Abstract

Retinal lesions induce a topographic reorganization in the corresponding lesion projection zone (LPZ) in the visual cortex of adult cats. To gain a better insight into the reactivation dynamics, we investigated the alterations in cortical activity throughout area 17. We implemented in situ hybridization and real-time polymerase chain reaction to analyze the spatiotemporal expression patterns of the activity marker genes zif268 and c-fos. The immediate early gene (IEG) data confirmed a strong and permanent activity decrease in the center of the LPZ as previously described by electrophysiology. A recovery of IEG expression was clearly measured in the border of the LPZ. We were able to register reorganization over 2.5–6 mm. We also present evidence that the central retinal lesions concomitantly influence the activity in far peripheral parts of area 17. Its IEG expression levels appeared dependent of time and distance from the LPZ. We therefore propose that coupled changes in activity occur inside and outside the LPZ. In conclusion, alterations in activity reporter gene expression throughout area 17 contribute to the lesion-induced functional reorganization.

Introduction

In mammals, the precisely organized cortical maps remain malleable in response to changes in sensory input throughout life (Kaas 1991; Buonomano and Merzenich 1998). Depriving a small area of primary visual cortex from its retinal input can cause a dramatic reorganization of its topography (Eysel et al. 1980; Kaas et al. 1990; Heinen and Skavenski 1991; Chino et al. 1992; Gilbert and Wiesel 1992; Chino 1995; Chino, Smith, et al. 1995; Das and Gilbert 1995a, 1995b; Eysel and Schweigart 1999; Dreher et al. 2001). Immediately after inducing small binocular homonymous retinal lesions, neurons in the cortical lesion projection zone (LPZ) become silenced, whereas neurons at the border of the LPZ remain responsive but display enlarged receptive fields that are slightly shifted toward the intact retina surrounding the lesion. Upon longer recovery periods, much larger receptive field shifts take place, which can result in the complete filling-in of the LPZ. Central retinal lesions of more than 5° in the visual field, however, have been shown to result in an LPZ consisting of a permanently deprived center surrounded by a reorganizing border (Kaas et al. 1990; Heinen and Skavenski 1991; Chino et al. 1992; Gilbert and Wiesel 1992; Donoghue 1995; Chino, Smith, et al. 1995; Darian-Smith and Gilbert 1995; Schmid et al. 1996; Calford et al. 2000).

It is generally assumed that this functional reorganization is based on alterations in the excitation/inhibition balance at the cortical level (Jones 1993; Das and Gilbert 1995b; Wörgötter et al. 1998; Arckens et al. 1998, Arckens, Schweigart, et al. 2000). On the other hand, cortical reorganization has been questioned on the basis of a permanently reduced metabolic signal in the LPZ of primates (Horton and Hocking 1998) and the lack of evidence for shrinkage of the initially silent cortical region when examined by functional magnetic resonance imaging (fMRI; Smirnakis et al. 2005; but see Calford et al. 2005; Giannikopoulos and Eysel 2006). Furthermore, until now investigations mainly focused on the activity changes in the center and the border of the LPZ. There was little interest in the cortical regions representing the nonlesioned peripheral retina. Peripheral area 17 has been considered to be normal because it is not directly deprived of visual input. But this axiom has never been thoroughly tested. So far, only time-dependent fluctuations in excitatory and inhibitory neurotransmitter levels have been described far outside the LPZ (Massie, Cnops, Jacobs, et al. 2003; Massie, Cnops, Smolders, et al. 2003; Qu et al. 2003).

We therefore initiated a detailed investigation of short- and long-term activity changes by charting the expression of the activity reporter genes zif268 and c-fos throughout the binocular part of area 17 in subjects with matching retinal lesions in the 2 eyes. Zif268 and c-fos are immediate early genes (IEGs) known to be rapidly and transiently induced by a variety of stimuli and have been frequently used as markers for neuronal activity in the brain of different species (Sagar et al. 1988; Dragunow and Faull 1989; for review, see Sheng and Greenberg 1990; Worley et al. 1991; Hoffman et al. 1993; Sagar and Sharp 1993; Kaczmarek and Chaudhuri 1997; Herdegen and Leah 1998; Lehner et al. 2004). We first carried out in situ hybridization for zif268 to determine the exact size and location of the unresponsive zone in area 17 after different survival times. By means of real-time polymerase chain reaction (PCR), we then determined quantitative differences in the mRNA level of the IEG c-fos along the dorsoventral as well as the posterior–anterior (P--A) axis of area 17, allowing the combined analysis of the LPZ as well as far peripheral area 17. We here report the shrinkage of the unresponsive cortex over a distance of 2.5–6 mm within 3 months based on the detection of a permanently low IEG expression level in the center of the LPZ and a recovery of IEG levels in the border of the LPZ. An important fluctuation in IEG levels in far peripheral area 17 dependent on time postlesion as well as distance from the LPZ is also documented.

