Molecular Correlates of Topographic Reorganization in Primary Visual Cortex Following Retinal Lesions

Shusei Obata1, Junko Obata, Aniruddha Das and Charles D. Gilbert

The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adult visual cortex undergoes substantial functional change as a result of alterations in visual experience. Binocular retinal lesions lead to a reorganization of the visuotopic map in primary visual cortex. Associated with this change is a strengthening of an existing plexus of long-range horizontal connections by sprouting of axon collaterals and synaptogenesis. To explore the molecular substrate of this change, we studied the expression of potential factors involved in neural plasticity in the area of reorganization. We found elevation in a number of factors as early as 3 days following the lesion, including neurotrophins BDNF, NT3, NGF and the insulin-like growth factor IGF-1. Associated with the changes in neurotrophin levels was an elevation in their receptors. We also measured elevation of transcription factors, CaMKII, MAP2 and synapsins. These experiments provide evidence for a signal transduction cascade associated with cortical reorganization.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Experience-dependent cortical plasticity is a well-documented characteristic of the developing brain. Even in adulthood, however, it is possible to instill profound changes in receptive field properties and in cortical functional architecture as a result of manipulation of the sensory periphery. This has been documented in a wide range of sensory systems. In somatosensory cortex, digit amputation or sensory nerve transection leads to a shift in the cortical representation of the affected digit to the adjacent, intact digits (Rasmusson, 1982Go, 1988Go; Merzenich et al., 1983aGo,bGo, 1984Go; Calford and Tweedale, 1988Go, 1991Go). In the visual system, lesioning the retina leads to a shrinkage in the cortical representation of the lesioned part of retina and an expansion in the cortical represenation of the part of the retina surrounding the lesion (Gilbert et al., 1990Go; Kaas et al., 1990Go; Chino et al., 1991Go, 1992Go; Heinen and Skavenski, 1991Go; Gilbert and Wiesel, 1992Go; Darian-Smith and Gilbert, 1995Go). At the level of individual cells, this topographic alteration is associated with a large shift in receptive field position. While the synaptic mechanisms of these changes are not fully understood, in the visual system the changes are most likely attributable to the long-range intrinsic horizontal connections formed by cortical pyramidal cells (Gilbert and Wiesel, 1992Go; Darian-Smith and Gilbert, 1995Go; Das and Gilbert, 1995Go). The exuberance of the connectivity within the plexus of horizontal connections allows information from large parts of the visual field to be fed to cells at a particular cortical site, thus allowing cells to alter their representation within the visual field. While these connections exist normally and are maintained throughout life, the change associated with topographic reorganization requires them to be strengthened from a subthreshold modulatory role to a suprathreshold, driving influence. The increase in strength of the horizontal connections following retinal lesions involves a sprouting of axon collaterals and synaptogenesis (Darian-Smith and Gilbert, 1994Go). The retinal lesion model presents an ideal system for characterizing the components of the cascade of molecular events leading to the elaboration of these connections. One has the ability to delineate a well-defined area of cortex that is affected by the retinal lesion, which we term the `cortical scotoma'. Initially the cortical scotoma represents an area that is silenced by the retinal lesion, but over a period of months it becomes responsive to visual stimuli, and is an area of active functional and structural alteration. Within the same cortical area it is possible to compare the expression of genes in the region of ongoing reorganization with the surrounding tissue, which is relatively quiescent and stable.

In this work we examine the expression of candidate molecules, including early immediate genes, neurotrophins, growth-associated proteins and synaptic vesicle-associated proteins. Many of these substances have been implicated in developmental plasticity. Rather than being suppressed to minor levels following the critical period of development, our evidence suggests that they may be recruited to fulfill the demands of alterations in synaptic weights associated with experiencedependent plasticity in adulthood.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Retinal Lesions

