We opened the sutured eye, rinsed it with ophthalmic saline, and treated it with Tear-Gel. To keep the eye lids open and the eyes in their forward position, two Minuten pins (Carolina Biological) were inserted through the conjunctiva at the point where it joins the sclera. One pin was positioned above the pupil and the other below so that the pupils were bilaterally symmetric and not dilated. Both pupils were visible from in front of the rat and were not obviously misaligned. Finally, the cortex was penetrated with the recording pipette, taking care to avoid rupture of visible blood vessels.

View larger version (24K):
[in this window]
[in a new window]
| FIG. 2.
Contrasts sensitivity of the 4 pathways of a single P37 rat averaged more than 30 trials for each eye. Each pathway shows the 30-trial average signal ( ) or noise ( ) at each value of contrast. Crossed pathways produce greater signal relative to the noise than the uncrossed pathways. Nondeprived pathways produced a greater signal to noise ratio than did the corresponding deprived pathways.
|
|
Recording
We used Ag-AgCl2 electrodes in glass pipettes containing 0.9% NaCl. The electrodes had a tip diameter of ~8 µm and an impedance of 0.5 to -3.0 M
. The pipette tip was placed in layer II/III by lowering it vertically 600 µm below the surface and then retracting 50 µm to remove the surface dimple. Cortical field potentials from each hemisphere were amplified differentially, displayed on an oscilloscope and fed to a digitizer. We reduced the halothane dose to 0.9%, and collected data when a state of light anesthesia appeared. Good recordings were obtained when electroencephalegram (EEG) lacked obvious slow waves below 10 Hz, inspired halothane was 0.9-1.2%, expired CO2 was 4.3-4.7%, rectal temperature was 37.5 ± 0.5, there was no brain bleeding or bruising, there was little brain swelling, respiration was unlabored, feet and mouth parts were pink, spontaneous breathing was delayed for several seconds when the respiration pump was momentarily turned off, and a pinch of the hind toes evoked a weak withdrawal reflex that was confined to the stimulated limb. When this favorable state appeared, it often persisted for more than an hour, which was more than sufficient time to collect the data. We were unable to obtain the required state of the EEG or to record pattern evoked responses in rats younger than P23 at the time of recording (P19 suture age).

View larger version (43K):
[in this window]
[in a new window]
| FIG. 3.
Response amplitude as a function of contrast. Data are grouped by age, pathway, and deprivation condition. Age is postnatal days at the time of monocular eye suture. All rats were sutured for 5 days prior to recording. : signal (6 Hz component). : noise (average of 4 and 8 Hz component). Error bars are ± SE.
|
|

View larger version (15K):
[in this window]
[in a new window]
| FIG. 4.
Monocular deprivation index grouped by age and pathway. Age categories are 20 days wide and are the same as used in the ANOVA. Circles: individual data. Horizontal lines: group means. Vertical lines: ±SE. The youngest animal was sutured at P19, the oldest at P86. These are the same animals as shown in Fig. 3.
|
|

View larger version (33K):
[in this window]
[in a new window]
| FIG. 5.
Recording locations. Brain diagrams are traced and simplified from Paxinos and Watson (1986) . Vertical lines indicate pipette tracks.
|
|

View larger version (31K):
[in this window]
[in a new window]
| FIG. 6.
Response amplitude following dark rearing from birth to about P90. Conventions as in Fig. 3. Rats were aged P84, P86, and P89 at time of recording.
|
|

View larger version (27K):
[in this window]
[in a new window]
| FIG. 7.
Response amplitude when monocular deprivation followed dark rearing from birth to about P90. Eyelid suture lasted 5 days beginning at P87, P90, and P94.
|
|