Materials and Methods

Animals and Tissue Preparation

All adult cats (n = 15, age between 12 K.U.Leuven and 20 months) were housed in the Animal Facilities of our Institute (K.U.Leuven, Belgium), exposed to a normal light environment (14:10 h light:dark) with access to food and water ad libitum. All animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the Institutional Ethical Committee of the K.U.Leuven. All efforts were made to minimize animals’ discomfort and to reduce the number of animals.

Three animals served as normal controls (N) and received no visual manipulation. Twelve animals received homonymous central retinal lesions by photocoagulation (LOG-2 Xenon light photocoagulator, Clinitex Inc., Denver, CO) under ketamine/xylazine anesthesia (0.5 mL Ketalar, 0.2 mL Rompun, intramuscularly [i.m.]). Nictitating membranes were retracted with phenylephrine hydrochloride (5%), and pupil size was stabilized with atropine sulfate (1%). Circular lesions with sharp borders and a ≈ 10° diameter were centered over the area centralis, as verified by fundus photography (Vandenbussche et al. 1990). This type of lesioning destroys all retinal cell layers (Eysel et al. 1981). The retinally lesioned subjects recovered for different survival times after the lesion: 14 days (14d, n = 3), 1 month (1m, n = 3), 3 months (3m, n = 3), or 8 months (8m, n = 3). A survival time less than 14 days was not included because of the effect of retinal edema in the first days after the lesions (Giannikopoulos and Eysel 2006). Fourteen days were chosen as the first time point because previous investigations have shown a maximal molecular response at that time point (for review, see Arckens 2006). The 8-month time point was chosen because connectional changes have so far only been documented for this long survival time (Darian-Smith and Gilbert 1994, 1995).

Normal as well as experimental animals were between 12 and 20 months of age at the time of the sacrifice. In cat the critical period ends at approximately 6–8 months after birth (Cynader et al. 1980), we therefore consistently excluded cats younger than 12 months from our investigations. Prior to sacrifice all animals were maintained overnight in total darkness followed by a 45-min light stimulation to induce maximal IEG expression in visual cortex (Arckens, Van der Gucht, et al. 2000; Van der Gucht et al. 2002). All cats were sacrificed with an overdose of pentobarbital (Nembutal, 60 mg/kg, intravenously) under deep ketamine anesthesia (Ketalar, 10 mg/kg, i.m.). Brains were immediately dissected, instantly frozen by immersion in dry ice cooled isopentane (Merck Eurolab, Leuven, Belgium), and stored at −70 °C. Coronal sections of 200 μm (for RNA extraction) and 25 μm (for in situ hybridization) were cut on a cryostat (Microm HM 500 OM, Waldorf, Germany).

In Situ Hybridization

As before, in situ hybridization for zif268 was applied to identify the position and the size of the unresponsive portion of the LPZ of area 17 on a series of coronal sections of each retinal lesion cat. A probe complementary to the nucleotides encoding amino acids 2–16 of the rat zif268 gene (5′-ccgttgctcagcagcatcatctcctccagyttrgggtagttgtcc-3′) was used (Arckens, Van der Gucht, et al. 2000; Van den Bergh et al. 2003; Leysen et al. 2004). The in situ hybridization experiment was performed as described previously (Arckens, Van der Gucht, et al. 2000). Briefly, after postfixation with 4% paraformaldehyde in phosphate-buffered saline (0.1 M, pH 7.4), slide-mounted sections of cat brain were dehydrated and delipidated. Sections were incubated overnight at 38 °C with hybridization solution containing the 3′-end terminal transferase 33P-dATP labeled probes specific for zif268. The next day, sections were thoroughly washed with 1× standard saline citrate buffer at 43 °C, dehydrated, and exposed to an autoradiographic Bio Max film (Kodak, Zaventem, Belgium). After 3 weeks, the film was developed following standard procedures. For image production from the autoradiograms, an HP Precisionscan Scanjet 5300C was used. Digital files were adjusted for brightness and contrast in Adobe Photoshop (version 9.0.2).