All surgeries and lesions were performed in cats anesthetized with sodium pentothal (20 mg/kg, maintained on 0.25 mg/kg/h) and paralyzed with pancuronium bromide (1.2 mg/h). Retinal lesions were made at corresponding positions in the two retinas as described previously (Darian-Smith and Gilbert, 1995Go). The lateral gyrus of the cat cerebral cortex was exposed, giving access to primary visual cortex (area V1, area 17) for electrophysiological recordings. Multiple unit extracellular recordings were made with tungsten electrodes, and receptive fields were mapped on a tangent screen with a hand-held projector over ~12 mm antero-posterior on the dorsal surface of area 17. In general, the mapping was done on one hemisphere in order to leave the second hemisphere unexposed to control for possible artifacts of cortical manipulation. Because the lesions were symmetrical in the two hemifields, the boundaries of the cortical scotomata that were mapped in one hemisphere could be extended to symmetrical positions in the opposite hemisphere. The sites for the retinal lesions were determined by recording from the desired site of the center of the cortical scotoma while stimulating with the guide beam of the laser. The lesions consisted of a series of adjacent lesions made by a diode ophthalmic laser (Iris Instruments) each at an energy of 800 mW for 1 s. After making the lesion, the boundary of the lesion was determined by back-projecting the lesion onto the tangent screen using a fundus camera (Zeiss). The diameter of the cortical scotoma was generally 3–9 mm, and was marked by inserting DiI-coated tungsten microelectrodes into identified cortical sites. The post-lesion survival periods included time points at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 3 months, 1 year and ~2 years.

Tissue Preparation

Under sodium pentothal anesthesia, cats were perfused with PBS, followed by 4% paraformaldehyde in 0.5 x PBS, 4% paraformaldehyde plus 15% sucrose in 0.5 x PBS and 25% sucrose. Blocks of visual cortex including the cortical scotomata were removed and immersed in 30% sucrose overnight. The blocks were then frozen in Tissu-tek (Miles) on dry ice and preserved at –80°C. Each block was cut tangentially into 20 µm sections by Cryostat (Zeiss). The sections were collected in 30% ethylene glycol (Sigma) plus 30% sucrose (Sigma) in 0.1 M sodium phosphate buffer, pH 7.4, and preserved at –30°C (Watson et al., 1986Go).

Immunohistochemistry

The source and working concentration of the antibodies are as follows: anti-BDNF rabbit serum (Chemicon, 1:600); anti-CaMKII mouse monoclonal antibody (specific to the alpha subunit, Boehringer Mannheim cat. no. 1481 703, 0.6 µg/ml); anti-CREB N-terminal sequence, rabbit serum (Dr Michael Greenberg, 1:800); anti-P-CREB rabbit serum (Dr Michael Greenberg, 1:600); anti-Egr1 rabbit serum (Santa Cruz Biotech., sc-110, 0.2 µg/ml); anti-GAP43 mouse monoclonal antibody (Boehringer Mannheim, 1.3 µg/ml); anti-cat GAP43 rabbit serum (Dr David Parkinson, 1:300); anti-GFAP rabbit IgG (Sigma, 6.4 µg/ml); anti-IGF-I sheep serum (Chemicon, 1:100); anti-MAP2 mouse monoclonal antibody (Sigma, HM2, 1:2000); anti-NGF rabbit serum (Chemicon, 1/100); anti-low affinity NGF receptor, p75, rabbit serum (Chemicon, 1:200); anti-NT3 rabbit serum (Chemicon, 1:600); anti-Synapsin I rabbit serum (Dr Andrew Czernik, 9 µg/ml); anti-Synapsin II rabbit serum (Dr Andrew Czernik, 4.7 µg/ml); anti-Synaptophysin mouse monoclonal antibody (Dr Andrew Czernik, 1:2000); anti-TrkA rabbit IgG (Santa Cruz Biotech., sc-118, 0.25 µg/ml); anti-TrkB rabbit IgG (Santa Cruz Biotech., sc-012, 0.3 µg/ml); antitruncated TrkB (TK-) rabbit IgG (Santa Cruz Biotech., sc-119, 1 µg/ml); anti-TrkC rabbit IgG (Santa Cruz Biotech., sc-117, 0.3 µg/ml).

