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The Journal of Neuroscience, December 15, 1999, 19(24):10829-10842
Synaptic Density in Geniculocortical Afferents Remains Constant after Monocular Deprivation in the Cat
Michael A. Silver and Michael P. Stryker W. M. Keck Center for Integrative Neuroscience and Neuroscience Graduate Program, Department of Physiology, University of California, San Francisco, California 94143-0444
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Monocular eyelid closure in cats during a critical period in development produces both physiological plasticity, as indicated by a loss of responsiveness of primary visual cortical neurons to deprived eye stimulation, and morphological plasticity, as demonstrated by a decrease in the total length of individual geniculocortical arbors representing the deprived eye. Although the physiological plasticity appears maximal after 2 d of monocular deprivation (MD), the shrinkage of deprived-eye geniculocortical arbors is less than half-maximal at 4 d and is not maximal until 7 d of deprivation, at which time the deprived arbors are approximately half their previous size. To study this form of plasticity at the level of individual thalamocortical synapses rather than arbors, we developed a new double-label colocalization technique. First, geniculocortical afferent arbors serving either the deprived or nondeprived eye were labeled by injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin into lamina A of the lateral geniculate nucleus. Then, using antibodies to synaptic vesicle proteins, we identified presynaptic terminals within the labeled arbors in layer IV of the primary visual cortex. Analysis of serial optical sections obtained using confocal microscopy allowed measurement of the numerical density of presynaptic sites and the relative amounts of synaptic vesicle protein in geniculocortical afferents after both 2 and 7 d of MD. We found that the density of synapses in geniculocortical axons was similar for deprived and nondeprived afferents, suggesting that this feature of the afferents is conserved even during periods in which synapse number is reduced by half in deprived-eye arbors. These results are not consistent with the hypothesis that a rapid loss of deprived-eye geniculocortical presynaptic sites is responsible for the prompt physiological effects of MD.
Key words: monocular deprivation; ocular dominance plasticity; presynaptic terminal; synaptic vesicle protein; cortical plasticity; primary visual cortex; lateral geniculate nucleus
INTRODUCTION
Cells in the visual cortex lose their response to an eye that was deprived of vision during a critical period in early life (Wiesel and Hubel, 1963b
). This phenomenon, called ocular dominance plasticity, has been studied as a model for experience-dependent changes in the CNS for over 30 years, but fundamental questions concerning the mechanisms underlying the physiological and anatomical plasticity induced by monocular deprivation (MD) remain unanswered. Two days of MD during the critical period for plasticity in kittens causes a robust and saturating shift in visual responsiveness such that primary visual cortical neurons respond preferentially to the nondeprived eye and very poorly or not at all to the deprived eye (for review, see Hensch, 1996
One possible mechanism for the rapid loss of cortical responsiveness to the deprived eye caused by 2 d of MD is the loss of presynaptic neurotransmitter release sites from the geniculocortical axon branches that are destined to be eliminated. If presynaptic sites are specifically removed from some branches of deprived-eye arbors, their absence might provide a signal for the retraction of those branches. This hypothesis could be tested by measuring the numerical synaptic density (the number of presynaptic sites per unit area of geniculocortical axon) in deprived geniculocortical afferents after 2 or 7 d of MD.
The predicted result of this hypothesis is as follows: 2 d of MD should result in a decrease in numerical synaptic density in deprived-eye afferents. Specifically, if presynaptic sites are selectively removed from deprived-eye axon branches that are destined to be retracted, then the deprived-eye synaptic density should become ~50% of its former value. With longer periods of MD, we know that many of the branches of the deprived-eye geniculocortical axons retract. If it is only those deprived-eye axon branches which are devoid of presynaptic sites that are withdrawn, then the synaptic density of the deprived-eye projection will return to normal. Because there is little morphological change in the geniculocortical arbors serving the nondeprived eye over this time course (Antonini and Stryker, 1996
More generally, one may hypothesize that the temporal mismatch between the loss of visual responsiveness to deprived-eye stimulation and the morphological withdrawal of deprived-eye geniculocortical afferent arbors may be accounted for by a loss of half of their synapses after 2 d of MD (although not necessarily only from those branches that will eventually be retracted), resulting in a transient halving of the numerical density of their presynaptic sites, followed by a homeostatic restoration of synaptic density to its long-term stable value, once half the branches of deprived-eye arbors have fully retracted after 7 d of MD. To test this hypothesis, we developed a method for identifying presynaptic sites in deprived and nondeprived geniculocortical afferents after MD, for measuring their relative numerical density, and for measuring the relative amounts of synaptic vesicle protein (SVP) per unit area of axon. Our results clearly reject the hypothesis and reveal instead a remarkable conservation of synaptic density throughout a period of dramatic axonal rearrangement, resulting in a twofold difference between the total numbers of presynaptic sites in deprived and nondeprived axons.
Some of these data have been published previously in abstract form (Silver and Stryker, 1997
MATERIALS AND METHODS
Labeling of geniculocortical afferents and monocular deprivation. A total of 10 kittens were used for this study. All of them were from the breeding colony at the University of California, San Francisco, and had normal pigmentation. All procedures were performed in accordance with the Society for Neuroscience's Policy on the Use of Animals in Neuroscience Research and were approved by the Committee on Animal Research, University of California, San Francisco.