The retinotopic maps of Tusa et al. (1978) and Rosenquist (1985) were used as reference maps to define the extent of the unresponsive part of area 17 in degrees of the visual field along the dorsoventral and posterior–anterior axis of the brain.

Tissue Sampling

For the real-time PCR experiments, all tissue samples were taken from the binocular segment of area 17, either along the dorsoventral axis (Fig. 1A–C, white arrow) or else the posterior–anterior axis of area 17 (Fig. 1A,B,D, black arrow). For each axis, we implemented a different manner of tissue sampling (Fig. 1C,D). Along the dorsoventral axis, we collected RNA from the 3 discrete regions within area 17 from each of the frontal sections (Horsley–Clarke level posterior [P] 5.0). We will from this point on refer to these 3 regions as the center of the LPZ (C), the border of the LPZ (B), and far peripheral area 17 (P; Fig. 1C). Figure 1 illustrates how these regions span the whole of binocular area 17 with the far peripheral region approaching the borders of area 17 with area 20a. As significant differences were observed for the c-fos mRNA expression levels between these 3 area 17 regions, we then decided to study the possible existence of an activity gradient along the posterior–anterior axis of the brain between Horsley–Clarke coordinates P7 and anterior (A) 12 (Fig. 1D). Along the P–A axis, the visual field in area 17 is more elongated and stretches over a longer cortical surface, thereby allowing a finer spatial sampling as a function of visual degrees in the field (Fig. 1B). We isolated RNA from a series of frontal sections with an approximate interval of 800 μm between Horsley–Clarke coordinates P7 and A12, given that area 17 is only large enough between these coordinates to ensure correct tissue sampling and to avoid tissue sampling from nearby nonvisual cortex anterior from A12 (Fig. 1D). Using the cat visual cortex map of Rosenquist (1985) as a guide, we collected RNA on the area 17 side of the vertical meridian. We consistently punched one sample from each frontal section along the posterior–anterior axis of the brain (Fig. 1D).

Figure 1.

Projection of the visual field and regions of RNA collection onto area 17 (A, B). Visualization of the LPZ (in gray) onto a visual field map and on a medial view of the left hemisphere of the cat brain. White arrows indicate the analyzed region of the visual field/area 17 along the horizontal meridian (HM) and the black arrow along the vertical meridian (VM). Note how the binocular visual field has been analyzed over its maximal dimensions. (C) In situ hybridization for zif268 in the primary visual cortex 3 months postlesion. Indication of the 3 regions of interest along the dorsoventral axis for a given frontal brain section (Horsley–Clarke coordinate posterior 5 or P5.0): the permanently unresponsive center of the LPZ (C), the far peripheral part of area 17 (P), and the intermediate transiently deprived border of the LPZ (B). (D) Along the posterior–anterior axis, RNA was isolated from a series of coronal brain sections (approximate interval of 800 μm). Using the cat brain atlas of Rosenquist (1985) as a guide, we persistently punched samples of equal size (≈ 3 mm2) adjacent to the VM. The VM is indicated as a white line, the border between the unresponsive zone and the spared periphery is indicated with a white arrowhead and the black boxes indicate the regions of tissue sampling. Scale bar: 1 mm.

Figure 1.

Projection of the visual field and regions of RNA collection onto area 17 (A, B). Visualization of the LPZ (in gray) onto a visual field map and on a medial view of the left hemisphere of the cat brain. White arrows indicate the analyzed region of the visual field/area 17 along the horizontal meridian (HM) and the black arrow along the vertical meridian (VM). Note how the binocular visual field has been analyzed over its maximal dimensions. (C) In situ hybridization for zif268 in the primary visual cortex 3 months postlesion. Indication of the 3 regions of interest along the dorsoventral axis for a given frontal brain section (Horsley–Clarke coordinate posterior 5 or P5.0): the permanently unresponsive center of the LPZ (C), the far peripheral part of area 17 (P), and the intermediate transiently deprived border of the LPZ (B). (D) Along the posterior–anterior axis, RNA was isolated from a series of coronal brain sections (approximate interval of 800 μm). Using the cat brain atlas of Rosenquist (1985) as a guide, we persistently punched samples of equal size (≈ 3 mm2) adjacent to the VM. The VM is indicated as a white line, the border between the unresponsive zone and the spared periphery is indicated with a white arrowhead and the black boxes indicate the regions of tissue sampling. Scale bar: 1 mm.

