Sensory Regulation of Immediate-early Genes c-fos and zif268 in

Sensory Regulation of Immediate-early Genes c-fos and zif268 in Monkey Visual Cortex at Birth and Throughout the Critical Period

The postnatal development of ocular dominance columns (ODCs) in monkey visual cortex provides an exquisite model for studying mechanisms of experience-guided neuronal plasticity. While the presence of columns at birth in Old World monkeys is now well established, it remains unclear whether cortical neurons at this early stage are capable of modulating gene expression in response to changing sensory conditions. Using a set of monocular deprivation and stimulation protocols, we examined activity-driven expression of the immediate-early genes (IEGs) c-fos and zif268 during the critical period of development. We observed well-delineated patterns of ODCs produced by sensory regulation of both IEGs throughout the critical period, starting as early as the first postnatal day. The expression levels are similar in layers II/III, IVC and VI throughout development, with no selective decline in the thalamorecepient layer (layer IVC) of adult monkeys. A narrow strip of non-columnar c-Fos expression was observed at the border of layers IVC and V. Our results show that neurons in monkey visual cortex are equipped at birth with the molecular machinery for coupling sensory inputs to active genomic responses and that this responsivity extends throughout the critical period. The findings are discussed within the context of a possible role for IEGs in sensory-driven cortical plasticity during development. Introduction The developing visual system of primates provides an excellent model for the study of activity-dependent neuronal plasticity. The geniculocortical afferents in these animals transmit visual information from both eyes into layer IV of primary visual cortex (area 17 or V1). Here, an initially desegregated pattern of eye inputs is sculpted by activity-driven mechanisms into an interdigitated arrangement of vertical ocular dominance columns (ODCs) (Hubel et al., 1977; Rakic, 1977; LeVay et al., 1978; Katz and Shatz, 1996). The plasticity in ODC architecture is especially striking during the so-called critical period of cortical development when alterations in sensory input can significantly affect neuronal morphology and function as well as the spatial characteristics of columns (Shatz and Stryker, 1978; LeVay et al., 1980; Constantine et al., 1990; Antonini and Str yker, 1993; Goodman and Shatz, 1993). The neuronal changes that occur in activity-guided plasticity are in turn believed to be orchestrated by specific patterns of gene expression (Kandel and O’Dell, 1992; Kaczmarek, 1993; Wallace et al., 1995). The discovery that neural activity can induce the expression of a large number of genes — many of which code for transcription-regulating proteins — has focused attention on the so-called immediate-early genes* (IEG) and their role in regulating long-term changes in brain function ( Morgan and Curran, 1991; Hughes and Dragunow, 1995; Kaczmarek and * In adherence to the usual convention, specific genes and their mRNA products are designated by italics (e.g. c-fos and zif268), whereas the proteins encoded by the genes are written in roman type with a capital letter (e.g. c-Fos and Zif268).

Leszek Kaczmarek1,2, Shahin Zangenehpour2 and Avi Chaudhuri2 Nencki Institute, Warsaw, Poland and 2Department of Psychology, McGill University, Montreal, Canada 1