Sections were stained by the avidin–biotin technique, using the Vectastain ABC kit (Burlingame, CA). The sections were washed three times in phosphate buffer, and incubated in a blocking solution containing 5% normal horse serum and 0.01% Triton X-100 in phosphate buffer for 2 h at 4°C. The sections were then incubated in phosphate buffer, which contained an antibody diluted at appropriate rate, 5% normal horse serum and 0.01% Triton X-100, for 3 days at 4°C. Anti-Synaptophysin and anti-Egr1 were incubated in the buffer without Triton X-100. After washing with phosphate buffer three times, the sections were incubated in buffer with appropriate Vectastain biotinylated second-antibody diluted in 1:200 concentration, 5% normal horse serum and 0.1% Triton X-100, for 1.5 h at 4°C. After washing three times, they were incubated in ABC reagent for 1.5 h at 4°C . After washing the reagent, they were color-developed with 0.05% diaminobenzidine and 0.01% H2O2. Sections were mounted on slides and coverslipped with Gel/MountTM (Biomeda), DPX mountant (BDH) or Gurr fluoromount mountant (BDH).

Photomicrographs of stained sections were taken with a digital camera (Kodak DCS200ci) and stored on a PC. Images were composed with Photoshop. The protocol for making higher-magnification, sideby-side views of staining in the area of the cortical scotoma and in the surrounding normal cortex were as follows. The photomicrographs were taken from the same sections under identical conditions, shifting the field of view by a few millimeters between one exposure and the next. The images were paired before further image processing. Hence any difference between the two areas is derived directly from the sections, and is not an artifact of image analysis.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To study the time course of expression of the factors that may contribute to the topographic reorganization of primary visual cortex following retinal lesions, we made binocular retinal lesions in 11 cats which survived for varying periods of time, from 3 days to 2 years. The cortical representations of the lesioned parts of the retinas, which we refer to as the cortical scotomata, ranged in diameter from 3 to 9 mm. For some animals we mapped the cortex before making the lesion in order to determine the boundary of the lesioned area, and the boundaries were marked by dye injection. These animals were also mapped at the end of the survival period in order to document the extent of the reorganization. In general we mapped one hemisphere and left the skull over the contralateral hemisphere intact so that we could control for possible effects of cortical manipulation. The lesions extended for considerable distances along isoelevation lines so that they included both hemifields, and would affect both hemispheres symmetrically. Sections from both hemispheres showed very similar patterns of staining, indicating that the patterns observed reflected the difference in neural activity caused by the lesion and not by exposing the cortex. In addition, we used one non-lesioned cat as a control, and none of the control sections showed the pattern of elevation of antibody staining seen in the experimental animals, but instead showed uniform staining.

Growth Factors and Receptors

There was significant elevation in the levels of anti-BDNF antibody staining extending throughout the cortical scotoma. For the experiments where the cortical scotoma was mapped electrophysiologically, we were able to see a close coincidence between the boundary of staining and the edge of the scotoma. The greatest elevation in staining was seen in the superficial cortical layers. Deep layers were also affected, but to a more limited extent. As shown in Figure 1Go, the anti-BDNF labeling extended across the full length of the cortical scotoma in the superficial layers. The strongest label was seen in the center of the scotoma, where it was also found in the deeper cortical layers. The increase in BDNF expression was seen at all time points examined, from 3 days to 2 years survival (Fig. 2Go). At the earlier times (3 days and 1 week), and with larger cortical scotomata, the elevation in staining was more pronounced towards the inner boundary of the scotoma than at the center (Figs 2A,B, 3AGoGo). Not uncommonly, one side of the scotoma was more affected than the opposite side. At later times the staining was highest at the center. No staining was observed in the absence of the primary antibody.



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Figure 1. Change in anti-BDNF staining in primary visual cortex at 87 weeks post-lesion. The highest levels of expression were found in the superficial cortical layers, extending across the full extent of the scotoma (indicated by the double-headed arrow). Increased levels in the deep layers were also seen in the central part of the scotoma.