View larger version (11K):
[in this window]
[in a new window]
| FIG. 8.
Monocular deprivation index in dark reared and dark reared monocularly deprived rats. Squares: individual data, crossed pathway. Circles: individual data, uncrossed pathway. Horizontal lines: group means. Vertical lines: ±SE. DD: dark reared rats. DDMD: Dark reared then monocularly deprived rats. These are the same rats as shown in Figs. 6 and 7.
|
|
Visual stimulus
The stimulus was a sinusoidal contrast grating of constant luminance displayed on a 19 in. video monitor, 30 cm in front of the eyes. Exactly 10 cycles of the grating appeared on the screen yielding a spatial frequency of 0.13 cycles per degree. The grating reversed phase 6 times per second (3 Hz). The stimulus was presented in a series of 10 s trials; in each trial, the grating contrast started at 1% and increased logarithmically to 70%. Sixty trials were run, switching eyes after each trial. Twenty seconds elapsed between trials. The interleaving of right and left eye stimulation controlled for minor, unnoticed variations of anesthesia state. The experiment was temporarily halted if the state of anesthesia noticeably deteriorated. Deterioration was indicated by a decrease in the high-frequency component of the cortical EEG or an increase in high-amplitude, low frequency components.
Data collection
Responses were recorded simultaneously from both hemispheres using two symmetrically placed pipettes. Prior to each experiment, electrode placement in the binocular portion of primary visual cortex was verified by recording the sweep VEP of the uncrossed pathway of the nondeprived eye (left cortex, left eye). If a response was not obtained, the electrodes were repositioned. Relocation of the recording pipette was always matched stereotaxically by a mirror symmetric relocation on the opposite side. In dark reared animals both eyes were deprived, and this preliminary test could not be applied. To ensure that the ocular occluders were effective, several rats were given a series of trials with both eyes occluded; no responses were observed.
When data collection was complete, the fluid in the recording pipettes was replaced with a solution of 2% pontamine blue in 0.5 M acetate buffer (pH 5.6). Then current (2-10 µA, electrode negative) was passed until a blue spot appeared in the cortex at the pipette tip. The rat was killed with 5% halothane and perfused transcardially with phosphate buffer saline (PBS) followed by 4% paraformaldehyde or 10% formalin. The brain was removed and stored in 4% paraformaldehyde or 10% formalin until sectioned at 30 µm on a cryostat. Sections containing pontamine blue marks were photographed and compared with Paxinos and Watson's (1986) rat atlas for verification of recording sites.
Data analysis
The display and analysis were controlled by an Apple II computer and custom hardware described previously in Norcia et al. (1989)
. The amplitude and phase of the evoked response were calculated with a discrete Fourier transform. The amplitude of the 6 Hz component was defined as the "signal" because this was the rate of phase reversal of the stimulus. The average amplitude of the field potentials at 4 and 8 Hz was defined as "noise." The data consisted of a signal value and a noise value for each value of contrast.
Each animal yielded four data sets of 30 trials each: 1) right eye right cortex (deprived uncrossed), 2) right eye left cortex (deprived crossed), 3) left eye left cortex (nondeprived uncrossed), and 4) left eye right cortex (nondeprived crossed). To describe the average response as a function of contrast for each animal, we averaged all 30 trials for each data set. For statistical analysis, the mean (30 trials) signal to noise ratio (S/N) in the crossed pathway and in the uncrossed pathway of each eye was used as the index of response strength. Changes in the signal to noise ratio were mainly due to changes in signal; the noise varied little as a function of age, pathway, or stimulus contrast.
An index, D, of the monocular deprivation effect was constructed by dividing the S/N of the deprived eye (Sd/Nd) by the S/N of the nondeprived eye (Snd/Nnd) and then taking the common logarithm of the ratio, that is D = log (Sd/Nd / Snd/Nnd). The purpose of taking the logarithm is to cast the deprivation scores into an approximately normal distribution for running parametric tests of statistical significance. A deprivation effect is indicated when D < 0. D was calculated separately for the crossed and uncrossed pathways of each rat. For the purpose of statistical analysis with analysis of variance (ANOVA), the D scores were categorized into four 20-day age bins based on age at suture: P15- P34, P35-P54, P55-P74, and P75-P94. As seen in Fig. 4, the youngest animal successfully tested was P19 at the time of suture, and animals were not evenly distributed within the age categories.
 |
RESULTS |
Pattern-evoked potential
Responses to the counterphase (reversing) grating occurred only if the concurrent EEG lacked obvious low frequencies and had strong signals above 10 Hz. Figure 1 illustrates this point with results from two single trials a few minutes apart, each having a duration of 10 s. These data were collected with both eyes open after the formal experiment on this animal had been completed. On trial 65, the EEG showed robust high frequencies and lacked slow waves; concomitantly there was a clear response to the counterphase grating. As contrast increased from 1 to 70%, the 6 Hz component of the EEG rose above the noise, and the phase of the response became locked to the stimulus. The growth of the 6 Hz component can actually be seen in the raw EEG record. On trial 68 the high-frequency components of the EEG were weaker and slow waves were more prominent. On this trial there was little, if any, response.