Chaudhuri, 1997). In particular, two IEG members that code for transcription factors — c-fos and zif268 — have been shown to be regulated by visual input in cortical neurons ( Worley et al., 1991; Beaver et al., 1993; Chaudhuri and Cynader, 1993; Kaminska et al., 1996). In adult monkeys, for example, monocular visual deficit reveals columnar patterns of Zif268 staining (Chaudhuri et al., 1995; Markstahler et al., 1998). While it is known that Old World monkeys are born with segregated ODCs (Des Rosiers et al., 1978; Horton and Hocking, 1996, 1997), it remains unclear whether neurons within these columns are capable of activity-dependent IEG expression during development. Recent reports on New World monkeys have questioned the contribution of Zif268 to ODC formation (Silveira et al., 1996; Fonta et al., 1997). These studies failed to reveal columns of Zif268 expression after monocular deprivation during the critical period, although they were clearly visible in adults (Silveira et al., 1996; Fonta et al., 1997; Markstahler et al., 1998). On the other hand, the expression of both c-fos and zif268 in cat and rodent visual cortex has been found to be higher during the critical period than in adults ( Rosen et al., 1992; Mower, 1994; Kaminska et al., 1995; Kaplan et al., 1996). Thus, the conjecture that both c-fos and zif268 may play important roles in postnatal cortical development has not been supported to date by evidence that these IEGs are linked to one of the most intriguing developmental events in primates, namely ODC formation and stabilization. To clarify the role of IEGs in this process, it is necessary to investigate their expression profiles in response to visually driven neural activity during development. In this paper, we present evidence that both c-fos and zif268 expression in Old World monkeys can be induced within ODCs by monocular visual impulse as early as the first postnatal day. Furthermore, the constitutive expression of zif268 can be down-regulated by monocular visual blockade to reveal ODCs at various times during the critical period. These results show that the developing visual cortex, starting as early as the first postnatal day, has at its disposal IEG products that are regulated by visual stimulation and which may be available to direct plastic changes in an experience-dependent manner.

Materials and Methods Animals and their Treatment Six infant male vervet monkeys (Cercopithecus aethiops) of 0, 6, 11, 40, 50, and 103 days of postnatal age (postnatal day, PD) and three male adults were used for this study. The PD 0, PD 6, PD 40 and two adult animals were subjected to a reverse occlusion procedure. An opaque eye patch was attached with Velcro to cover one eye for 3 h, after which it was removed and transferred to the second eye. The second eye was covered for 30 min. These durations were chosen because they fall within the temporal window of c-fos/zif268 protein and mRNA accumulation following sensory stimulation (Chaudhuri, 1997; Chaudhuri et al., 1997; Kaczmarek and Chaudhuri, 1997). The PD 11, PD 50, PD 103 and one adult monkey were first bincocularly dark-adapted for 3 h and then

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Figure 1. Columnar expression of c-fos and zif268 mRNA and protein in adult monkey primary visual cortex. Monocular visual stimulation for 30 min produced elevated mRNA staining within ODC. Immunostaining for the protein products revealed a similar set of columns, though after 2.5 h of monocular stimulation. The staining intensity for c-Fos protein was consistently weaker than of Zif268. monocularly exposed to ambient light for 2.5 h. All animals were fully awake during the exposure period and restrained in a primate chair, except for the infants which were kept in their playpen. All animals were killed after the exposure period with an overdose of sodium pentobarbital (2 mg/kg, i.v.) and perfused transcardially with phosphate-buffered saline (PBS). The brains were then removed, blocked along the lunate sulcus and frozen by immersion in a 2-methylbutane/ dry-ice bath. This protocol was used for one set of animals (PD 0, PD 6, PD 40 and three adults) to allow for detection of both c-fos mRNA and protein as well as to permit cytochrome oxidase (CO) staining. In a second set of animals (PD 11, PD 50, PD 103 and one adult), PBS perfusion was followed by perfusion with 4% cold paraformaldehyde (PFA) in PBS and then with PBS alone again. We have previously found this protocol to be optimal for c-Fos immunocytochemistry. Brain sections were then cut on a cryostat at 15 µm (for in situ hybridization) or 20 µm thickness (for immunocytochemistry, CO and Nissl staining), mounted on polylysine-coated glass slides, and preserved at –80°C until further histological processing.

In Situ Hybridization (ISH) Radiolabeled antisense probe selective for c-fos was obtained by [33P]UTP incorporation during cRNA synthesis with T7 RNA polymerase using plasmid HWFA D71 harboring human c-fos (ATCC no. 108362) as a template. To visualize the distribution of zif268 mRNA, the probe was obtained with [33P]UTP (NEN–Du Pont) terminal labeling of a 45mer oligonucleotide corresponding to amino acids 2–16 (Moratalla et al., 1992). Glass-mounted sections were thawed at room temperature for 10