 


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Figure 2. Changes in BDNF and Synapsin I expression in transverse sections over a range of time (3 days to 2 years) following binocular retinal lesions. Elevation in BDNF expression was seen within 3 days following the lesion, as shown at top left. The elevation was maintained at all time points shown, although the difference was not as pronounced at the intermediate times (4 weeks). The expression begins in the superficial layers and, at the longest times, extends down to the deep layers as well. Synapsin I expression mirrors that of BDNF both in terms of tangential and laminar distribution. Scale marker = 1 mm.

 


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Figure 3. Survey of expression of trophic factors, BDNF, NT3, NGF and IGF-1, at 1 week following the lesioning procedure, all taken from tangential sections of the same hemisphere at 1 mm, for comparison of relative distributions. In this hemisphere, those factors showing elevation had similar distributions, concentrated towards the posterior and, to a lesser extent, the anterior thirds of the cortical scotoma. Similar patterns were seen for the four factors at all survival time. Scale marker = 1 mm.

 
Other growth factors showed similar patterns of staining. Anti NT-3 and NGF antibodies showed a pattern of elevation in staining at 1 week similar to that of the anti BDNF antibody in the same animal (Fig. 3B,CGo). Also, IGF-1 immunoreactivity was increased, as shown in Figure 3DGo.

The change in BDNF staining is shown at higher magnification in Figure 4Go. The photomicrographs shown indicate an elevation in diffuse staining surrounding the cell somata. Although the somata also appear to be darker, closer inspection reveals the dark profiles to consist of punctate staining near the surfaces of the cell somata. The stained cells include a wide range of cell sizes, including the largest pyramidal cells.



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Figure 4. Comparison of the cellular distributions of trophic factors, BDNF, NT3, NGF and IGF-1, taken from the opposite hemisphere of those shown in Figure 1Go, at 87 weeks following the lesioning procedure. The primary change in the trophic factors staining in the cortical scotoma relative to the surrounding region was both in the cell somata and in the surrounding diffuse staining. Scale marker = 0.1 mm.

 
The level of expression of neurotrophin receptors appear to mirror that of the neurotrophins themselves. Low-affinity NGF receptor p75, TrkB and TrkC all showed increased staining (Fig. 5B,C,EGo). Interestingly, TrkA, the high-affinity NGF receptor, showed no obvious difference in staining intensity in the scotoma (Fig. 5AGo). The truncated form of the TrkB receptor also showed uniform levels of staining throughout (Fig. 5DGo). The change of TrkB and TrkC expression was seen in the neuropil of the superficial cortical layers, as shown when comparing Figure 6C with 6D and 6E with 6FGo. There were fewer TrkBand TrkC-positive cells than those positive for the neurotrophins themselves (compare Fig. 4 with Fig. 6GoGo), and the cell staining was considerably more sparse for TrkB than for TrkC and p75.



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Figure 5. Survey of expression of the receptors for neurotrophic factors, Trk A, p75 (low-affinity NGF receptor), TrkB, truncated TrkB (TK-) and TrkC, taken from the same hemisphere and the approximately same depth as the sections shown in Figure 3Go. p75, TrkB and TrkC were elevated and showed the same distribution as neurotrophins in Figure 1Go. TrkA showed virtually no change, and the truncated form of the TrkB receptor showed uniform staining throughout, indicating that the predominant change was in the active form of the receptor. Scale marker = 1 mm.

 


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Figure 6. Comparison of the cellular distributions of p75, TrkB, TrkC and MAP2 in the same area shown in Figure 4Go. TrkB and TrkC in the cortical scotoma showed strong elevation in neuropil in layers 2 + 3. TrkB showed staining in a smaller proportion of the cell somata than BDNF. The distribution of MAP2 staining seemed quite similar to that of TrkB, seen in the large pyramidal cell bodies and neuropil. Scale marker = 0.1 mm. Laminar boundaries are indicated at right.