Quantitative Real-Time PCR

All quantitative real-time PCR experiments have been done as described by Cnops et al. (2007). The RNA extraction was performed with the Versagene RNA purification kit (Gentra, Biozym, Landgraaf, The Netherlands) according to the manufacturer's instructions. After biophotometric analysis (Eppendorf, VWR International, Leuven, Belgium), RNA samples of identical quantity were reverse transcribed with GeneAmp RNA PCR products containing oligo d(T) primers (Applied Biosystems, Foster City, CA) at 42 °C for 60 min, 99 °C for 5 min, and 4 °C for 10 min. For the real-time PCR experiments, we used primers (Eurogentec, Seraing, Belgium) and TaqMan probes (Applied Biosystems), which were designed with the Primer Express program (Applied Biosystems), based on the cat sequence of c-fos (Forward primer: 5′-tgggctctcctgtcaatgc-3′; Reverse primer: 5′-cagtcaccgttgggataaagttg-3′; and TaqMan probe: 5′-caggacttctgcacggatctggcc-3′) or gapdh (Forward primer: 5′-tggaaagcccatcaccatct3-′; Reverse primer: 5′-caacatactcagcaccagcatca-3′; and TaqMan probe: 5′-ccaggagcgagatcccgcca-3′). The cDNAs were subjected to PCR utilizing the ABI Prism 7000 SDS apparatus in a 25-μL reaction of 1× Absolute QPCR Mix (Westburg, Leusden, The Netherlands) with primers at final concentration of 300 nM and probes of 200 nM. Serial dilutions of control cDNA for generating standard curves were run in duplicate for each gene, whereas target samples were run in triplicate on the same well-plate under standard amplification settings (1× 50 °C for 2 min, 1× 95 °C for 10 min, 40× 95 °C for 15 s, and 60 °C for 1 min). To compare samples between different runs, we included a reference control in every well-plate. Data were expressed relative to this reference control (Wong and Medrano 2005). Analysis was carried out using ABI Prism 7000 SDS software. C-fos quantities were normalized to the endogenous control gapdh to account for variability in initial mRNA concentrations and differences in reverse transcription efficiency. The relative amount of transcript was quantified by the comparative cycle threshold (Ct) method. To confirm reproducibility, we performed real-time PCR analysis on each cat (n = 3) at least 2 times. Statistical analysis of data was accomplished with the nonparametrical Kruskal–Wallis test and post hoc Wilcoxon test.

Results

Zif268-Based Detection of the Unresponsive Zone in Area 17

By means of in situ hybridization for zif268, we visualized the part of area 17 unresponsive to visual stimuli due to retinal lesions and we evaluated the position of its outer borders along the dorsoventral (D–V) axis (Fig. 2) and the posterior–anterior axis of the brain (Fig. 3). We determined the impact of postlesion survival time on the extent of this unresponsive cortical zone. Series of coronal sections demonstrate a fairly homogenous distribution of zif268 mRNA throughout area 17 of normal subjects (Figs 2 and 3). Central retinal lesions however induced a distinct decrease in zif268 expression in the topographically matching posterior, dorsal portions of area 17 of each experimental animal (Figs 2 and 3). The position of the border of this unresponsive zone as detected by a switch to high zif268 expression levels clearly shifted with survival time along the D–V and P–A axis of the brain, thereby creating a smaller unresponsive zone as well as an adjacent transiently unresponsive border zone characterized by a time-dependent restoration of zif268 expression. This recovery of zif268 expression was maximal at about 3 months postlesion since after that time-point no further significant shift was discernible, leaving the center of the LPZ permanently unresponsive to visual stimulation (Figs 2 and 3). Clearly, the outer rim of the LPZ recovered normal IEG levels.

Figure 2.

Effect of recovery time on the size of the unresponsive zone of area 17 along the dorsoventral axis as detected by in situ hybridization for zif268: Whereas normal subjects (N) exhibited a homogenous zif268 expression along area 17, the retinal lesion cats (RL) displayed a lower expression in the dorsally situated LPZ (= C+B) in comparison with the ventrally situated far peripheral parts of area 17 (P). By comparing the 14 days (14d) RL with the 1 (1m), 3 (3m), and 8 months (8m) RL, we noticed that the border between low central and high peripheral zif268 expression shifted approximately 2.5 mm dorsally into the LPZ with time. This resulted in size shrinkage of the deprived zone, but the LPZ was not observed to recover completely even out to 8 months. Consequently, we divided the LPZ into a permanently deafferented center (C) and a transiently deprived border zone (B). The border of the unresponsive zone in area 17 after a survival time of 14 days is indicated (black arrowhead) and extrapolated onto the 1m, 3m, and 8m RL. The border at longer survival times is indicated with a gray arrowhead. The white lines represent the vertical meridian (VM: posterior [P] 7.0 and P5.0) or the area centralis (AC: P4.0). Scale bar: 2 mm.

