The excised skull flap was replaced over the tectum. ... The skull flap was then lifted off. During the ..... In CA1 of hippocampus, the temporal window for LTP.
J Comp Physiol A (1992) 171:207-212
Joun
l of Neural, and
PtanC~y
9 Springer-Verlag 1992
Xenopus exhibits seasonal variation in retinotectal latency but not tecto-isthmo-tectal latency Warren J. Scherer and Susan B. Udin Department of Physiology, State University of New York at Buffalo, Buffalo, NY 14214, USA Accepted May 4, 1992 1. The tectum of Xenopus receives visuotopic input from both eyes. The contralateral eye's projection reaches the tectum directly, via the optic nerve. The ipsilateral eye's projection reaches the tectum indirectly, via the nucleus isthmi and isthmotectal projection. 2. Because of the multi-synaptic nature of the ipsilateral pathway, there is an inherent delay between the time that information from the contralateral eye reaches the tectum and the time that information from the ipsilateral eye arrives at the tectum. The length of the intertectal delay is a function of the latencies of the contralateral and ipsilateral pathways. 3. The length of this intertectal delay has functional, as well as developmental, implications with regard to the role of N-methyl-D-aspartate receptors in tectal cell activity and development of orderly synaptic connections. 4. We have found that the latencies of the contralateral and ipsilateral pathways exhibit a seasonal variation, increasing during the winter months. The increases of both latencies during the winter were of similar magnitude, indicating that there were no significant changes in intertectal delay. The seasonal alteration in contralateral latency was not affected by dark-rearing and was affected to only a minor extent by a week-long alteration of ambient temperature. Summary.
Key words: Tectum
- Binocular
-
Xenopus
-
layed to the tectum through a midbrain structure, the nucleus isthmi, by way of the retino-tecto-isthmo-tectal (ipsilateral) projection. Because of the multi-synaptic nature of the tecto-isthmo-tectal pathway in Xenopus, there is a delay between the onset of visually-elicited contralateral and ipsilateral input to the tectum. Information from the ipsilateral eye arrives at the tectum later than information from the contralateral eye. The magnitude of the intertectal delay may be important for the development and function of orderly topographic maps. During the course of measuring the contralateral and ipsilateral latencies, it was noted that they exhibited a seasonal variation, lengthening during the winter months. If a significant seasonal variation in the latencies of all or some of the projections were to exist, this variation could alter intertectal delay. In order to test whether significant seasonal variations in latency exist, and if so, whether intertectal delay varies, we measured tectal response latencies to visual stimuli (light flashes) presented through either the ipsilateral or contralateral eye. Laten-
Retina
Seasonal
variation - Latency
Introduction
In the African clawed frog Xenopus laevis, each eye relays a visuotopic representation to both lobes of the optic tectum (Fig. 1). Each eye projects directly to the opposite tectum via the retinotectal (contralateral) projection while visual information from the ipsilateral eye is reAbbreviation." N M D A (N-methyl-D-aspartate) Correspondence to." S.B. Udin
Tectum
Nucleus Isthmi
Fig. 1. Projections from the right eye are relayed directly to the left tectal lobe and indirectly to the right tectal lobe. The left optic nerve was cut and contralateral latencieswere recorded from the left tectal lobe while ipsilateral latencies were recorded from the right tectal lobe
W.J. Scherer and S.B. Udin: Xenopus binocular latencies
208
cies measured during the winter (January and February) and summer (July and August) months were compared to determine if latency was affected by season and if this change had any effect on intertectal delay.
Materials and methods
Experimental animals. Xenopus laevis were obtained from a labreared colony. Animals aged 8 to 12 months after metamorphosis were used. Light-reared animals were housed in a laboratory with large windows and ample access to natural light. In addition to natural light, the animals were exposed to fluorescent illumination during working hours ( ~ 8:00 AM- 6:00 PM) at least 5 days per week. The water in the animals' tanks was ~ 18 ~ during the winter and 28 ~ during the summer. Dark-reared animals were put into complete darkness as tadpoles and were not exposed to light until approximately 2 days prior to the recording session. Light- and dark-reared animals were fed identical diets and received similar care.
Surgical procedures. Twenty-four h prior to the recording session, the frog was anesthetized and the left optic nerve was transected in order to facilitate detection of the weak ipsilateral units in the right tectal lobe by eliminating the stronger contralateral units. Both tectal lobes were exposed. The excised skull flap was replaced over the tectum. The skin was sutured, and the animal was allowed to recover. On the day of recording, succinylcholine (Sigma, 0.6 mg/g body weight) was used to paralyze the animal. Animals were paralyzed rather than anesthetized because general anesthesia prevents reliable recording of single ipsilateral units. Also, general anesthesia increases latency substantially, rendering measurements unreliable (unpublished observations). The skin overlying the tectum was topically anesthetized with lidocaine (1 mg/ml in methanol, Sigma) prior to reflecting it. The skull flap was then lifted off. During the recording session, the skin of the left foot (where the ground lead is attached) and the area surrounding the exposed tectum were anesthetized with lidocaine. Lidocaine was periodically applied to these areas during the recording session. Following the recording session, animals were euthanized by subcutaneous injection of 2% ethyl m-aminobenzoate (Sigma).