 
Early Immediate Genes and Associated Signal Transduction Factors

Several transcription factors have been shown to be regulated after brief periods of increasing neural activity, including C-Fos, Egr1 (also known as Zif268 or NGFI-A) and CREB. In our cortical scotomata, the most pronounced changes concerning these factors were seen for CREB, which showed elevation within the area of the cortical scotoma. The period over which we observed this elevation was comparable to that seen for BDNF (Figs 3A and 7AGoGo). As expected, CREB was localized primarily in the nucleus (Fig. 8A,BGo). While N-CREB itself was elevated both at early and late times following the lesion (3 days and 87 weeks), the phosphorylated form, P-CREB, was only elevated after much longer intervals (Fig. 8DGo), presumably after the recovery of visually driven activity within the cortical scotoma. Another transcription factor, Egr1, also showed some elevation (Fig. 7BGo).



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Figure 7. Survey of expression of proteins potentially related to plasticity, CREB, Egr1, CaMKII, MAP2, GAP43, Synapsin I, Synapsin II and Synaptophysin, from the same hemisphere and the same area at 1 week shown in Figures 2 and 4GoGo. The early immediate gene egr1 (also known as zif268 and NGFI-A) was elevated in a region similar to that seen for the neurotrophins, as was the transcription factor CREB and the protein kinase CaMKII. MAP2 showed a clear elevation in expression, but any change in GAP43 was minor, if any. Of the synaptic vesicle-associated proteins, Synapsins I and II were elevated, but synaptophysin showed little change. GFAP was elevated. Scale marker = 1 mm.

 


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Figure 8. Staining by N-CREB, an antibody against the N-terminal sequence of {alpha}CREB, and P-CREB, an antibody against phosphorylated CREB (Ginty et al., 1993Go), at 3 days and 87 weeks following lesioning. Note that N-CREB showed elevation at the early time point, but P-CREB did not. At the longest survival, both N-CREB and P-CREB showed elevation. Scale marker = 0.1 mm.

 
CaMKII phosphorylates various proteins, including CREB, and is thought to have an important role in signal transduction. CaMKII showed strong elevation in the superficial cortical layers within the cortical scotoma, as shown Figure 7CGo. The elevation of CaMKII expression was most apparent in the neuropil, and included layer 1 (Fig. 9AGo).



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Figure 9. Comparison of the cellular distributions of CaMKII and Synapsin I in the same area as Figure 4Go. CaMKII showed elevation in superficial layers. The tangential and cellular distribution of Synapsin I was similar to that of BDNF. Scale marker = 0.1 mm. Laminar boundaries indicated at right.

 
Growth-associated Proteins and Microtubular Proteins

Growth-associated proteins have been linked to neurite extension in developing systems, and various forms of MAP2 have been shown to exist at elevated levels also during development. In the cortical scotoma, we did observe a short-lived and minor elevation in the level of GAP43, but this effect appeared to be minor. MAP2 expression, however, showed considerable elevation, as shown in Figure 7EGo. Interestingly, the laminar and cellular distribution of the increase in MAP2 was quite similar to that observed for TrkB, with the most darkly stained cells being large pyramidal cells (Fig. 6EGo).