180 Visual Induction of c-fos and zif268 in Development • Kaczmarek et al.

min and post-fixed in 4% PFA for 10 min. The PFA solution, as with all others used in this protocol, was made with Milli-Q purified (Millipore) water treated to diethyl pyrocarbonate (DEPC) overnight and autoclaved. The sections were then treated to standard ISH protocols, as described previously (Chaudhuri et al., 1997; Kaminska et al., 1997). A fter processing, the slides were dehydrated in graded ethanols (70, 95, 100%) containing 0.3 M ammonium acetate, air dried and exposed to Hyperfilm β-max (Amersham) for 21 days at room temperature. Immunocytochemistry (ICC) Slide-mounted sections were placed in a solution containing rabbit polyclonal antibodies selective either for c-Fos (Santa Cruz Biotechnology, cat. no. SC52x at 1/10 000 dilution or Calbiochem at 1/400 dilution) or Zif268 (gift from R. Bravo, Bristol–Myers–Squibb, Princeton, NJ) at 1/10 000 concentration in PBS with 2.0% normal goat serum and processed according to standard procedures, as described previously (Chaudhuri et al., 1995). The colorimetric reaction used nickel-enhanced diaminobenzidine (DA B) to produce a dark blue stain within immunoreactive neurons. The primar y antibody was absent in control sections which were otherwise processed identically. Cytochrome Oxidase Histochemistry and Nissl Staining Cytochrome oxidase histochemistr y was performed according to a previously described protocol (Dyck and Cynader, 1993). Brief ly, sections were fixed for 5 min in 4% PFA in 0.05 M phosphate buffer (pH 7.2) followed by two rinses in buffer for 2 min each. The slides were then placed in 0.05 M phosphate buffer containing 0.01 g/ml sucrose, 0.5

Figure 2. Effects of monocular deprivation on Zif268 protein expression during the critical period. Coronal sections of monkey primary visual cortex at various time-points spanning the critical period show different degrees of ocular dominance staining. Zif268 immunostaining failed to reveal ODC at PD 0 after 3 h of monocular deprivation. The columns are barely noticeable at PD 6. At later time-points (PD 40 and 90), a set of vertically oriented columns can be clearly seen to extend the full cortical thickness. The immunopositive columns are visible because of Zif268 down-regulation in complementary columns that represent the deprived eye. Scale bar: 0.5 mm. mg/ml nickel ammonium sulfate, 0.25 mg/ml DAB, 0.15 mg/ml cytochrome c, 0.1 mg/ml catalase and 2.5 mM imidazole (all reagents from Sigma). The reaction was stopped by immersion in phosphate buffer, after which the sections were dehydrated and cover slipped. Adjacent sections were also stained for Nissl substance with 1% cresyl violet. Image Analysis Autogradiograms of striate cortex sections processed for ISH visualization of c-fos mRNA were digitally captured and subjected to image analysis. The aim was to compare the optical density in layers II/III, IVC and VI within positively stained columns. This procedure was performed on autoradiographic data obtained from two infant monkeys (PD 6, PD 40) and two of the adults that were subjected to short-term light exposure (30 min) — i.e. all animals that were selectively exposed for c-fos mRNA induction. Optical density was obtained within a 5 × 5 pixel (85 × 85 µm) sampling window that was placed at the center of each column at each of the three laminar levels. A total of five neighboring columns in each animal was sampled in this manner.

Results ISH and ICC Detection of c-fos and zif268 Products in Adult Visual Cortex There have been no prior reports of c-fos expression in monkey visual cortex. Therefore, prior to investigating IEG expression profiles in neonates, we initiated a study on the effects of visual stimulation on c-fos expression in adult animals and compared it with another immediate-early gene, zif268, whose mRNA and protein expression in monkey visual cortex had already been characterized (Chaudhuri et al., 1995, 1997). To separately reveal mRNA and protein induction, we employed different periods of light exposure — 30 min for mRNA and 2.5 h for protein — following a brief (3 h) period of dark adaptation (see Materials and Methods for details). Figure 1 shows the staining patterns that were produced by ISH and ICC staining for the mRNA and protein products respectively of the c-fos and zif268 genes. In all cases, a clear columnar pattern of staining was obtained. One notable difference, however, was that the intensity of c-Fos immunostaining was clearly less than that of Zif268. Indeed, we never obser ved any instances of c-Fos immunostaining that were as intense as that found for Zif268. As a result, c-Fos-immunopositive columns, though clearly discernible, consistently showed reduced contrast in all layers.