On postnatal day 26-30 (P26-30), the anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin (Pha-L) (Gerfen and Sawchenko, 1984
1 · hr
A bilateral craniotomy was performed above the lateral geniculate nuclei (corresponding approximately to Horsley-Clarke stereotaxic coordinates AP 1-AP 8 and ML 6-ML 10). The dura was retracted, and a tungsten microelectrode was inserted vertically into the cortex overlying the LGN. As the electrode was lowered into the brain, the eyes were monocularly stimulated with a flashlight, and electrophysiological visual responses were recorded. A penetration was considered to traverse the binocular portion of the LGN if monocular visual responses corresponding to the known response properties of the eye-specific LGN laminae were observed (Sanderson, 1971
After a recording site was identified, the tungsten electrode was removed, and a glass pipette (tip diameter 10-15 µm) filled by suction with a solution of 2.5% Pha-L (Vector, Burlingame, CA) in sodium PBS was lowered along the same trajectory. Visual responses were recorded through the tip of the pipette, and the pipette was advanced until contralateral visual responses were obtained. Pha-L was then iontophoretically injected into lamina A of the LGN using a 2000 V compliance current source device (Stoelting, Wood Dale, IL) that delivered 7 sec pulses of 8 µA positive current at 50% duty cycle for 4 min. Approximately three separate Pha-L injections were made in each LGN. After the scalp incision was sutured, kittens received topical application of lidocaine ointment to the wound margins as well as subcutaneous injections of the analgesic butorphenol (total dose = 0.2-0.4 mg/kg). Animals were administered oral amoxicillin (14 mg/kg) twice a day for several days after the surgery. Monocular lid suture was performed under isoflurane anesthesia for some animals on either P33 (7 d MD) or P38 (2 d MD).
Perfusion and tissue processing. After a 10-14 d period during which the Pha-L was transported to the geniculocortical afferents in layer IV of the cortex, kittens received an intraperitoneal injection of pentobarbital (100 mg/kg) to induce deep anesthesia on P40 and were perfused transcardially with 1 l of ice-cold 0.1 M sodium phosphate buffer, pH 7.4, followed by 1 l of ice-cold 4% paraformaldehyde in the same buffer. The brain was blocked in the coronal plane at AP + 10 mm, and the LGNs and primary visual cortices of both hemispheres were removed from the skull in a single block of tissue. After 1 hr of post-fixing at 4°C, each primary visual cortex was dissected from the block. The pia was removed from each tissue block using fine forceps, and the tissue was embedded in 5% agar in distilled water and sectioned on a Vibratome. The block containing the LGNs was cut into 80-µm-thick coronal sections, and the cortical blocks were sectioned at 70 µm in the coronal plane. Because the location of the injection sites and cortical labeling were unknown until after the immunohistochemistry was performed, the entire LGN and primary visual cortex of each hemisphere were completely sectioned during the 16-48 hr after perfusion. Sections were collected in 20 mM potassium phosphate with 150 mM sodium chloride [potassium PBS (KPBS), pH 7.4].
Immunohistochemistry. Standard diaminobenzidine (DAB) immunohistochemistry was performed to determine the location of Pha-L-labeled neurons within the LGN. Although every section of the LGN was processed with DAB, only every seventh section of the visual cortex was processed this way. This allowed the determination of the anterior and posterior boundaries of the Pha-L-labeled geniculocortical afferents in the visual cortex but still left most of the sections containing Pha-L antigen available for double-label immunofluorescence. For DAB immunohistochemistry, sections were incubated for 1 hr at room temperature in a blocking solution consisting of 20 mM KPBS, 2.5% bovine serum albumin (Sigma, St. Louis, MO), 0.5% Triton X-100, 3% normal rabbit serum (Vector), and 0.05% thimerosal (Sigma). They were then transferred to a primary antibody solution consisting of the blocking solution and a goat anti-Pha-L antibody (Vector; used at 1:1000 dilution) and incubated overnight at 4°C. After three 10 min washes with KPBS, standard procedures were followed using the Vector ABC peroxidase anti-goat IgG kit. KPBS was used in all solutions except the final three 10 min washes preceding the DAB reaction. Tris buffer (10 mM) with 150 mM sodium chloride was used for these washes and for the DAB reaction itself. The DAB reaction solution contained 0.5 mg/ml DAB, 6.95 mg/ml nickel ammonium sulfate, and 0.01% hydrogen peroxide. After the DAB reaction, sections were mounted from tap water onto gelatinized slides, cleared with xylenes, and coverslipped. An animal was only used for this study if both LGNs contained Pha-L-labeled neurons located in the A lamina of the LGN. In addition, labeled neurons had to be located in the region of the LGN in which the binocular portion of the visual field is represented. Label resulting from injection sites in the monocular segment of the LGN or in the medial intralaminar nucleus was excluded from further analysis. Pha-L that was anterogradely transported to axon terminals in primary visual cortex often labeled distinct ocular dominance columns in layer IV. After the anterior-posterior boundaries of the cortical label were determined, the remaining sections from this portion of primary visual cortex were used for double-label immunofluorescence.
