through the optic nerve; this sect,ionvxs typical of all the 16-1~msections observed with respect,l.o giulglion cell degeneration. ,411t,lte retinas were stained with hematoxylin and eosin. 3. Results Figure 1 shows typical ERG tracings from the normal and denervated eyes as a function of stimulus intensity in the dark-adapted state. The ERGS (a-wave, b-wave
ELECTRORETINOGRAM
AFTER
OPTIC
NERVE
SECTION
229
FIG. 1. Simultaneous recordings of ERGS in response to varying light intensities. Upper trace of each pair in all figures; control eye. Lower trace of each pair in all figures; denervated eye. Numbers be&de tracings indicate light intensities in relative logarithmic unite. Amplitude calibration, 100 pV; time mark, 10 maec. Note greater sensitivity and change in time scale for the bottom row of ERGS. In all pictures the flash occum at the beginning of the sweep and positive is upward.
and wavelets) are indistinguishable with respect to their form, amplitudes, latencies, and rates of rise. Figure 2 is a plot of the ERG amplitude versus stimulus intensity. At all intensities the amplitudes of the a- and b-waves are identical within experimental error. The average amplitude of the b-wave (ten experiments) measured from the trough of the a-wave was 604-+24 ,V and 579 f24 ~LV for the control and denervated 700
:
600 -
500
-
400
-
300
-
5
200 -
Log
Fm. 2. Dark-adapted a-wave and b-wave eye; 0, denervated eye; Q , superimposable Vertical lines represent S.D.
intensity
amplitudes responses.
as a function of stimulus intensity. 0, Control Each point is the average of ten experiments.
HAHi:Y
030
S. WISKLEI:
eyes, respect,ively, in response to the maximal light intensity. The maximal amp1itut-l~~ of the a-wave was 155&20 PV and 152+21 PY for the control and denerva,tccl eye>. respectively. Figure 3 showsERGS from both eyes in responseto varying rates of flicker stimulation (log 1 = 0.6). At low stimulus frequencies both the a- and b-waves are seen. while at rates greater than lO/sec only a positive potential is seen. The form of the ERGS from the two eyes is nearly identical with respect to the latencies and amplitudes of the potentials.
&&A I/C&C
II
ZO/sec
5/set
I O/set
4o/sec
60/set
FIG. 3. ERGs in response to repetitive light stimulation Amplitude calibration, 100 rV; time mark, 10 msec. Note
1
over the range of l/set to 60/set. Log Z = 0.6. change in time scale for bottom row of ERGS.
UFigure 4 showstypical ERGS from the normal and denervated eyes during various levels of background illumination from one experiment. No significant differences were found between the two eyes. The ERG undergoesmarked changeswith increasing background illumination. In responseto the highest intensity flash (log I = 1.2) at each background level, the rate of rise of the a-wave decreased(resulting in a less acute deflection from the baseline) as the background intensity was increased. The sharp upward rise of the b-wave was present at all background levels. However, in the Log intensity
FIG. 4. ERGs are shown in the dark and during various levels of background illumination. background of -4 is the lowest background intensity. Upper calibration refers to top row while calibration refers to all other rows. Amplitude calibration, 100 pV; time scale, 10 msec.
A log lower
140 -
I20 -
100 -
60-
-0,8
0
0.6
I.2
Log interwty
FIG. 5. Intensity-response curves of the amplitude of the a-wave in the dark- and light-adapted states. Test flashes are superimposed on a steady state of background illumination. Numbers on the right indicate background illumination in relative logarithmic units. Each point is the average of five experiments. 0, Control eye; 0, denervated eye; Q, superimposable responses.
Log
intensity
Fro. 6. Intensity-response curves of the amplitude of the b-wave in the dark- and light-adapted states. Test flashes are superimposed on a steady state of background illumination. Numbers on the right indicate background illumination in relative logarithmic units. Each point is the average of five experiments. Only the dark curve contains S.D. 0, Control eye; 0, denerrated eye; 0, superimposable responses.