Electrophysiological recording and data collection. As mentioned previously, prior to recording, the left optic nerve was transected. This procedure allowed us to record exclusively contralateral responses from the left tectum and ipsilateral responses from the right tectum. Without this procedure, it is difficult to isolate ipsilateral activity from contralateral activity because of the enormous disproportion between contralateral units and ipsilateral units (approximately 50: 1) (Udin and Fisher 1985). Recording was performed using Wood's metal electrodes tipped with gold and platinum (Dowben and Rose 1953). Signals were amplified 1000 x using an A - M Systems Model 1800 microelectrode amplifier. Recordings were performed at approximately 25 ~ during both winter and summer. A drop of mineral oil was placed on the tectal surface to prevent dehydration. A rostrolateral tectal locus representing a receptive field on the tangent screen was chosen and the electrode was lowered into position until a slight dimpling of the tectal surface was observed. The electrode holder was then gently tapped to advance the electrode into the neuropil. In the case of ipsilateral responses, this procedure assured that all units recorded were from the deeper lamina of crossed isthmotectal axons. Contralateral units were recorded in the left tectal lobe and ipsilateral units in the right lobe. Contralateral and ipsilateral latency measurements were made on separate trials; in each animal, data were collected from up to 5 locations in the ipsilateral lobe and 5 locations in the contralateral lobe, for a maximum of 10 data sets per frog. Once the electrode was in place, a light illuminating a tangent screen located 20 cm
from the frog was flashed using a Uniblitz shutter in order to determine if visually induced activity was present. If activity was heard on the audio monitor, the receptive field center was located by moving a 5 ~ x 10~ black rectangle over the surface of the screen. If no activity was detected, the depth of penetration was changed by approximately 10 um. If activity still could not be detected, a new electrode penetration was made at a distance of 100 lam from the previous penetration site. When a receptive field was identified, a spot of light 30 cm in diameter was centered on the receptive field. The light intensity at that position was assessed using a photodiode placed in the center of the receptive field on the screen and was adjusted such that the same flux was presented to all analyzed cells. The photodiode and black rectangle were then removed from the screen. The amplified electrophysiological signal was analyzed using a MacADIOS analog-to-digital board interfaced to a Macintosh Ilcx computer. The MacAdios Manager data collection program was triggered for a single sweep to permit the setting of a threshold for discriminating the activity of the single unit with the largest spikes. This method was used for detecting ipsilateral responses on the right tectal lobe and contralateral responses on the left tectal lobe. To begin the experiment, the shutter and computer were triggered simultaneously using the shutter driver/timer. Each experiment consisted of l0 sequences of 600 ms of dark followed by 5 s of light. Data consisting of the numbers of spikes/ms that exceeded threshold height for each run were collected for 200 ms following the light O F F and were stored in sets of 200 bins of 1 ms duration. The sum of all 10 runs was stored as a total post-stimulus time histogram. Contralateral latency is defined as the time delay between light offset and first bin containing 5 or more spikes per l0 stimulus presentations for units recorded in the tectum contralateral to the intact eye. Ipsilateral latency is defined similarly for units recorded ipsilateral to the intact eye. Intertectal delay was computed by subtracting the latency of the each ipsilateral response from the mean latency of the contralateral responses for animals of each age range. Intertectal delay therefore is a measure of tecto-isthmo-tectal conduction time.