Figure 2.

Effect of recovery time on the size of the unresponsive zone of area 17 along the dorsoventral axis as detected by in situ hybridization for zif268: Whereas normal subjects (N) exhibited a homogenous zif268 expression along area 17, the retinal lesion cats (RL) displayed a lower expression in the dorsally situated LPZ (= C+B) in comparison with the ventrally situated far peripheral parts of area 17 (P). By comparing the 14 days (14d) RL with the 1 (1m), 3 (3m), and 8 months (8m) RL, we noticed that the border between low central and high peripheral zif268 expression shifted approximately 2.5 mm dorsally into the LPZ with time. This resulted in size shrinkage of the deprived zone, but the LPZ was not observed to recover completely even out to 8 months. Consequently, we divided the LPZ into a permanently deafferented center (C) and a transiently deprived border zone (B). The border of the unresponsive zone in area 17 after a survival time of 14 days is indicated (black arrowhead) and extrapolated onto the 1m, 3m, and 8m RL. The border at longer survival times is indicated with a gray arrowhead. The white lines represent the vertical meridian (VM: posterior [P] 7.0 and P5.0) or the area centralis (AC: P4.0). Scale bar: 2 mm.

Figure 3.

Effect of recovery time on the size of the unresponsive zone of area 17 along the posterior–anterior axis as detected by in situ hybridization for zif268: Whereas the normal cat (N) exhibited a homogenous zif268 expression over area 17 along the posterior–anterior axis, the retinal lesion cats (RL) displayed a lower expression in the centrally located LPZ (= C+B) in comparison with the far peripheral zone (P). By comparing the 14 days (14d) RL with the 1 (1m), 3 (3m), and 8 months (8m) RL, we noticed a posterior shift of the border of the unresponsive zone with time. The border can be set near Horsley–Clarke coordinate anterior 3 (A3) 14d postlesion but moves to posterior 3 (P3) after 3–8 months, indicating a maximal reorganization of 6 mm along the posterior–anterior axis. The border of the deprived zone along the posterior–anterior axis is shown as a black arrowhead. Scale bar: 2 mm.

Figure 3.

Effect of recovery time on the size of the unresponsive zone of area 17 along the posterior–anterior axis as detected by in situ hybridization for zif268: Whereas the normal cat (N) exhibited a homogenous zif268 expression over area 17 along the posterior–anterior axis, the retinal lesion cats (RL) displayed a lower expression in the centrally located LPZ (= C+B) in comparison with the far peripheral zone (P). By comparing the 14 days (14d) RL with the 1 (1m), 3 (3m), and 8 months (8m) RL, we noticed a posterior shift of the border of the unresponsive zone with time. The border can be set near Horsley–Clarke coordinate anterior 3 (A3) 14d postlesion but moves to posterior 3 (P3) after 3–8 months, indicating a maximal reorganization of 6 mm along the posterior–anterior axis. The border of the deprived zone along the posterior–anterior axis is shown as a black arrowhead. Scale bar: 2 mm.

Along the D–V axis, we determined the reorganization distance in millimeters at Horsley–Clarke coordinate P4, the position of the area centralis projection where the extent of reorganization can be most accurately verified. We measured a shift of zif268 expression over approximately 2.5 mm (Fig. 2). Along the P–A axis, we observed that 14 days after the induction of central retinal lesions, the border of the unresponsive area could be located near A3.0 (Fig. 3). After a longer survival time, the border had moved more posterior into the LPZ near P3.0. Our in situ results therefore indicate that the border could shift over a maximal distance of 6 mm (Fig. 3).