Zif268 Staining of ODCs During the Critical Period Prior studies in New World monkeys failed to show ODCs during the critical period by means of Zif268 immunostaining. We were curious whether this finding had general validity across primates and therefore set out to investigate it in Old World monkeys. We followed two different approaches that employed stimulation parameters and staining protocols developed from our studies in adult animals. In the first, we proceeded with short-term monocular deprivation on light-adapted animals because this had been previously shown in adult monkeys to effectively down-regulate Zif268 protein expression in visually deprived columns (Chaudhuri et al., 1995). As Figure 2 shows, a 3 h period of monocular visual blockade produced well-delineated columns at various time-points during the critical period. The clearest columns could be obser ved at PD 40 and 90. At earlier time-points, the columns, though discernible, were less clear. This is evident at PD 6 where the columns were just barely distinguishable. No ODCs could be observed at PD 0 following monocular visual deprivation. In the second approach, we examined Zif268 protein induction in ODCs after monocular visual stimulation. While the Zif268 columns of Figure 2 showed that this protein can be effectively down-regulated by visual deprivation, the counter issue of whether Zif268 can be rapidly induced by sensory stimulation during the critical period remained open. Infant monkeys were now exposed to brief monocular visual stimulation (2.5 h) but only after a period of prior dark-adaptation (3 h). As with the findings in adult monkeys (Fig. 1), this procedure also produced Zif268 up-regulation within visually stimulated columns in infant animals. Figure 3 shows that Zif268 protein columns can be clearly seen at all three time-points spanning the critical period. We also observed similar staining patterns for c-Fos protein. That is, columns of c-Fos-immunopositive neurons were clearly evident at PD 11, PD 50 and PD 103 after monocular visual stimulation. However, given the weak intensity of c-Fos immunostaining in monkey visual cortex, the columns were generally of poor contrast and therefore difficult to capture by photomicroscopy at low magnification. We obtained similar staining patterns with both anti-c-Fos antibodies.

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Figure 3. Effects of monocular stimulation on Zif268 protein expression during the critical period. Coronal sections of monkey primary visual cortex at three developmental time-points reveal columns of Zif268 immunostaining. Animals were subjected to 2.5 h of monocular light stimulation after a brief period (3 h) of dark adaptation. The vertical immunopositive columns are visible because of Zif268 up-regulation by monocular stimulation.

Figure 4. Sensory-guided expression of c-fos and zif268 mRNA in early postnatal development. (A) In situ hybridization reveals c-fos and zif268 mRNA up-regulation in ODCs after 30 min of monocular visual stimulation. The vertical columns are well delineated at both PD 0 and PD 6. (B) Flattened sections from PD 6 were treated to Zif268 immunostaining and c-fos and zif268 in situ hybridization. Darkly stained columns within the rectangular region of each section were manually delineated after sections were aligned using fiducial marks. The marked columns from the zif268 mRNA (gray) and Zif268 protein (black) stained sections show two complementary sets when they are digitally superimposed (left inset). Similarly, the c-fos mRNA and Zif268 protein columns form spatially non-overlapping profiles (right inset).

Columnar Induction of c-fos and zif268 mRNA in Early Postnatal Period Given that prior studies showed anatomical and 2-deoxyglucoselabeled ODCs at very early postnatal periods in monkeys (Rakic, 1976; Hubel et al., 1977; Des Rosiers et al., 1978; Horton and Hocking, 1996), we were surprised that monocular deprivation produced discernible Zif268 protein columns at various time-points during the critical period but not at PD 0. We therefore asked whether neurons within ODCs are nevertheless capable of an inductive IEG response following visual stimulation at such early time-points. To compare both stimulation and deprivation effects in the same animal, we took advantage of the reverse-occlusion procedure. In the first step of this approach, one eye is dark adapted for 3 h and the other eye is left open. This is followed by reversing the eye patch and stimulating the previously occluded eye for 30 min only. This procedure has previously been used in adult monkeys to show one set of ODC columns by way of ICC staining for Zif268 protein and the complementary set of