Immunofluorescence. Agar embedding medium was removed from the sections to decrease the volume they occupied in the antibody solutions. The blocking solution was as described above except that 3% horse serum (Vector) was added. Sections were blocked for 1 hr at room temperature and then transferred to blocking solution containing goat IgG anti-Pha-L antibody (Vector; dilution of 1:500) and one of the following primary antibodies: mouse IgG monoclonal anti-synaptophysin (Boehringer Mannheim, Indianapolis, IN; dilution of 1:10), mouse IgG monoclonal anti-synaptotagmin antibody (Matthew et al. 1981
Sections were incubated in primary antibodies for 48 hr at 4°C, washed three times for 10 min each in KPBS, and transferred to a secondary antibody solution containing biotinylated horse anti-mouse IgG (Jackson, West Grove, PA; dilution of 1:200) and Cy3-conjugated rabbit anti-goat IgG (Jackson; dilution of 1:100) in blocking solution. After overnight incubation at 4°C, sections were washed three times in KPBS for 10 min each and then transferred to a solution containing Cy5-conjugated streptavidin (Jackson; dilution of 1:100) in KPBS with 0.05% thimerosal and then incubated overnight at 4°C. After a final series of three washes in KPBS for 10 min each, sections were mounted on gelatinized microscope slides from tap water. A mounting medium consisting of 5% n-propyl gallate (Sigma) and 10% (v/v) 0.1 M sodium PBS in glycerol was used, and coverslips were sealed with clear nail polish.
Confocal microscopy and image processing. Image collection and processing were performed as described in . Images were collected as pairs of sequential optical sections separated by 1 µm in a stack. Presynaptic sites were considered to be colocalized if each pixel in the cluster of SVP label in the reference section was located entirely within the boundaries of the corresponding Pha-L-labeled axon branch in the reference section.
Quantification of colocalization. Colocalization indices were designed to allow for quantitative comparisons of either the number of colocalized presynaptic sites per unit area of labeled axon (numerical synaptic density) or the average intensity of these sites per unit area of labeled axon (SVP density). For numerical synaptic density, the index is simply the number of colocalized presynaptic sites in the field divided by the number of Pha-L-positive pixels in the portions of the geniculocortical axon branches localized to the reference section. For SVP density, the colocalization index is defined as:
where p = the total summed intensities of the SVP pixels within colocalized presynaptic sites in the field, a = the number of Pha-L positive pixels in the portions of the geniculocortical axon branches localized to the reference section, and u = the average intensity of the SVP label in the neuropil (the entire field except for cell somata and blood vessels). Essentially, this measure gives the average intensity of SVP label per unit area of labeled axon, corrected for many sources of variability that are unrelated to monocular deprivation (.
Because all of the Pha-L injection sites were located entirely within lamina A of the LGN, the Pha-L-labeled geniculocortical axons in a given field represent only the eye contralateral to the hemisphere containing the field. In each monocularly deprived animal, one hemisphere contained only deprived-eye Pha-L-labeled geniculocortical axons, whereas the other hemisphere contained only nondeprived-eye Pha-L-labeled axons. For animals that were not deprived, one hemisphere had labeled axons representing the contralateral left eye, and the other had labeled axons serving the contralateral right eye. Each field generated one value for the numerical synaptic density and one value for SVP density, and the fields were averaged to compare deprived versus nondeprived (or left vs right) synaptic densities or SVP densities for each animal. Additionally, all deprived and all nondeprived fields were averaged across animals to allow for statistical comparisons incorporating the entire data set.
Descriptive statistics. Individual fields vary widely in the number of Pha-L-labeled axon branches that they contain. Colocalization indices computed from fields that have dense axon labeling are more reliable measures of colocalization than fields that have sparse labeling. Therefore, when averaging fields to compute a group colocalization index, each field was weighted by the number of Pha-L-positive pixels localized to the reference section for that field, as was the SD (Silver and Stryker (Silver and Stryker, Hunter and Schmidt, 1990
Weighted averages of deprived and nondeprived fields were compared using a contrast index, defined as:
This contrast index would have a value of 0 if the average colocalization indices for the deprived eye and the nondeprived eye were equal. If all geniculocortical presynaptic sites were located exclusively within nondeprived-eye geniculocortical axons, the contrast index would be equal to
1. Similarly, the contrast index would have a value of +1 if the cortex contained only deprived-eye geniculocortical presynaptic sites. Because the numerator of the contrast index contains a difference between the deprived-eye average and nondeprived-eye average and each average has its own weighted SEM, the SE of the contrast index is the SE of the difference (Hays, 1963
To compare changes in synaptic density and SVP density after 2 d of MD to the physiological plasticity that takes place after this period of deprivation, we used existing data obtained from normal kittens of ages P37-P45 from Stryker (1989)
Because a prominent feature of ocular dominance plasticity after 7 d of MD is the retraction of deprived-eye geniculocortical axon branches (Antonini and Stryker, 1993b
Analysis of synaptic density and SVP density data using each animal as its own control. Because every animal used in this study received bilateral Pha-L injections in lamina A of the lateral geniculate nuclei, it was possible to compare numerical synaptic density or SVP density values in the left hemisphere of a single animal with the corresponding values in the right hemisphere. For monocularly deprived animals, this is equivalent to comparing deprived-eye afferents with nondeprived-eye afferents, because lamina A neurons receive exclusively monocular retinal input from the contralateral eye. Synaptic density or SVP density for all the fields in a single hemisphere for a given SVP antibody were averaged, and the average was weighted by the number of Pha-L-positive pixels in each field. The hemisphere averages were compared using the contrast index described above to compute a single contrast index for each SVP antibody in each animal. These data were separated into normal, 2 and 7 d MD groups, and the group mean of the single animal contrast indices (combining all three SVP antibodies) was computed using a weighted average. This group average and the group SD were weighted by the total number of Pha-L-positive pixels in all the fields for each animal for each SVP antibody.