Quantitative Analysis of c-fos Activity Changes

In a second set of experiments, we have quantified the molecular activity changes after short-term and long-term survival times (Figs 1C and 4). Our quantitative results revealed a small yet statistically significant level of c-fos expression in the central part that was less than that of far peripheral area 17 (Table 1A, row 1; P < 0.01). This difference was however much more explicit in the visual cortex of retinally lesioned cats. Indeed, the retinal lesions induced a remarkably decreased c-fos expression in the LPZ in comparison with far peripheral area 17 in each animal (Fig. 4, Table 1A, rows 2–5; all P < 0.05). Further, c-fos expression in the center of the LPZ remained low, there was hardly any recovery in this zone even out to 8 months postlesion (Table 1B, C-column). In contrast, we observed a gradual increase in c-fos levels in the border of the LPZ with postlesion survival time (Table 1A, C/B-column, Table 1B, B-column rows 5–10). However, even at 8 months postlesion, normal levels were not restored in the border (Table 1B, B-column rows 1–4; all P < 0.01). Moreover, there were also clear modifications in far peripheral area 17. At short survival times (14d and 1m), this part of area 17 exhibited significantly lower activity levels than the corresponding region of area 17 in normal cats just like the whole LPZ (Table 1B, P-column rows 1–2; all P < 0.01). Yet at long survival times (3m and 8m), far peripheral area 17 regained the same activity level as the corresponding peripheral region in normal subjects (Table 1B, P-column rows 3–10). Only the border zone and far peripheral area 17 thus exhibited a time-dependent recovery of c-fos expression, and we observed the highest c-fos level 3 months postlesion in these 2 regions.

Table 1

Statistical analysis of the real-time PCR c-fos expression levels

 Dorsoventral axis
 
  Posterior–anterior axis
 
 C/B C/P B/P  C/B C/P B/P 
 0.108 0.004 0.394  0.142 0.020 0.959 
 14d RL 0.310 0.002 0.002  14d RL 0.132 0.026 0.132 
 1m RL 0.818 0.04 0.026  1m RL 0.065 0.002 0.002 
 3m RL 0.002 0.002 0.025  3m RL 0.002 0.002 0.065 
 8m RL 0.002 0.002 0.026  8m RL 0.004 0.004 0.055 
  
 N/14d RL 0.004 0.002 0.002  N/14d RL 0.002 0.008 0.008 
 N/1m RL 0.004 0.002 0.009  N/1m RL 0.002 0.013 0.414 
 N/3m RL 0.004 0.009 0.818  N/3m RL 0.002 0.108 0.491 
 N/8m L 0.004 0.002 0.485  N/8m RL 0.002 0.008 0.008 
 14d RL/1m RL 0.132 0.065 0.589  14d RL/1m RL 0.310 0.065 0.041 
 14d RL/3m RL 0.026 0.002 0.002  14d RL/3m RL 0.180 0.002 0.004 
 14d RL/8m RL 0.818 0.009 0.015  14d RL/8m RL 0.394 0.937 0.873 
 1m RL/3m RL 0.937 0.002 0.026  1m RL/3m RL 0.589 0.065 0.394 
 1m RL/8m RL 0.132 0.132 0.132  1m RL/8m RL 0.002 0.093 0.010 
 3m RL/8m RL 0.026 0.132 1.000  3m RL/8m RL 0.002 0.002 0.010 
 Dorsoventral axis
 
  Posterior–anterior axis
 
 C/B C/P B/P  C/B C/P B/P 
 0.108 0.004 0.394  0.142 0.020 0.959 
 14d RL 0.310 0.002 0.002  14d RL 0.132 0.026 0.132 
 1m RL 0.818 0.04 0.026  1m RL 0.065 0.002 0.002 
 3m RL 0.002 0.002 0.025  3m RL 0.002 0.002 0.065 
 8m RL 0.002 0.002 0.026  8m RL 0.004 0.004 0.055 
  
 N/14d RL 0.004 0.002 0.002  N/14d RL 0.002 0.008 0.008 
 N/1m RL 0.004 0.002 0.009  N/1m RL 0.002 0.013 0.414 
 N/3m RL 0.004 0.009 0.818  N/3m RL 0.002 0.108 0.491 
 N/8m L 0.004 0.002 0.485  N/8m RL 0.002 0.008 0.008 
 14d RL/1m RL 0.132 0.065 0.589  14d RL/1m RL 0.310 0.065 0.041 
 14d RL/3m RL 0.026 0.002 0.002  14d RL/3m RL 0.180 0.002 0.004 
 14d RL/8m RL 0.818 0.009 0.015  14d RL/8m RL 0.394 0.937 0.873 
 1m RL/3m RL 0.937 0.002 0.026  1m RL/3m RL 0.589 0.065 0.394 
 1m RL/8m RL 0.132 0.132 0.132  1m RL/8m RL 0.002 0.093 0.010 
 3m RL/8m RL 0.026 0.132 1.000  3m RL/8m RL 0.002 0.002 0.010 