columns by way of ISH staining for zif268 mRNA (Chaudhuri et al., 1997). This approach therefore has the advantage of making visible both sets of ODCs and allowed us to distinguish between sensory deprivation effects (by way of Zif268 protein down-regulation) and stimulation (by way of zif268 mRNA up-regulation) in the same animal. As Figure 4A shows, both c-fos and zif268 mRNA staining revealed a columnar pattern of IEG expression at both early time-points (PD 0 and 6). We then examined the spatial distribution of these columns in f lattened sections (PD 6) to see whether both mRNA profiles stained the same set of columns and if these in turn could be distinguished from a complementary set demarcated by Zif268 immunostaining. Figure 4B shows three sections of the opercular surface, each revealing a columnar pattern. The positively stained columns were manually delineated and digitally overlaid (Chaudhuri et al., 1997). An analysis of both mRNA-stained profiles showed that the columns were indeed spatially superimposed. However, as the left inset shows, zif268 mRNA columns (gray) were spatially comple-

Figure 5. In situ hybridization results with c-fos RNA probe in monkey striate cortex. (A) Representative autoradiograms taken from a 6 day old (PD 6) and an adult animal showing ocular dominance columns with signal in layers II/III, IVC and VI. (B) Densitometric analysis of c-fos mRNA expression in two developing animals (PD 6 and 40) and two adults. The optical density, represented in arbitrary units (a.u.), is found to be similar across the three laminar segments. Most notably, the signal in layer IVC of adults is not reduced in comparison to other layers. Error bars represent standard deviation (n = 5).

mentary to the Zif268 protein columns (black). Similarly, c-fos mRNA-stained columns did not coincide with the Zif268 protein columns (right inset).

Laminar Patterns of c-fos Expression During the Critical Period Given that both c-fos and zif268 are rapidly expressed by visual stimulation in developing vervet monkeys, we next examined the laminar patterns of expression to see whether differences existed among the major cortical strata. We focused our attention on the laminar patterns of c-fos induction because of prior reports in cat visual cortex of an interesting difference in the profile between developing and adult animals, especially with respect to the thalamorecepient layer IVC, and the potential implications for neocortical plasticity (Mower, 1994; Kaplan et al., 1996). Figure 5A shows the ISH results that were obtained with an antisense c-fos RNA probe hybridized to PD 6 and adult vervet monkey striate cortex. The laminar profiles of mRNA expression at both ages appeared to be qualitatively similar. Specifically, c-fos mRNA levels were abundant in layers II/III, IVC and VI, independent of the age of the animal. Laminar assignments were made by comparison of autoradiographic data to adjacent sections that were treated to Nissl staining. Densitometric analysis was performed on autoradiograms from two adults and two animals from the critical period (PD 6 and 40). These results confirmed that signal strength was similar in all three strata regardless of age (Fig. 5B). Furthermore, we found no evidence of a selective decline in c-fos expression within layer IVC of

adult monkeys in comparison to those in the critical period of development. It may be appreciated from Figure 5A that mRNA autoradiograms do not provide sufficient resolution to allow visualization of fine spatial detail. We therefore examined c-Fos immunostaining patterns in order to permit a more detailed laminar analysis during development. Although c-Fos immunostaining is generally weak in monkey neocortex, we found that sufficient signal is present at higher magnification to display laminar features that could be correlated with other histological stains. Figure 6 shows the c-Fos immunostaining profiles for three infant monkeys aged PD 0, 6 and 40. A notably distinct feature of developing striate cortex is the presence of a narrow strip of c-Fos expression located in the middle of the cortical plate. To explore this issue further, we compared c-Fos immunostaining with CO histochemistr y. The thin strip of elevated c-Fos immunostaining coincided with a similar strip in CO-stained sections (small black arrowheads in accompanying CO profiles