Calculation of absolute synapse spacing. To compare the data in this paper with previous estimates of synaptic density of the geniculocortical projection based on electron microscopic measurements, we estimated the absolute density of presynaptic sites in geniculocortical afferents of normal animals. The length of individual segments of Pha-L-labeled geniculocortical axons located within the reference section was estimated by determining the length of the major axis of an ellipse whose dimensions were chosen to best fit the shape of the traced axon segment. The parameters of the ellipse were computed using the Analyze Particles algorithm in NIH Image. The major axis lengths were summed for each field and divided by the number of colocalized presynaptic sites in that field to produce an estimate of synaptic spacing for a single field in units of micrometers of geniculocortical axon per colocalized presynaptic site. These values were then averaged across all fields to obtain a measure of absolute spacing of geniculocortical presynaptic sites. The average and the SD were weighted by the sum of the lengths of the major axes in each field. Sample size was 30 fields from a total of three normal animals.
Statistical analysis. To determine whether two means were significantly different from each other, two-tailed Student's t tests were used with a criterion probability of p = 0.05. Corrections for multiple comparisons were made using the formula pcorr = 1
RESULTS
Labeling of presynaptic sites in geniculocortical afferents
To determine the effects of monocular deprivation on the distribution of presynaptic sites in geniculocortical afferents, a technique for identifying individual presynaptic sites located within deprived and nondeprived afferents was required. To accomplish this, focal injections of the anterograde neuronal tracer Pha-L (Gerfen and Sawchenko, 1984
Examples of synaptophysin, synaptotagmin, and SV2 label and an example of Pha-L-labeled axons are shown in Figure 1. For all SVP antibodies, the pattern of labeling was consistent with that expected for presynaptic markers (Fig. 1A,D,E). Dense punctate label was observed in the layer IV cortical neuropil, and staining was absent from neuronal somata and blood vessels. Figure 1C is a false color overlay of a synaptophysin image and a Pha-L image collected from the same field. Several yellow punctate areas of apparent colocalization that may represent presynaptic sites within Pha-L-labeled geniculocortical axons are apparent.
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Figure 1. Synaptic vesicle protein antibodies can be used to label presynaptic sites within Pha-L-labeled geniculocortical afferents. Images are single confocal microscope optical sections of immunofluorescent label in layer IV of P40 kitten primary visual cortex. A, False-color image of synaptophysin immunofluorescence. The pattern of labeling is similar to that expected for a synaptic vesicle marker. There is a high density of labeled neuropil punctate structures that are likely to correspond to presynaptic terminals and an absence of label in cell bodies. B, False-color image of geniculocortical afferents labeled by an injection of the anterograde neuronal tracer Pha-L into lamina A of the lateral geniculate nucleus. Geniculate neurons located at the injection site took up the Pha-L and transported it to their axon terminals in layer IV of primary visual cortex. Thin axonal ramifications and varicosities can be visualized. C, False-color overlay of the images in A and B. Yellow represents regions of apparent colocalization of synaptophysin and Pha-L. Not all of the yellow puncta correspond to presynaptic sites within Pha-L-labeled geniculocortical afferents. This is because the large number of synaptophysin-labeled presynaptic sites within axons not labeled with Pha-L make the probability of false positive colocalization artifact very high if only single optical sections are considered. Special object-based techniques were used to decrease the contribution of false positive artifact to the colocalization measurements (see Materials and Methods). D, Synaptotagmin immunofluorescence. The overall pattern of labeling is similar to that observed for synaptophysin (A). E, SV2 immunofluorescence. Again, the label is consistent with that observed with other synaptic vesicle markers. F, False-color overlay of GAD65 (red) and Pha-L (green) immunofluorescence. GAD65 is an isoform of glutamic acid decarboxylase, which is preferentially localized to presynaptic terminals in GABAergic inhibitory neurons. Because the geniculocortical projection is entirely excitatory, it does not contain any GAD65 labeling. Because the overall pattern of GAD65 immunoreactivity is roughly similar to that of synaptophysin, synaptotagmin, and SV2, it can be used to estimate the amount of artifactual apparent colocalization. Because there is no actual overlap of the GAD65 and Pha-L patterns, any apparent overlap must be caused by false colocalization artifact. Scale bars, 5 µm.