Synaptic Vesicle-associated Proteins

Associated with the sprouting of axon collaterals and with synaptogenesis, one would expect an increase in the expression of structural proteins associated with synapses, presuming that the area of reorganization contained a net increase in synaptic density. We chose markers known to be associated with synaptic vesicles, synapsins I and II and synaptophysin. Synapsins I and II showed the same expression pattern, with a pronounced elevation within the cortical scotoma, and the greatest effects at the longest survival times following the lesion. As shown in Figures 2G, 7 and 3AGoGoGo, the distribution of synapsin I was nearly identical to that observed for BDNF. The laminar and cellular distribution of the change was also similar to that seen for BDNF, concentrated in the neuropil of the superficial layers. Synaptophysin, on the other hand, showed no difference in intensity (Fig. 7HGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurotrophins have been implicated in developmental plasticity. It has been suggested that in the visual cortex, the competitive interaction between inputs coming from the two eyes may be regulated by NGF (Maffei et al., 1992Go), BDNF (Cabelli et al., 1995Go) and/or NT-4 (Riddle et al., 1995Go). The effects of monocular deprivation can be blocked by infusion of neurotrophins or potentiated by blocking neurotrophin action via antibodies (Maffei et al., 1992Go; Carmignoto et al., 1993Go; Berardi et al., 1994Go; Cabelli et al., 1995Go; Fiorentini et al., 1995Go; Riddle et al., 1995Go; Griesbeck et al., 1996Go). Both dendritic (McAllister et al., 1995Go, 1997Go) and axonal (Cohen-Cory and Fraser, 1995Go) growth is regulated by neurotrophins in the developing brain. Given that the functional reorganization following retinal lesions involves growth of axon collaterals (Darian-Smith and Gilbert, 1994Go), it seemed plausible that neurotrophins might be involved.

Our results suggest that, indeed, by modification of visual experience in adult animals, the subsequent changes in cortical topography may involve an increase in expression of neurotrophins including BDNF and NT-3. The response in neurotrophin expression is quite rapid, increasing in the region of the cortical scotoma within 3 days following the lesion (the earliest time point in our study). The role of BDNF in modulating activity is seen in rat barrel cortex where infusion of BDNF leads to an enlarged area of activation with whisker stimulation (Prakash et al., 1996Go) and in facilitation of hippocampal synapses (Kang and Schuman, 1995Go). These effects are quite rapid, with changes occurring within 1 h. The recovery in the cortical scotoma shows both rapid changes near the margin of the scotoma, possibly reflecting an unmasking of existing connections, and a slow period of recovery towards the center of the scotoma, involving synaptogenesis (Gilbert and Wiesel, 1992Go; Darian-Smith and Gilbert, 1995Go). The BDNF elevation may be involved in both shortand long-term effects, by facilitating existing synapses and creating new ones. The fact that neurotrophins are elevated relatively soon after making a lesion, and that neurotrophins can induce sprouting within hours in some systems, leaves open the question of why the cortical reorganization takes several months to cover the area of recovery. It may be that the effects in developing systems have a different time course than those seen in adult animals. Interestingly, in our experiments the increase in neurotrophin expression appears to come in two waves, one within days, with a dip at 4 weeks, and then a second increase in expression at 3 months, extending for more than a year. Perhaps only the second wave is associated with synaptic proliferation. The fact that the elevation of neurotrophin levels is sustained, and can be seen even 2 years following the lesion, may reflect the fact that even though visually driven activity returns to the area of the cortical scotoma, the level of activity never returns fully to that of the surrounding cortex, and some imbalance of activity persists (Das and Gilbert, 1995Go).

Associated with the elevation of the neurotrophins was an elevation of their receptors. Among the receptors, TrkB and TrkC were significantly elevated, although in a smaller proportion of cells than those showing elevation of neurotrophin expression. The low-affinity receptor, p75, was elevated significantly in almost all cells, but TrkA appeared to be relatively unaffected. This is consistent with earlier work showing that infusion of NGF elevates the expression level of p75 but not of TrkA (Cavicchioli et al., 1989Go; Verge et al., 1992Go). The neurotrophin receptors, including TrkB, TrkC and p75, have been shown to be present in the visual cortex and to be regulated during visual system development, and may play a role in plasticity during the critical period (Allendoerfer et al., 1994Go). Overall, our results suggest that the NGF/TrkA system is less involved in the cortical reorganization than the BDNF/TrkB or NT-3/TrkC system. The effects of NGF elevation could be mediated, however, by p75. The fact that in our experiments no change was seen in the truncated TrkB receptor, which may be involved in guiding growing axons (Frisen et al., 1992Go, 1993Go), and that most of the observed change was in the active form, suggests that the full receptor is responsible for the recovery within the cortical scotoma.