232
BARRY
S. WINKLER
dark-adapted response the peak of the b-wave was round and smooth, while with low levels of background illumination the b-wave had several peaks. Further increases in background illumination resulted in a flat peak and, finally, at the highest background illumination there was a single sharp peak. The latencies of the a- and b-waves for a given stimulus intensity were not changed with increasing background illumination. The peak time of the b-wave, however, decreased markedly as the background intensity increased.
Nembutol
IOmg/kg
FIG. 7. ERGS in response to intravenous administration of Nembutal(l0 mg/kg). The tracings of the upper row were obtained shortly before injection of the drug. The indicated times (e.g., 45”) give the time in seconds after the start of the injection. Log I = 0. Amplitude calibration, 100 pV; time scale 10 meet.
The amplitude of the a- and b-waves decreased with increasing background illumination. Figures 5 and 6 show the amplitudes of the a- and b-waves, respectively, as a function of stimulus intensity in the dark- and light-adapted states. At all background levels the curves for both eyes are superimposable over the entire range of stimulus intensities. The a-wave shows little if any adaptation at low background intensities but then it adapts rapidly and fails to be evoked as the strength of the background illumination is further increased. In contrast to the a-wave, the b-wave appears to adapt at all background levels, although these particular curves are complex and not easily interpretable. Part of this complexity relates to the fact that the amplitude measurements are being made on different components or subunits of the b-wave as is suggested by the striking changes in the form and peak time of the b-wave in the light-adapted state. These adaptation curves are qualitatively identical to those reported by Dowling (1967) for the a- and b-waves of the rat ERG.
ELECTRORETINOGRARIAFTEROPTICNERVESECTION
233
The effect of Nembutal Following intravenous administration of Nembutal(l0 mg/kg), there was a sudden and marked increase in the amplitudes of the a- and b-waves (Fig. 7). The enhancement and prolongation of the a-wave resulted in an increase in the apparent latency of the b-wave, a finding in agreement with that of Noel1 (1958). In addition, as has been previously shown (Yonemura, Kawasaki and Tsuchida, 1966) the augmentation of the a- and b-waves was accompanied by an attenuation of the wavelets. These effects of Nembutal were identical in both the normal and denervated eyes.
An 80% reduction in the total number of ganglion cells was observed in the denervated retinas. This value was obtained by counting the number of ganglion cells in the 15-p”’ section through the optic nerve in 12 normal and denervated eyes. Comparisons were always made between the control and denervated eyes from the same animal. This average value of 80% includes retinas observed 2-7 months after the transection of the nerve. There were essentially no significant differences between the 2-month and the “r-month retinas with respect to the per cent degeneration of ganglion cells. Thus, degeneration was not dispersed in time during this period. The inner nuclear and outer nuclear layers appeared identical in the normal and denervated eyes. A variety of cell types were observed in the ganglion cell layer of the normal retina. They were classified as small, medium and large. Cell counts showed they represented about 60,25 and 15%, respectively of the total number of cells observed in the 15-pm section through the optic nerve. In the central region of the normal retina (Plate 1) the ganglion cells constituted an uninterrupted row in which the small ganglion cells were easily the most numerous. The ganglion cells decreased continually in number per unit area as the ora serrata was approached. All cell types were found in the periphery. The large cells were oval in shape and possessed a distinct nucleus. The small and medium cells were round and their nuclei were not easily seen. The denervated retinas lacked the close packing of ganglion cells in their central region (Plate 2). It was consistently observed that practically all the large and medium sized ganglion cells were missing in the denervated retinas, leaving the small cells as the only surviving cell type. Furthermore, the width of the inner plexiform layer in the denervated retinas was approximately 2576 smaller than its counterpart in t’he normal retjina. 4. Discussion The results of the present st’udy suggest that an intact optic nerve is not a prerequisite for the generation of the ERG of the rabbit. The ERGS of the control and denervat’ed eyes were observed under wide and diverse testing procedures that included : (1) responses to varying light intensities in the dark- and light-adapted states; (2) responses t,o repetitive stimulation; and (3) responses to low doses of Nemhutal. At no time in any experiment were the ERGS of t’he two eyes significantly different. Jacobson and Gestring (1958) found that Nembutal, a central nervous system depressant, evoked a larger ERG in the normal cat eye as compared to the optic