Results We
compared
the
response
latencies
of
lab-reared
Xenopus f r o g s d u r i n g t h e w i n t e r ( J a n u a r y a n d F e b r u a r y ) a n d s u m m e r ( J u l y a n d A u g u s t ) m o n t h s . A s e x p e c t e d , the
'70
6O
5O
'4o
g 2O 10 0
LightReared
DarkReared
18~
Light-
Dark-
Reared
Reared
28~
Fig. 2. Comparison of the average retinotectal and isthmotectal latencies of light-reared, dark-reared, and temperature-adjusted groups during summer and winter :t: S.D. I , Contralateral latency; [~, Ipsilateral latency
W.J. Scherer and S.B. Udin: Xenopusbinocular latencies
209
Table 1. Latency to onset response to light OFF Contralateral Summer Summer Dark-reared Summer 18~ C Winter Winter Dark-reared Winter 28~ C
tpsilateral
12
Intertectal Delay
33.4+ 3.1 (65) 41.54-3.5 (39) 34.14- 1.7 (22) 42.6+3.5 (20)
8.2+3.5 8.5:t:3.5
35.74- 1.1 (17) 42.94-3.8 (15)
7.24-2.9
43.3:t: 3.7(11) 53.1+3.4(12) 48.44- 10.8 (11) 58.04-7.8 (17)
9.8+3.0 9.64-7.8
I0
@
o'l
40.0+ 3.8 (25) 48.6+6.5 (14)
8.6+6.5
0 a u
I
Values are given as means + S.D. in ms (number of units) |
ipsilateral latencies were longer than the contralateral latencies during both seasons. Our data indicate that during the winter months, contralateral and ipsilateral latency increase in Xenopus. The data comparing summer and winter show that both contralateral (P 0 . 0 5 , Student's t-test) indicating that the intertectal delay remains unchanged. Because the ipsilateral and contralateral latency measurements are independent of one another, one cannot simply average the differences of corresponding pairs of ipsilateral and contralateral latency measurements. Instead, we determined the average contralateral latency within a group and subtracted it from each ipsilateral latency within the same group. These differences of ipsilateral and average contralateral latency were then used to compare intertectal delay between groups. To test whether the seasonal change in contralateral latency was related to variations in the light-dark cycle of the animals, we c o m p a r e d the contralateral latencies of Xenopus reared in a normal light-dark cycle with those reared in the dark. We found that during both the summer and winter, contralateral latency did not differ significantly in light-reared as opposed to dark-reared animals (Student's t-test, summer P > 0.1, winter P > 0 . 0 5 , see Fig. 4 and Table 1). In order to determine if the seasonal effects on contralateral latency could be reversed by changes in ambient temperature, we altered the temperature of the tank water and then recorded latency measurements 7 to 10 days later. We were interested in whether frogs living in cool water in the summer would have retinotectal laten-
0
~0
I00 T~me,
150
200
msec
J
12
8
6
4
9.
0 0
50
i00 T:Lme,
150
200
msec
Fig. 3. Post-stimulus-time histogram comparing the overlap of the
averaged retinotectal and isthmotectal firing for summer and winter Xenopusafter stimulation at time 0 ms. Spike numbers were collected in 1 ms bins, and the number of spikes/bin was summed for l0 successive OFF sequences. Resultant numbers from all penetrations in summer or winter light-reared animals were averaged. Vertical lines indicate mean latencies of the summer measurements and are used to help visualize the displacement of the mean winter latency measurements. The time corresponding to an average spike number of 5 was chosen as the latency threshold for each group, m, Contralateral response; [], Ipsilateral response
cies that were similar to winter values and whether frogs living in w a r m water during the winter would have retinotectal latencies similar to those measured during the summer. The water temperature during the winter was increased f r o m 18 ~ C to 28 ~ C. Likewise, during the summer, the water temperature was decreased to 18 ~ C. The chosen alterations in temperature were based on
W.J. Scherer and S.B. Udin: Xenopus binocular latencies
210
ing of binocular inputs in Xenopus?In the adult frog, the retinotectal and isthmotectal maps are in alignment with one another. However, during development, the visuotopic map formed by the retinotectal projection is essentially complete before the isthmotectal map is established (Udin and Fisher 1985; Grant and Keating 1986). It is hypothesized that the developing isthmotectal axons are dependent upon temporally-correlated activity cues from the contralateral eye in order for the isthmotectal map to correctly align to the retinotectal map. In Xenopus, visual activity is crucial for the development of orderly isthmotectal topography (Grant and Keating 1986)9 Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, located on tectal cell dendrites (McDonald et al. 1989), may mediate the cellular events responsible for the formation of orderly tectal connections (Scherer and Udin 1989). However, it has been shown that NMDA receptor activation requires temporal correlation of convergent afferent activity9 That is to say, there is a temporal window during which the afferents must fire if NMDA receptors are to be activated. For binocular projections in Xenopus, the convergent afferent pathways correspond to the retinotectal and isthmotectal inputs, originating from each eye, that synapse onto tectal cells. If the inputs to a given tectal cell originate from a part of each retina that is viewing the same position in space, then the activity at the tectal cell will be temporally correlated, and the isthmotectal synapse will be stabilized. If the inputs originate from a part of each retina that is not viewing the same position in space, then the inputs are not temporally correlated and the isthmotectal synapse will not be stabilized, or may be actively destabilized. There are two models, both of which require temporally correlated activity, which could explain the role of NMDA receptors in the development of orderly isthmotectal topography in Xenopus(Fig. 4). The first model
average water temperatures recorded during the summer and winter. Latencies of animals shifted to 18~ C during the summer differed significantly from those of winter animals (P < 09 Student's t-test). Likewise, latencies of animals shifted to 28 ~ C during the winter differed significantly from those of summer animals (P