The synaptic vesicle protein antibodies used in this study label most if not all of the synapses in layer IV of the cortex. However, only ~6% of the synapses made onto layer IV spiny stellate neurons in cat visual cortex are of thalamic origin (Ahmed et al. 1994
To minimize the contribution of this artifact to the colocalization data, customized object-based techniques were used to accurately select only those labeled synapses actually located within Pha-L-labeled afferents. These techniques measure colocalization by determining the location of each Pha-L-labeled geniculocortical axon branch and each labeled presynaptic site in three-dimensional space. The method is fully described in and will only be briefly summarized here. All image processing procedures were performed blind with respect to whether the field contained deprived or nondeprived Pha-L-labeled axons. After tracing and excluding pixels within non-neuropil structures (cell bodies and blood vessels) from further analysis, the SVP images were thresholded such that the brightest 10% of the pixels were above threshold. The use of a relative threshold allowed for quantitative comparison of fields from different animals and corrected for several sources of interfield variability including quality of perfusion, antibody penetration, and gain and black settings on the confocal microscope. This relative threshold is only valid if MD has no effect on the relative levels of overall SVP label in ocular dominance columns in primary visual cortex. We have quantitatively tested this and have found that the amount of overall SVP label is not dependent on position within deprived or nondeprived ocular dominance columns in layer IV after 2 or 7 d of MD (Silver and Stryker (Silver and Stryker, 1998
Colocalization analysis of GAD65 validates object-based colocalization analysis
To assess the validity of our procedures, we used the apparent colocalization of GAD65 with geniculocortical axons as a biological control. GAD65 is an isoform of the GABA synthetic enzyme glutamic acid decarboxylase that is localized primarily to presynaptic terminals of GABAergic inhibitory neurons (Silver and Stryker, Kaufman et al. 1991
To compare the amount of colocalization measured for synaptophysin with Pha-L-labeled axons to that for GAD65 with Pha-L-labeled axons, we computed colocalization indices for a Pha-L field collected from one location with either a synaptophysin or GAD65 field from a different location (still within layer IV of primary visual cortex). This "shuffled" condition (by analogy to electrophysiological cross-correlation studies) provides a quantitative estimate of the amount of colocalization expected for two labels based on random overlap. If the colocalization index for two labels collected from the same field (the "experimental" condition) is significantly greater than the shuffled colocalization index, then the two labels are considered to be colocalized. If the experimental colocalization index is less than the shuffled index, the two labels are anticolocalized (that is, they label distinct sets of structures). These data can also be expressed in the form of a contrast index (the difference between the experimental and shuffled colocalization indices divided by the sum of these indices). This contrast index has a positive value for colocalized antigens and a negative value for anticolocalized antigens. Figure 2 indicates that synaptophysin is colocalized with Pha-L-labeled geniculocortical afferents, whereas GAD65 is anticolocalized with these afferents. The difference between the synaptophysin and GAD65 contrast indices was statistically significant (p < 0.01, two-tailed t test; six experimental and six shuffled fields analyzed for each antigen). This result validates the object-based colocalization analysis by showing that it excludes the false colocalization of a molecule not contained within the geniculocortical afferents.
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Figure 2. Quantitative comparison of the amount of colocalization of synaptophysin or GAD65 with Pha-L-labeled geniculocortical afferents in P40 kittens. The amount of apparent colocalization of synaptophysin with Pha-L expected based on random overlap of the two labels was computed by performing colocalization analysis on a synaptophysin field and a Pha-L field obtained from separate and nonoverlapping regions of layer IV (referred to as the shuffled condition). This was compared with the actual colocalization index, measured by carrying out an identical analysis on synaptophysin and Pha-L fields collected from the same location (the experimental condition). The colocalization index was defined as the sum of synaptophysin pixel intensities in presynaptic sites colocalized with Pha-L-labeled axons normalized by the amount of Pha-L label in the field (SVP density) (see Materials and Methods). Shuffled and experimental colocalization indices were also computed for GAD65 and Pha-L. Experimental and shuffled colocalization indices were compared in the form of a contrast index. As expected, synaptophysin was colocalized with Pha-L-labeled geniculocortical afferents, and GAD65 was anticolocalized with these afferents. The object-based colocalization procedure used in this study can easily discriminate between synaptophysin and GAD65 colocalization contrast indices (p < 0.01, two-tailed t test). Error bars indicate SEM.
Synaptic density and SVP density in normal animals
Synaptic density and SVP density data were first collected from control animals with normal visual experience to determine the baseline variability in these measures. Every animal in the data set received Pha-L injections in lamina A of the LGN of both hemispheres. These injections labeled geniculocortical afferents in each cortical hemisphere that served only the eye contralateral to the injection site. Therefore, it was possible to estimate the variability of the data by comparing the colocalization indices in the left hemisphere with the colocalization indices in the right hemisphere. Each confocal microscope field generated a single value for the colocalization index. A contrast index (the difference between the average of the colocalization indices for the left hemisphere fields and the average for the right hemisphere fields divided by the sum of these averages) provided a standardized measure for comparing the two hemispheres. If the average colocalization indices for the two hemispheres were equal, the contrast index would have a value of 0.
Colocalization was quantified as either numerical synaptic density (the number of presynaptic sites normalized by the amount of Pha-L label in the field) or SVP density (the summed intensities of the colocalized SVP pixels normalized by the amount of Pha-L label in the field) (see Materials and Methods). Figure 3 shows the contrast indices for synaptic density and SVP density for three different synaptic vesicle proteins: synaptophysin, synaptotagmin, and SV2. In all six cases, the average left hemisphere colocalization indices were not significantly different from the average right hemisphere values (p > 0.1, two-tailed t test; sample sizes: synaptophysin, 30 fields from three animals; synaptotagmin, 20 fields from two animals; SV2, 20 fields from two animals).