The optimal state of the EEG was more easily produced with a mixture of halothane and nitrous oxide than with other anesthetic agents we tested. In pilot experiments we tried and rejected urethane, chloral hydrate, ketamine/xylazine, fentanyl/fluanisone, sufentanil/fluanisone, pentobarbital, and methohexital. Only halothane and nitrous oxide permitted minute to minute adjustments of anesthesia level. The proper EEG occurred at a halothane dose of 0.9-1.2% carried in a 65:35% mixture of NO2-O2. In animals younger than P24 at the time of recording (P19 suture), we failed to obtain the proper EEG and also failed to obtain reliable evoked responses. We were, however, able to obtain occasional weak responses contralateral to the nondeprived eye in some animals between P19 and P24 at the time of recording.
Crossed and uncrossed responses
In the formal experiment, the stimulus was always presented monocularly and responses were recorded simultaneously in both hemispheres. Crossed responses were responses recorded in the cortex contralateral to the stimulated eye, and uncrossed responses were recorded in the cortex ipsilateral to the stimulated eye. Perhaps because there are more crossed than uncrossed fibers in the rat visual pathway, amplitudes of the crossed responses could be as much as threefold greater than the amplitudes of the uncrossed response (Fig. 2).
The contrast threshold and response amplitude can be estimated from the averaged data for each animal (e.g., Fig. 2). In this study we did not analyze the threshold data, instead concentrating on the amplitude. We neglected the threshold for two reasons. First, it was not our aim to measure the contrast sensitivity in the rat. Therefore we did not repeat the measurements at values of spatial frequency other than 0.13 cycles/deg. Second, at 0.13 cycles/deg the contrast threshold does not appear to be very sensitive to the effects of monocular deprivation. Indeed, the contrast threshold was typically about 7% independent of age, pathway, or deprivation. The exception was when there was little or no response to the grating and therefore no threshold at all. This type of exception was frequently seen in young monocularly deprived animals.
Monocular deprivation effect
Average response amplitude was strongly influenced by 5 days of monocular deprivation during the sensitive period. We measured the monocular deprivation effect separately for the crossed and uncrossed pathways. As seen in the example of Fig. 2, the signal from the nondeprived eye is greater than the signal from the deprived eye in both crossed and uncrossed pathways; the noise is similar in both pathways. Figure 3 shows the 30 trial means and standard errors for each age group. Figure 4 shows the index of the monocular deprivation effect, D, for both individuals and groups, D = log (Sd/Nd / Snd/Nnd). In the crossed pathway, D was <0 in 12 of 13 rats in the two youngest age groups. In the uncrossed pathway, D was <0 in 11 of 13 animals. The individual scores in Fig. 4 indicate that the monocular deprivation effect occurred in animals as young as P19 and in animals as old as P50. There was no consistent monocular deprivation effect in the two older age groups (n = 9), in which the youngest animals were sutured at P55. Recording locations were successfully marked with pontamine blue for 14 of 22 rats summarized in Figs. 3 and 4, and in all cases the pipette tip was within layer II/III of the binocular area of V1 (Fig. 5).
We tested the statistical significance of the effect of monocular deprivation using ANOVA. The dependent variable was the index of the monocular deprivation effect, D. The two independent variables were age and pathway. Pathway was categorized as either crossed or uncrossed. Age was categorized into four 20-day bins starting at age P15. The data and its categorization are shown in Fig. 4. The effect of age on the monocular deprivation effect was significant (P = 0.006). The effect of the pathway was also significant (P = 0.03), i.e., the monocular deprivation effect was larger in the uncrossed pathway. The age by pathway interaction was not significant (P = 0.9). Bonferroni paired comparisons were performed between the groups deprived between P75 and P94 and each of the three younger age groups. The difference was significant for the P15 to P34 group (P = 0.007) and P35 to P54 group (P = 0.009), but not significant for the P55 to P74 group (P = 0.8).
Dark rearing
The amplitudes of sweep VEPs from rats reared in total darkness (DD) for about 90 days (Fig. 6) were similar to the amplitudes from nondeprived eyes in monocularly sutured rats (Fig. 3). Thus, lack of visual experience, by itself, did not diminish the amplitude of the sweep VEPs. The monocular deprivation effect reflects the unequal use of the two eyes.
Monocular deprivation following dark rearing
Dark rearing had a marked impact on plasticity. Monocular eyelid suture following 90 days of dark rearing (DDMD) produced a clear monocular deprivation effect in both crossed and uncrossed pathways (Fig. 7). This contrasts markedly with the absence of a monocular deprivation effect in LD rats of similar age (Figs. 3 and 4). The amplitudes of the sweep VEPs in the deprived pathways of the DDMD rats were reduced to a level well below the amplitudes following dark rearing alone (Fig. 7 cf. Fig. 6). Comparing the DDMD rats with the DD rats, the deprivation index was significantly more negative in the DDMD animals than in the DD animals (Fig. 8; t-test, 1-tail, P < 0.003, n = 3, crossed and uncrossed averaged). In this test the right eye was the "nominal" deprived eye in the DD rats and the "actual" deprived eye in the DDMD rats. The recording locations of all six dark reared rats were successfully marked, and all were within the binocular portion of the primary visual cortex.