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nerve-severed eye. They suggested that, there is a center in the braiu modulating thr* activity of the retina. However, it appears that the effects and conclusions reported by these authors regarding the importance of an intact’ optic nerve are not, supported l)y the findings of this or other studies. Arden, Granit and Ponte (1960) injected Nembutal in low doses to decerebrate cats and found an increased response of both single flashes and flickering wavelets. Since these experiments (Arden et al., 1960) were done on decerebrate cats. the investigators found no support for the conclusion of Jacobson and Gestring (1958) that increased b-waves after pentobarbitone administration required intact central connections through the optic nerve. In this study, injection of Nembutal produced an enhancement of the a- and bwaves in the normal and denervated eyes, controverting the findings of Jacobson and Gestring (1958). Although the actual presence of centrifugal fibers was not under investigation in this study of the rabbit ERG, it is concluded that activity from these fibers, if they are present, does not appear to influence the ERG. ERGS have been recorded previously from retinas possessing functionally and structurally impaired ganglion cells (Granit and Helme, 1939; Noell. 1953). The essential conclusion from these studies is that the ganglion cells do not generate any component of the ERG. Although Noel1 (1953) utilized the same procedure to isolate ganglion cell activity as in this study, he did not report the extent of ganglion cell degeneration or the precise conditions under which the ERG was observed. This investigation provided a more rigorous test for determining the influence of the ganglion cells on the ERG. Determination of this influence requires that the ganglion cells be destroyed and functionally inactivated. It was assumed that transection of the optic nerve and waiting for retrograde degeneration of the ganglion cells would fulfill this requirement. Histological examination of retinas 2-7 months after the transection proved, however, that 20% of the ganglion cells still survived. Uppermost in my mind was the reason for the survival of these cells. Incomplete transection was a possibility, but unlikely. The surgery was performed with the utmost care. The resistance to degeneration may be related to the age of the animal. Stone (1966) reported that sectioning one optic tract in 11-21-day-old kittens caused complete retrograde degeneration of the aflected ganglion cells within 24 days, while in the adult cat the ganglion cells showed no signs of degeneration after 10 weeks and required 2 years to degenerate completely. Therefore, it is likely that degeneration would have been more complete for the same time interval following transection of the nerve in younger rabbits. As a result of the incomplete destruction of the ganglion cells one could ask, “Do the surviving 20% in any way influence the ERG?” The experimental protocols contained a variety of tests in order that any influence of the ganglion cells, regardless of the subtlety of this influence, would be detected. In fact, no significant differences between the ERGS of the normal and denervated eyes were ever observed. Hence, in agreement with earlier studies, it is concluded that the ganglion cells do not appear to affect the generation of the a- and b-waves and wavelets of the ERG. ACKNOWLEDGMENTS This investigation was supported in part by a Training Grant (5 TO1 GM00341) from National Institute of General Medical Sciences, U.S. Public Health Service. I wish to thank Dr W. K. Noel1 for his encouragement and criticisms during the course of this work, and Mrs R. Albrecht for her assistance in preparing the histological sections.
PLATE
of ganglion
PLATE
1. Photomicrograph through cells. Retina stained with
2. Photomicrograph
through
the central hematoxylin
region of a normal and cosin.
a comparable
but from a denervated retina. Close packing of ganglion absent due to degeneration of the ganglion cells. Retina
sectlon
of the
retina
illustrating
central
regon,
the close pa&in:!
as shown
m Hate
cells as seen in the normal retina is noticeably stained with hematoxglin and rosin.
I,
ELECTRORETINOGRAM
AFTER
OPTIC
NERVE
SECTION
235
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