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Figure 3. Relative numerical synaptic density and SVP density in geniculocortical afferents in P40 kittens with normal vision. To estimate the variability in the colocalization measurements used in this study, a comparison of left hemisphere and right hemisphere values was performed in normal control animals. Colocalization indices were expressed as either numerical synaptic density (the number of colocalized presynaptic sites normalized by the amount of Pha-L label in the field) or SVP density (the sum of the intensities of the SVP pixels in colocalized presynaptic sites normalized by the amount of Pha-L label in the field). Quantitative comparisons of the average of left hemisphere colocalization indices and the average of the right hemisphere values were made in the form of a contrast index, defined as (left
Comparison with previous methods for measuring ocular dominance plasticity
Because these synaptic density and SVP density measurements have not been used before to study ocular dominance plasticity, it was important to compare the values and variability obtained using these techniques with other methods for quantifying ocular dominance plasticity that are more established. The CBI (Reiter et al. 1986
Anatomical plasticity after MD has been assessed by measuring changes in the lengths of geniculocortical arbors (Antonini and Stryker, 1993b
Effects of 2 d of MD on synaptic density and SVP density in deprived and nondeprived geniculocortical afferents
A decrease in synaptic density or SVP density in deprived geniculocortical axons might account for the striking mismatch between anatomical and physiological effects of 2 d of MD. Two days of MD is sufficient to cause a robust and saturating physiological ocular dominance shift such that most primary visual cortical neurons lose their responses to stimulation of the deprived eye (for review, see Hensch, 1996
One hypothesis is that loss of some presynaptic sites in deprived-eye geniculocortical arbors and/or addition of presynaptic sites to nondeprived-eye arbors is responsible for the physiological ocular dominance shift observed after 2 d of MD. More specifically, one may propose that the loss of presynaptic sites from half of the branches of deprived-eye arbors is the signal that leads to the eventual loss of those branches. Because there is thought to be little retraction of deprived-eye geniculocortical arbors and no elaboration of nondeprived-eye arbors taking place during this time, measurement of relative synaptic density of the deprived versus nondeprived afferents provides a direct test of the general hypothesis. Relative synaptic densities and SVP densities were computed for deprived and nondeprived afferents and expressed as a contrast index, the value of which would be negative if deprived-eye density were reduced relative to nondeprived-eye density after 2 d of MD.
Figure 4 shows that 2 d of MD had no effect on the relative numerical synaptic density in deprived and nondeprived geniculocortical afferents (p > 0.2, two-tailed t test; synaptophysin, 30 fields from three animals; synaptotagmin, 20 fields from two animals; SV2, 20 fields from two animals). The results were identical for all three SVP markers used in this study. Therefore, addition or removal of presynaptic sites in the geniculocortical projection is not responsible for the physiological ocular dominance shift produced by 2 d of MD. Although synaptic density measurements reflect the number of presynaptic sites per Pha-L-positive pixel, SVP density is the total summed intensities of all colocalized SVP pixels normalized by the number of Pha-L-positive pixels. Therefore, SVP density is sensitive to potential changes in the size of presynaptic sites and to the amount of synaptic vesicle protein per site. SVP density in deprived afferents was equal to that in nondeprived afferents for all three SVP antibodies tested (p > 0.2, two-tailed t test), indicating that the amount of SVP label per presynaptic site is unaffected by 2 d of MD.
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Figure 4. Relative numerical synaptic density and SVP density in deprived and nondeprived geniculocortical afferents after 2 d of MD. P38 kittens were monocularly deprived for 2 d, and averages of synaptic density and SVP density were separately determined for deprived and nondeprived geniculocortical axons. Deprived and nondeprived afferents were compared using a contrast index. For all six conditions, the contrast index was not significantly different from 0 (p > 0.2, two-tailed t test). To calculate our confidence in these negative results, statistical power analysis was used to determine minimum detectable differences. These represent deviations of the contrast index from 0 for two populations that would have been detected for a particular pair of samples from these populations given the sample sizes, the sample variances, and a choice of confidence level. Horizontal bars indicate minimum detectable difference levels for a confidence level of 90%. Data on the effects of 2 d of MD (deprivation beginning P37-P40) on the ocular dominance of cortical neurons from the thesis of Hensch (1996)
These results fail to show a statistically significant difference between deprived and nondeprived afferents in synaptic density or SVP density after 2 d of MD. The lack of significance of a difference is not equivalent, however, to the positive assertion that synaptic density or SVP density are identical in deprived and nondeprived arbors. If the synaptic density or SVP density were actually different in deprived and nondeprived afferents, the present data set might not reveal this difference if the sample sizes were too small or the data were too variable. Statistical power analysis (Cohen, 1988
= 0.10), we determined the minimum detectable differences in numerical synaptic density or SVP density between deprived and nondeprived afferents after 2 d of MD. These ranged from 0.18 (for synaptophysin synaptic density) to 0.23 (for SV2 SVP density) when expressed on the same scale as the contrast indices (Fig. 4). For example, given our sample, we can be 90% certain that the true contrast index for synaptophysin synaptic density does not differ by >0.18 from a value of 0.
To compare these data with physiological measurements of the effect of 2 d of MD, data from the thesis of Hensch (1996)
Despite the profound physiological effect of 2 d of MD, the density of presynaptic sites on deprived-eye afferents is not reduced relative to that in nondeprived-eye afferents. Because the deprived-eye afferents probably have not shrunk by this time, their reduced efficacy is not accounted for by a loss of the presynaptic component of synapses.