The insulin-like growth factors have been implicated in nerve sprouting in the peripheral nervous system (Caroni et al., 1994Go) but are distributed throughout the central nervous system (Folli et al., 1994Go; De Pablo and De la Rosa, 1995Go). The elevation we see of IGF-1 may indicate a possible role in the sprouting itself, or in the subsequent increase in myelination (Ye et al., 1995Go). In addition to the sprouting of axon collaterals that is associated with reorganization in our system, there was also an increase in GFAP expression.

Although the BDNF elevation invariably occurred within the area of the cortical scotoma, it did not always extend throughout that region. This may reflect the pattern of recovery seen physiologically, where one often sees a faster regression of the boundary of the scotoma on one side than on the other. Interestingly, the laminar pattern of elevation also mirrors the laminar distribution of functional recovery as well as that of the horizontal connections, which tend to target cells in the superficial and deep layers, and are relatively absent in layer 4. The fact that the involvement of deep layers was less extensive than that of the superficial layers may reflect differences in receptive field size, deeper layers having much larger receptive fields, so that deep layer cells near the edge of the scotoma may have a part of their receptive fields left intact after the lesion.

Much of the literature on activity-dependent regulation of neurotrophins (as well as that on early immediate genes) points towards an association between increase in activity and neurotrophin expression. The situation is not so straightforward in this system. The lesions effectively silence the cells within the cortical scotoma (to visually driven activity, though there is still some spontaneous activity). It has been suggested that the relationship between activity and the regulation of synaptic weight, however, is normalized by the overall level of background activity in a system, sometimes referred to as a `sliding threshold' model (Bienenstock et al., 1982Go). In effect, the background level of activity modulates the level of activity in the inputs required for potentiation, so that by silencing the cells in the scotoma, the horizontal inputs, which are still active, become potentiated. Moreover, silencing of cells within the cortical scotoma could reduce tonic inhibition exerted on the cells in the surrounding region, leading to a net increase in activity for the cells projecting from parts of the cortex unaffected by the lesion to the center of the scotoma. In the end, the effect of synaptic plasticity in our system serves to correct an imbalance of activity between one cortical region and another, by strengthening the drive from cells in the active region to cells in the inactive region.

The mechanism of recovery in the cortical scotoma is likely to involve a strengthening of the long-range horizontal connections formed by cortical pyramidal cells (Gilbert and Wiesel, 1992Go; Darian-Smith and Gilbert, 1995Go). These connections run between cells with receptive fields in widely separated parts of the visual field, and therefore enable cells in the scotoma to receive input from parts of the retina surrounding the lesion. Though horizontal connections exist throughout cortex under normal circumstances, the shift in receptive field positions requires them to be strengthened, a process that may be mediated by the factors investigated here. Another possible morphological change, which has not yet been studied in this system, is an alteration in the dendritic fields of the target cells in the cortical scotoma. Neurotrophins have been shown to affect the growth of dendrites in slices of ferret visual cortex, though different neurotrophins appear to have antagonistic effects (McAllister et al., 1997Go).

Previous experiments on monocular deprivation also show an influence of the resultant change in activity on early immediate genes and the presumed targets of these genes. The ocular dominance/monocular deprivation system, both in developing and in adult animals, has provided an important model for studying not only the roles of these genes in activity-dependent plasticity, but also of regulation of GABA and glutamate synthetic enzymes and receptors (Jones et al., 1990Go; Simon et al., 1992Go; Hendry and Carder, 1992Go; Hofer et al., 1994Go; Cline et al., 1996Go). While monocular deprivation leads to an enhanced immunoreactivity of CaMKII, MAP2 and Zif268 are reduced (Hendry and Kennedy, 1986Go; Jones et al., 1990Go; Benson et al., 1991Go; Hendry and Bhandari, 1992Go; Chaudhuri et al., 1995Go). These three components studied in our system were elevated, suggesting a fundamental difference between an imbalance of activity induced between alternate ocular dominance columns and one induced between cortical regions serving different parts of the visual field. The salient difference between these experiments is that with the cortical scotoma, horizontal connections and superficial layer cells are involved, whereas with ocular dominance the primary effect is in layer 4.