Note: Statistical analysis of the differences in the c-fos expression levels in the permanently unresponsive center of the LPZ (C), the recovering border of the LPZ (B), and far peripheral area 17 (P) of RL with a survival time of 14 days (14d), 1 month (1m), 3 months (3m), and 8 months (8m) and the corresponding cortical regions in normal subjects (N), as accomplished with the Kruskal–Wallis test and post hoc Wilcoxon test. The P values are shown in the table.

Figure 4.

The relative c-fos mRNA concentrations along the dorsoventral axis: In the retinally lesioned animals (RL), the center of the LPZ (C) remains permanently unresponsive, whereas the border of the LPZ (B) undergoes a time-dependent recovery in c-fos expression. Interestingly, we also observed significant changes in far peripheral area 17 (P) in comparison with the corresponding cortical regions of normal cats (N). White circles denote individual cat data.

Figure 4.

The relative c-fos mRNA concentrations along the dorsoventral axis: In the retinally lesioned animals (RL), the center of the LPZ (C) remains permanently unresponsive, whereas the border of the LPZ (B) undergoes a time-dependent recovery in c-fos expression. Interestingly, we also observed significant changes in far peripheral area 17 (P) in comparison with the corresponding cortical regions of normal cats (N). White circles denote individual cat data.

An overall view of the activity course along the P–A axis based on c-fos mRNA levels showed that normal cats had a roughly constant activity level along this axis (Fig. 5). In contrast, animals with retinal lesions exhibited a clear activity gradient: c-fos expression was lowest in the center of the LPZ and augmented gradually toward anterior, far peripheral area 17. We could locate the border between the unresponsive zone and surrounding area 17 around A3.0 for 14-day subjects (Fig. 5). At longer survival times, the border had clearly shifted toward the center of the LPZ. A survival time of 1 month resulted in a position at P2.2. After a survival time of 3 months, the border was situated at P3.0. These P–A positions correlated well with the in situ findings. According to the real-time PCR data, we conclude that the recovery of c-fos levels along the P–A axis spanned approximately 6 mm of cortex (A3.0–P3.0), which had largely taken place within 1 month.

Figure 5.

C-fos expression as a function of time and space relative to the LPZ along the posterior–anterior axis: The mRNA concentrations of the activity marker c-fos as a function of distance from the LPZ between Horsley–Clarke coordinates posterior 7 (P7) and anterior 12 (A12) for normal cats (N) and retinal lesion cats (RL) with a survival time of 14 days (14d), 1 month (1m), 3 months (3m), and 8 months (8m). Black arrows indicate the border between the unresponsive and responsive regions of area 17. Previous immunocytochemical detection of glutamate and electrophysiological experiments has demonstrated that the border can be recognized by an activity peak (Arckens, Schweigart, et al. 2000; Giannikopoulos and Eysel 2006). We therefore have set the border at the first c-fos expression peak we encountered going from posterior to anterior. This border shifted from A3 at 14d postlesion to P3 at 3m postlesion and is therefore considered as the border of the LPZ (B), flanked on the left by the permanently unresponsive center of the LPZ (C) spanning from P7 to P3.8 and on the right by the far peripheral cortex (P) spanning from A3 to A12. White circles denote individual cat data.

Figure 5.

C-fos expression as a function of time and space relative to the LPZ along the posterior–anterior axis: The mRNA concentrations of the activity marker c-fos as a function of distance from the LPZ between Horsley–Clarke coordinates posterior 7 (P7) and anterior 12 (A12) for normal cats (N) and retinal lesion cats (RL) with a survival time of 14 days (14d), 1 month (1m), 3 months (3m), and 8 months (8m). Black arrows indicate the border between the unresponsive and responsive regions of area 17. Previous immunocytochemical detection of glutamate and electrophysiological experiments has demonstrated that the border can be recognized by an activity peak (Arckens, Schweigart, et al. 2000; Giannikopoulos and Eysel 2006). We therefore have set the border at the first c-fos expression peak we encountered going from posterior to anterior. This border shifted from A3 at 14d postlesion to P3 at 3m postlesion and is therefore considered as the border of the LPZ (B), flanked on the left by the permanently unresponsive center of the LPZ (C) spanning from P7 to P3.8 and on the right by the far peripheral cortex (P) spanning from A3 to A12. White circles denote individual cat data.