Models of possible presynaptic changes in geniculocortical afferents after 7 d of MD
After 7 d of MD, the total length of deprived-eye geniculocortical arbors is reduced by ~50% (Antonini and Stryker, 1993b
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Figure 5. Possible models of presynaptic site rearrangements in deprived-eye geniculocortical arbors after 7 d of MD. As shown in all three models, 7 d of MD causes a 50% reduction in the total length of individual deprived-eye geniculocortical arbors (Antonini and Stryker, 1993b
In the active synapse model, synapse elimination is even greater than retraction of deprived-eye axon branches. Therefore, 7 d of MD would cause a decrease in the total number of presynaptic sites per deprived arbor and would also decrease numerical synaptic density. The passive synapse model postulates that synapses and axon branches are removed at equal rates. In this model, 7 d of MD results in a decrease in the number of presynaptic sites per deprived-eye arbor. As deprived-eye axon branches are retracted, the presynaptic sites within these branches are eliminated. However, the distribution of presynaptic sites in the remaining axon branches is essentially unchanged. Therefore, in this model, the synaptic density of deprived-eye branches is unaffected by 7 d of MD. In the dynamic synapse model, presynaptic sites are redistributed within a shrunken arbor so that 7 d of MD does not result in any net synapse elimination. In this case, the number of presynaptic sites per deprived-eye arbor does not change, so the synaptic density increases.
By measuring the density of presynaptic sites within deprived-eye arbors after 7 d of MD, it is possible to discriminate among these three models. Although the colocalization analysis used in this study was not designed to measure absolute synaptic density, it can be used to compare the relative numerical synaptic densities in deprived and nondeprived geniculocortical afferents. If 7 d of MD does not alter the absolute synaptic density of nondeprived-eye afferents, a comparison of deprived-eye and nondeprived-eye synaptic densities is equivalent to measuring absolute synaptic density in the deprived-eye projection and represents a direct test of the models presented in Figure 5. Consistent with this possibility is the finding that 7 d of MD causes a 50% reduction in the total length of deprived-eye geniculocortical afferents but has little or no effect on nondeprived-eye axons (Antonini and Stryker, 1993b
Similarly, if changes in the synaptic density of nondeprived-eye afferents are in the opposite direction of the changes that occur in deprived-eye afferents, then a relative comparison of deprived and nondeprived afferents would yield the same results as an absolute measure of deprived-eye synaptic density, but the size of the effect would be larger. The only scenario in which a relative comparison of deprived versus nondeprived synaptic density would yield qualitatively different results from a measurement of absolute deprived-eye synaptic density is if the synaptic densities of deprived and nondeprived afferents both changed in the same direction (i.e., both increased or both decreased). Because there is ample evidence that ocular dominance plasticity is a result of competition between the populations of afferents representing the two eyes (Guillery, 1972
Effects of 7 d of MD on synaptic density and SVP density in deprived and nondeprived geniculocortical afferents
Depriving kittens of pattern vision in one eye by monocular lid suture for 7 d (P33-P40) had no effect on the relative numerical synaptic density or SVP density in deprived and nondeprived geniculocortical afferents (Fig. 6) (p > 0.95, two-tailed t test; synaptophysin, 30 fields from three animals; synaptotagmin, 20 fields from two animals; SV2, 20 fields from two animals).
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Figure 6. Relative numerical synaptic density and SVP density in deprived and nondeprived geniculocortical afferents after 7 d of MD. P33 kittens were monocularly deprived for 7 d, and averages of synaptic density and SVP density were computed independently from hemispheres containing either deprived or nondeprived Pha-L-labeled geniculocortical afferents. The same contrast index used in Figure 4 was used to quantitatively compare deprived and nondeprived axons. For all six conditions, the contrast index was not significantly different from 0 (p > 0.95, two-tailed t test). Horizontal bars indicate minimum detectable difference levels for a confidence level of 90%. The effect of 6-7 d of MD on relative total lengths of deprived and nondeprived individual geniculocortical arbors from Antonini and Stryker (1993b)
Because 7 d of MD produced a negative result for all measures of relative synaptic density and SVP density, statistical power analysis was used to determine the strength of these negative results. The analysis was identical to that used for the 2 d MD results. Minimum detectable differences in the 7 d MD samples ranged from 0.19 to 0.35 when expressed on the same scale as the contrast indices. To compare these results with previously published data on the effects of 6-7 d of MD on the geniculocortical projection (MD beginning P32-P36), data from Antonini and Stryker (1993b)
These results are most consistent with the passive synapse model presented in Figure 5. That is, relative synaptic density in deprived versus nondeprived geniculocortical axons is unchanged by 7 d of MD. Because deprived-eye afferents are known to undergo significant retraction of axon branches after 7 d of MD (Antonini and Stryker, 1993b
Analysis of the effects of MD using each individual animal as its own control
The data presented on the effects of MD on relative synaptic density and SVP density so far have involved the averaging of density measurements from fields containing labeled deprived-eye geniculocortical afferents from multiple animals and comparison of this average with the corresponding average from nondeprived-eye fields. Because every animal in our data set received Pha-L injections into lamina A of the LGN of both hemispheres, every monocularly deprived animal had Pha-L-labeled deprived-eye afferents in one hemisphere and labeled nondeprived-eye afferents in the other hemisphere. This allowed for the reanalysis of the data set using each animal as its own control. We reasoned that this analysis could decrease the variability of the data and increase the likelihood of detection of small changes in relative synaptic density or SVP density produced by MD.