Another important issue addressed by this study concerns the identity of the molecular intermediates by which activity is coupled to neurotrophin levels. One possibility is that second messengers, which have been implicated in synaptic plasticity, such as phosphorylated transcription factors (P-CREB, for example) or Ca2+, may detect levels of synaptic activity and induce gene expression (reviewed by Frank and Greenberg, 1994; Ghosh et al., 1994; Malenka, 1994; Ghosh and Greenberg, 1995). CREB regulates a number of genes, including egr1 (Sakamoto et al., 1991Go), CaMKII (Olson et al., 1995Go) and synapsin I (Sauerwald et al., 1990Go). CREB has been implicated in the formation of long-term memory (Tully et al., 1994Go, Yin et al., 1994Go, Huang et al., 1994Go; Bourtchuladze et al., 1994; Deisseroth et al., 1996Go), which may invoke similar mechanisms to the cortical reorganization seen after peripheral lesions. CREBor Ca2+-dependent protein kinases may regulate BDNF expression (Condorelli et al., 1994Go; Ghosh et al., 1994Go; Nibuya et al., 1996Go), either independently or in association. We did see elevated CREB and CaMKII expression contemporaneously with the BDNF elevation. It is not clear which is the proximal event, though neurotrophic factors have been shown to phosphorylate CREB (Ginty et al., 1993Go, 1994Go). In our system, P-CREB, the presumed active form of CREB, was expressed only after the initial phase of BDNF elevation. Perhaps other forms of CREB or other intermediates promote neurotrophin expression.

Some of the probes we used were intended to represent possible downstream effects of the action of the trophic factors. Synapsin I, which is a membrane protein of synaptic vesicles (De Camilli et al., 1983Go, 1990Go), showed exactly the same staining pattern as BDNF, and seems to parallel quite closely in time the expression of neurotrophins and their receptors. Another factor we examined is the growth-associated protein GAP43. This shows elevation in the LGN following retinal lesions, though not in the cortex (Baekelandt et al., 1994Go). We observed only minimal changes in GAP43 expression in the region of the cortical scotoma. MAPs, which can be substrates of various kinases, including CaMKII (Quinlan and Halpain, 1996Go), are possible intracellular regulators of process outgrowth and maintenance (reviewed by Tucker, 1990). MAP2c is expressed temporally in development and associated with plasticity in the adult. Our antibody was not MAP2c specific, reacting with MAP2a, b and c, but it is not unlikely that the observed change may have come primarily from changes in levels of MAP2c.

Clearly these experiments have only begun to trace the sequence of molecular changes responsible for reorganization of cortical topography. What is apparent, however, is that the functional and anatomical plasticity observed in cortex following peripheral lesions is associated with an upregulation of a broad range of factors in a very short time span and these factors are kept at high levels for an extended period of time. These factors represent some of the same constituents of the transduction cascade underlying developmental organization and plasticity, which are brought back into action for the purpose of adult, experience-dependent plasticity. Our experiments show that the cortical scotoma is a useful model system for studying the molecular mechanisms of adult cortical plasticity, and may be applicable not only to recovery of function after central nervous system damage, but also to experience-dependent functional alterations of cortical maps and to receptive field properties that occur normally.


    Notes
 
We thank Dr Michael Greenberg for anti-CREB antibodies, Dr David Parkinson for anti-cat GAP43 and Dr Andrew Czernik for anti-synapsin and anti-synaptophysin antibodies. We also thank Ella Leers and Rishi Goyal for technical assistance. This work was supported by NSF grant IBN 9630508 and by a fellowship from the Toyota Motor Corporation to S.O.

Address correspondence to Charles D. Gilbert, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. Email: gilbert{at}rockvax.rockefeller.edu.


    Notes
 
1 Current address: FP Div. No. 1, Toyota Motor Corporation, 1 Toyota-cho, Toyota, Aichi 471, Japan Back


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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