Figure 6. Laminar profiles of c-Fos protein induction during development. c-Fos immunostaining data from three infant monkeys at PD 0, 6 and 40 are shown along with cytochrome oxidase (CO) staining from an adjacent section. Columns of c-Fos expression are shown by angled black arrowheads. Layer IVC in the accompanying CO profiles is indicated by white arrowheads. The thin strip of elevated CO staining at the bottom of layer IVC is indicated by small black arrowheads. A similar strip of elevated c-Fos immunostaining was found in the infants at all three ages.

of Fig. 6). The thin c-Fos strip was evident in all infant animals, although the CO strip became less conspicuous by PD 40 and seemed to merge with the heavy staining in the rest of layer IVC (upper and lower boundaries of layer IVC are shown by white arrowheads in CO-stained sections). The columnar distribution of c-Fos was especially evident in the supragranular layers at PD 6 and 40 (black arrowheads). However, there are no hints of punctuated staining within the narrow band of c-Fos staining. Rather, this strip appeared to be continuous in all of the infant animals that we studied.

Discussion The two major findings of this study are as follows. First, an examination of c-fos and zif268 expression in primary visual cortex of developing monkeys by both mRNA and protein staining showed that their expression is regulated by visual input such that ocular dominance columns are visible after brief periods of monocular light exposure. The most important finding in this regard is that visual cortex of infant monkeys is capable of engaging genomic responses as early as the first postnatal day and that such responses within ocular dominance columns are preserved during postnatal development. Second, the developmental characterization of c-Fos expression showed that staining intensities among the major strata were similar in both infant and adult monkeys. Furthermore, we observed a narrow strip of c-Fos staining that was evident at early ages and which correlated with a similar strip of CO staining. This narrow strip did not show a columnar pattern after monocular light exposure, although columns were clearly visible in other cortical layers. Detection of c-fos mRNA and Protein Expression There have been no prior published reports of c-fos expression in monkey visual cortex. Thus to obtain veridical staining


patterns, it was necessary to proceed with a multi-faceted approach that involved different manipulations of light exposure and detection strategies. It was known that c-fos is expressed at low basal levels in the visual cortex of rats and cats and that in order to induce its expression, sensory stimulation had to be applied after a period of dark adaptation or rearing (Worley et al., 1991; Beaver et al., 1993; Mower, 1994; Montero and Jian, 1995; Kaminska et al., 1996; Kaplan et al., 1996; Konopka et al., 1998). The duration of visual deprivation in prior c-fos induction studies had typically been several days (although see Montero and Jian, 1995). We have now found that as little as 3 h of dark adaptation followed by light stimulation was sufficient for c-fos induction in the monkey. This result paralleled that which we had previously observed for the induction of another immediate-early gene, zif268 (Chaudhuri et al., 1997). This concordance of c-fos and zif268 induction has also been observed in other species and experimental conditions (Kaczmarek and Chaudhuri, 1997). The time course of zif268 induction in the monkey along with c-fos in other species suggested that visual stimulation for 30 min would be optimal for c-fos mRNA and 2 h for c-Fos protein visualization (Hughes and Dragunow, 1995; Chaudhuri et al., 1997; Kaczmarek and Chaudhuri, 1997). We obtained essentially the same expression profiles for c-fos at both mRNA and protein levels. The agreement between the mRNA and protein expression was important in validating the immunostaining results given the difficulty in obtaining adequate c-Fos protein detection in the monkey brain. In our study, we employed two different anti-c-Fos antibodies to verify specificity. Both antibodies produced similar, though weak, staining. It is not clear why Fos antisera consistently produces less than optimal staining in primate brain tissue compared to other mammals. One recent suggestion is the presence of unbound structural proteins that may interfere with specific binding of the antisera (Casto-Balderrama et al., 1998).