Numerical synaptic density or SVP density measurements were averaged for each hemisphere, and these averages were used to compute a contrast index for each SVP marker in each animal. Because the data presented in Figures 3, 5, and 6 do not indicate any differences among synaptophysin, synaptotagmin, or SV2, the results from these three SVP markers were combined and used to calculate average synaptic density or SVP density contrast indices for normal animals, 2 d MD animals, and 7 d MD animals (Fig. 7). Data from fields containing synaptophysin, synaptotagmin, and SV2 were pooled in the group averages. In all cases, there were no statistically significant differences between deprived and nondeprived (or left vs right in the case of the normal control animals) synaptic density or SVP density measurements [p > 0.5, two-tailed t test; seven contrast index values for each condition (three synaptophysin, two synaptotagmin, and two SVP), with each contrast index value incorporating data from 10 fields]. The group averages for all the contrast indices within a condition were weighted by the total number of Pha-L-positive pixels in the fields from each animal.
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Figure 7. Analysis of numerical synaptic density and SVP density data using each animal as its own control. Because every animal in this study received Pha-L injections in the LGN of both hemispheres, geniculocortical afferents representing the eye contralateral to the injection site were labeled in each hemisphere. This allowed intra-animal comparisons to be made of deprived and nondeprived geniculocortical axon synaptic density and SVP density. Some of the data presented in Figures 3, 4, and 6 were reanalyzed in this manner. Average synaptic density and SVP density values were computed separately for each SVP antibody in each hemisphere, and one contrast index was generated for each SVP antibody per experimental animal. This contrast index compared deprived and nondeprived afferents for animals that received MD and compared left and right hemisphere values for normal control animals. Open circles represent contrast indices for individual animals. In all cases, the group average of the contrast indices was not significantly different from 0 (p > 0.05, two-tailed t test). Error bars indicate SEs of the mean, and horizontal bars indicate minimum detectable difference levels for a confidence level of 90%. The group averages and minimum detectable differences for animals receiving 2 or 7 d of MD were indistinguishable from normal control animals.
Statistical power analysis of these negative results produced minimum detectable differences ranging from 0.10 to 0.23 using a confidence level of 90% (Fig. 7). Of the six comparisons tested, the two largest minimum detectable differences were for the synaptic density and the SVP density from normal animals. This suggests that any differences between deprived and nondeprived afferents after either 2 or 7 d of MD are well within the range of normal variability. Given that identical results were obtained for three independent SVP markers, it is very likely that numerical synaptic density and SVP density are equal in deprived and nondeprived geniculocortical afferents after either 2 or 7 d of MD.
Exclusion of additional potential sources of variability: ocular dominance columns, laminar position, and axon orientation
Several other analyses were performed to attempt to account for the variability in the synaptic density and SVP density data. Because primary visual cortex is organized into ocular dominance columns (Hubel and Wiesel, 1962
By photobleaching the confocal microscope field immediately after image collection, the position of the field could be accurately determined. Because the Pha-L injections in the LGN labeled a monocular population of geniculocortical afferents serving the eye contralateral to the injection site, ocular dominance columns were often observed in the pattern of Pha-L-labeled afferents in the cortex. An analysis of synaptic density and SVP density as a function of ocular dominance column position revealed no correlation between these measures (data not shown). Similarly, neither synaptic density nor SVP density was correlated with the depth of the field within the cortex (superficial vs deep layer IV) (data not shown).
Finally, because it was sometimes difficult to trace Pha-L-labeled axon segments that were oriented perpendicular to the plane of the optical section, we selected only those axon segments whose uninterrupted length within the reference section was at least 25 pixels, corresponding to a length of 3.3 µm. These long axon segments were within the plane of the reference section and very easy to identify and trace. Reanalysis of a portion of the data set consisting of only long axon segments produced the same results as analysis of the entire data set, although the variance was greater because of the smaller sample sizes (data not shown).
Distribution of geniculocortical presynaptic sites after MD of 2 or 7 d
Figure 8 summarizes the present findings on the distribution of presynaptic sites within geniculocortical afferents after MD. The top portion of the model indicates the known effects of MD on physiological visual responses of primary visual cortical neurons and on the morphology of geniculocortical afferents, whereas the bottom portion represents the distributions of presynaptic sites on deprived-eye geniculocortical afferents indicated by the findings of this study.
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Figure 8. The distribution of geniculocortical presynaptic sites after 2 and 7 d of MD. The data presented in this study suggest the following model: 2 d of MD results in no change in relative numerical synaptic density or SVP density in deprived and nondeprived geniculocortical afferents, despite the fact that 2 d of MD is sufficient to cause a saturating physiological ocular dominance shift (for review, see Hensch, 1996
Two days of MD cause a saturating loss of visual cortical responses to deprived-eye visual stimulation (for review, see Hensch, 1996
DISCUSSION
We have determined the effects of monocular deprivation on relative numerical synaptic density and SVP density in deprived and nondeprived geniculocortical axon arbors. Our results demonstrate that these measures are unchanged after 2 d of MD, although deprivation for this period of time has significant effects on the activity patterns of geniculocortical afferents and causes a physiological ocular dominance shift (for review, see Hensch, 1996