 |
DISCUSSION |
Summary of results
Our aim was to assess in the rat the susceptible period for the monocular deprivation effect and the effect of dark rearing on this period. Eyelid suture for 5 days beginning between P19 and P50 produced a monocular deprivation effect; that is, lid suture reduced the amplitude of the sweep VEP in the binocular visual cortex in response to stimulation of the deprived eye. Lid suture beginning at P55 or thereafter did not produce a consistent monocular deprivation effect. Rats reared in continuous darkness from birth to about P90 showed a robust monocular deprivation effect. Dark rearing by itself did not diminish the amplitude of sweep VEPs at the spatial frequency tested.
Our experiments did not establish an age of onset for the critical period. Following eyelid suture from P19 to P24 we observed a monocular deprivation effect, but we were unable to record reliable responses in rats younger than P24. This failure may derive from difficulty in establishing the proper level of anesthesia in young animals, or it may derive from some type of immaturity of cortical connections that prevents vigorous responses to a counterphase grating. Intracortical connections are probably required for normal receptive fields in the primary visual cortex (Allison et al. 1995
; Gilbert and Wiesel 1989
; Weliky et al. 1995
). Immaturity of receptive field organization, presumably because of the immaturity of intracortical connections, was observed in both rats and mice that are younger than their 5th postnatal week (Fagiolini et al. 1994
; Gordon and Stryker 1996
).
Comparison with previous results
Previous investigations of the monocular deprivation effect in rats and mice have reported a critical period that is shorter than observed here. Fagiolini et al. (1994)
found that 10-day deprivations beginning in the 4th postnatal week (at P23) produced a deprivation effect, but that deprivations beginning in the 5th week (at P33) no longer did so. In mice, Gordon and Stryker (1996)
found that lid suture beginning during the 4th week produced an effect, but that sutures in the 6th week (P36) did not. In our experiments, deprivations initiated throughout the 6th and 7th weeks (P37-P50), including four after P41, consistently showed a monocular deprivation effect (Fig. 4).
Several differences between our methods and those of previous investigators can easily account for the differences in the observed length of the critical period. First are the differences in anesthesia. Fagiolini et al. (1994)
used urethan, Gordon and Stryker (1996)
used barbiturate with chlorprothixene, and we used halothane with nitrous oxide. Because we found it difficult to obtain desynchronized EEGs while using urethan or barbiturates, we think it unlikely that our results could be obtained with these anesthetics. Second, our electrodes were probably always located in layers II/III, whereas the previous studies sampled cells from all cortical layers. Several investigators believe that neurons of layers II/III retain plasticity longer than do cells of layer IV (Daw et al. 1992
; LeVay et al. 1980
). In addition, long-term potentiation can be produced at later ages in layers II/III than in deeper cortical layers (Kirkwood and Bear 1994
). Third, we measured the amplitude of sweep evoked potentials rather than the ocular dominance of single units. The sweep VEP method stimulates the retina with a reversing grating and simultaneously records the summed current from a large population of visual neurons. The population responses are generated by postsynaptic potentials as well as action potentials. In contrast, the single unit method uses oriented edge stimuli and gives equal weight to cells that spike vigorously and those that spike weakly; it completely ignores cells that do not spike. Finally, the results of Gordon and Stryker are on mice, not rats. Whereas mice and rats are both rodents, they are different species, and there is no reason that their critical periods should be identical.
The two methods both yield important information, but the information they yield is not quite identical. Our data show that a monocular deprivation effect exists in the 6th and 7th postnatal weeks. An advantage of the sweep VEP method is that it permits rapid assessment of the monocular deprivation effect in individual animals. In addition, there is good correspondence between contrast thresholds determined with sweep VEPs and contrast thresholds determined psychophysically in conscious humans. Our observation that the sweep pattern evoked potential requires a desynchronized state of the EEG validates the opinion that it is closely linked to functional vision.
Multiple critical periods
Prior studies have shown that there are many plastic periods. For example, in the cat the plastic period for morphological segregation of thalamocortical afferents occurs earlier than the plastic period for ocular dominance in the extragranular layers (Daw et al. 1992
; LeVay et al. 1980
). The plastic period for specification of ocular dominance appears to be later than the plastic period for specification of directional selectivity (Berman and Daw 1977
). In the rodent somatosensory cortex, the plastic period for specification of the whisker barrels in layer IV is earlier than the plastic period for specification of receptive fields of the overlying neurons in layer II/III (Fox 1992
; Woolsey and Wann 1976
). To effectively search for the cellular, molecular, and biochemical bases of visual plasticity, it is necessary to have a detailed description of the various critical periods. Our study provides such a description for one of these critical periods.