Old World versus New World Monkeys Our observation that zif268 expression is modulated by visual input throughout the entire critical period appears to contradict the findings in New World monkeys (Silveira et al., 1996; Fonta et al., 1997). There are several possible reasons for this discrepancy, besides the obvious species differences. First, there are technical differences in the paradigm. The marmoset study (Fonta et al., 1997) used 2 weeks of monocular deprivation that was later shown to be too long to reveal Zif268-immunostained columns in adult members of this species (Markstahler et al., 1998). Second, our use of very short deprivation times (3 h) most likely ensures that staining patterns ref lect nascent architectural features rather than the possible consequences of prolonged deprivation in a highly plastic developing visual cortex. And finally, a number of New World monkey species display different degrees of ODC segregation (Florence and Kaas, 1992; Livingstone, 1996; Spatz, 1979) that may in turn ref lect developmental differences when compared to Old World monkeys.

Sensory Deprivation versus Stimulation Effects on zif268 Expression An interesting facet of our results was the notable absence of Zif268-immunostained columns after monocular visual deprivation at PD 0 and their gradual emergence to an adult-like pattern over the critical period. The lack of deprivation columns at PD 0 (Fig. 2) may be interpreted within the context of endogenously generated patterns of retina-driven cortical activity that has been shown to be present in other mammals in early development (Meister et al., 1991; Feller et al., 1997). Thus, a visual deficit at this early time-point may not be sufficient to disengage the genomic response from ongoing electrical activity in the cortex. Furthermore, our results support the notion that with further postnatal development, this intrinsic retinal contribution diminishes and cortical activity becomes progressively more reliant upon external visual input (Katz and Shatz, 1996). IEG-stained columns, on the other hand, were clearly visible at birth if a stimulation paradigm was followed instead (Fig. 3). Since IEG mRNA staining revealed one set of columns that were complementary to the protein stained set (Fig. 4B), this implies that mRNA expression in these columns resulted from sensory stimulation only. Thus, a clear distinction can be made between sensory deprivation versus stimulation protocols and their respective effects on gene expression, i.e. down- versus up-regulation. While monocular visual deprivation may not sufficiently down-regulate IEG expression below an intrinsic baseline in early development, our stimulation results at PD 0 and 6 show that light input can augment IEG expression over and above this constitutive level to reveal very well delineated columns by mRNA staining. The stimulation results are especially significant because they clarify the developmental milestones as to when neurons within existing columns can actively respond to changes in the visual environment. The presence of primate ODCs at birth, and thus prior to onset of any visual experience, is now well established (Horton and Hocking, 1996; Rakic, 1976, 1977). These anatomical studies are in agreement with a physiological demonstration of ODCs in which 3 h of visual deprivation at PD 0 showed columns by way of 2-deoxyglucose mapping (Des Rosiers et al., 1978). What we now show is that neurons within the visual cortex have the molecular machinery in place already at birth for coupling sensory inputs to active genomic responses. This is important because if IEG products do indeed participate in

experience-guided developmental plasticity, then a necessary requirement is their early modulation in response to changing sensory conditions. Laminar Patterns of c-fos Expression During Development Given the current belief that c-Fos has important, though poorly understood, roles in marshaling plastic changes in neocortex (Worley et al., 1990; Beaver et al., 1993; Mower, 1994; Kaczmarek and Chaudhuri, 1997), it was important to critically examine the expression profiles of this transcription factor during development in primates. In this study, we investigated sensory-driven c-Fos expression in monkey striate cortex with particular emphasis on laminar changes that occur during development. We have found that c-Fos expression in major strata of the primary visual cortex is similar in developing and adult monkeys (Fig. 5). This is surprising given that Mower and colleagues have reported high levels of c-Fos expression in the thalamorecepient layer (layer IV) as well as the supragranular (II/III) and one of the infragranular (VI) layers of kittens but not in adult cats, where c-Fos expression was largely confined to the supragranular and infragranular layers only (Mower, 1994; Kaplan et al., 1996). Given that layer IV is plastic in kittens but not in adults and, further, that neurons here are among the most active in adult visual cortex, the results suggested that c-fos induction was associated with plasticity and not just a simple response to neuronal activity. Thus, our finding that c-fos expression in the thalamorecepient layer IVC of the adult monkey at levels similar to the supra- and infragranular layers requires consideration with regard to its postulated role in plasticity. As with layer IV in the cat, layer IVC in the monkey is highly malleable during the critical period but not in the adult. Unlike the cat, however, reduced plasticity in this layer of the monkey is not accompanied by reduced c-fos expression. This suggests that the association between c-fos expression and plasticity is not an exclusive one and that sensory stimulation alone is effective. One explanation for the discrepant results in the two species (cat versus ver vet monkey) can be linked to differences in N-methyl-D-aspartate (NMDA) receptor distribution. In cats, NMDA receptor density becomes significantly reduced in layer IV after the critical period in comparison to other cortical layers (Bode-Greuel and Singer, 1989; Gordon et al., 1991). However, adult macaque monkeys retain these receptors in layer IVC at high levels that are similar to layers II/III and VI (Rosier et al., 1993; Huntley et al., 1994). We have also observed such a profile in the primary visual cortex of adult vervet monkeys (unpublished observations). The fact that high levels of NMDA receptor molecules is now shown to be accompanied by elevated c-fos expression in the relatively aplastic thalamorecepient layer of adult monkeys remains surprising. Two possible explanations may be offered. The molecular scenario guiding plasticity is obviously complex and requires interaction among multiple signaling pathways. It may be that in adult monkeys, the pathway involving NMDA receptors and c-fos activation remains functional in layer IVC neurons though some other critical link — possibly downstream to c-Fos itself — may be disrupted and in whose absence plastic changes fail to occur. Alternatively, c-fos expression in these neurons may be triggered by intracortical rather than geniculate afferents at synaptic sites with functional NMDA receptors. If it is the case that c-Fos expression is firmly linked to plasticity, then such circuits may remain malleable even


in the adult whereas geniculate afferents may have lost that feature because they no longer articulate with functional NMDA receptors and therefore cannot induce c-fos. Narrow Strip of c-Fos and CO Staining in Developing Striate Cortex The fine spatial detail offered by ICC staining revealed a narrow strip of c-Fos expression located at the border of layers IVC and V and seen only in infant monkeys. We observed the strip to be most intense at early time-points of the critical period. A distinctive feature of this strip was the lack of punctuated Fos staining, which suggested the absence of ODCs in this sublayer. Horton (1984) has also observed that this strip as seen with CO is never affected by monocular deprivation. The exact classification of this strip remains unclear. It has been proposed that the strip may belong to either the lower part of layer IVC or the upper part of layer V (Kennedy et al., 1985; Spatz et al., 1993). Kennedy et al. ( 1985) showed that the strip receives thalamocortical projections. Thus, this sublayer could be a site where considerable activity-dependent competition for cortical territory occurs among geniculocortical afferents and where anatomical consequences following alterations in sensory input should be especially evident. Nevertheless, we failed to observe any effects of monocular visual manipulations that typically produce interdigitated columns of staining in other cortical layers. We offer three possible explanations for these results. First, ODCs may not yet be firmly developed within this strip at early time-points in the critical period. Rather, there may be an intermingling of eye inputs that through activity-driven competition is later sculpted into an adult-like pattern of columns. Second, the strip may be driven by waves of spontaneous retinal activity that may indeed retain eye segregation (Katz and Shatz, 1996; Feller et al., 1997). If so, then the external light manipulations that we used in this study would have had no effect on neuronal activity and consequently on c-Fos levels. And finally, it may be that c-Fos expression in this strip is not driven by the retina but rather is the consequence of a cortical developmental regime that is unconnected to visual input.

Notes We thank Dina Matsoukas and Fariborz R.-Dehgan for help with histology and photography. This research was supported by grants from the Medical Research Council of Canada (MRC) and Natural Sciences and Engineering Research Council (NSERC) to A.C. as well as a NATO Linkage Grant to A.C. and L.K. A.C. is an MRC Scholar. Address correspondence to Avi Chaudhuri, Department of Psychology, McGill University, 1205 Dr. Penfield Avenue, Montreal, Quebec, Canada H3A 1B1. Email: [email protected].

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