Fourteen days after the induction of the retinal lesions, a plateau of normal c-fos expression was only observed far anterior (A9–A12). One month postlesion we detected normal c-fos activity values at more posterior locations (A3). For the experimental animals with a survival time of 3 months, the switch to a normal activity level was located even more posterior (P1.4). This implies that besides the shift of the border, there is also a wave of normal c-fos expression shifting into the LPZ with increasing postlesion survival time (Fig. 5).

For statistical analysis of the c-fos data along the P–A axis, we analyzed the data of the 3 major parts of area 17, with the center of the LPZ spanning from P7 to P3, the border of the LPZ from P3 to A3, and far peripheral area 17 from A3 to A12 (Figs 5 and 6). As in the D–V axis, we observed a far greater significant difference between central and peripheral c-fos levels in the experimental animals regarding to normal animals (Table 1C; all P < 0.05, Fig. 6). The c-fos activity in the center of the LPZ remained decreased (Table 1D, C-column). On the other hand, the border of the LPZ exhibited significant restoration of normal c-fos levels 3 months postlesion (Table 1C, C/B-column, Table 1D, B-column). Once again we witnessed a clear-cut activity difference between far peripheral area 17 of experimental and normal subjects (Table 1D, P-column rows 1 and 4, all P < 0.01). Rather surprisingly, the 8-month cats showed a recurrent decrease in molecular activity throughout area 17. The center and border of the LPZ as well as peripheral area 17 had a significantly decreased c-fos expression in comparison with cortex of normal subjects (Table 1D, row 4, P < 0.01).

Figure 6.

The relative c-fos quantities along the posterior–anterior axis: We divided the P–A activity gradient in its 3 major parts: the center of the LPZ (C), the border of the LPZ (B), and far peripheral area 17 (P; as in Fig. 5). The retinal lesion cats (RL) exhibit a decreased c-fos expression in the LPZ. Only the border of the LPZ recovers with increasing survival time. Furthermore, the central retinal lesions also induce c-fos alterations in peripheral area 17 regarding the peripheral cortex of the normal cat (N). White circles denote individual cat data.

Figure 6.

The relative c-fos quantities along the posterior–anterior axis: We divided the P–A activity gradient in its 3 major parts: the center of the LPZ (C), the border of the LPZ (B), and far peripheral area 17 (P; as in Fig. 5). The retinal lesion cats (RL) exhibit a decreased c-fos expression in the LPZ. Only the border of the LPZ recovers with increasing survival time. Furthermore, the central retinal lesions also induce c-fos alterations in peripheral area 17 regarding the peripheral cortex of the normal cat (N). White circles denote individual cat data.

Cortical Reorganization as a Function of Visual Angle

Figure 7 summarizes and visualizes the spatiotemporal evolution of the c-fos activity changes in area 17. Over time, its unresponsive zone is significantly reduced in size in all directions. After a survival time of 14 days, the unresponsive zone still had the expected size considering the dimensions of the retinal lesions of 10° because its borders were detected around 5° of visual angle (yellow outline) in the P–A axis as well as the D–V axis. After a survival time of 1 month, the border had shifted in position, moving more dorsal and posterior toward the center of the LPZ (green outline). At 3 and 8 months, the border had moved slightly more inward (red and blue outline, respectively), but the largest shift of the border clearly took place within 1 month. Concerning the dimensions of the reorganization, we noticed a border shift over a larger distance in millimeters for the P–A axis than for the D–V axis. Nonetheless, as a function of degrees in the visual field, the reorganization was quite comparable for the 2 axes.

Figure 7.

Spatiotemporal evolution of the deprived zone in area 17: Below, a medial sight view of the cat brain illustrates the retinotopic organization of the visual field in area 17 (adapted from Tusa et al. [1978]: Copyright [1978, J. Comp. Neurol.]; “Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley and Sons, Inc.”). The size of the deprived zone is shown in yellow for the 14 days retinal lesion cat (14d RL), in green for the 1 month (1m) RL, in red for the 3 months (3m) RL, and in blue for the 8 months (8m) RL. The figure gives a clear visualization of the time-dependent shrinkage of the inactivated cortex along the dorsoventral axis as well as the posterior–anterior axis. In the right lower inset, this reorganization is shown in the visual field. In the line graph, the activity gradient along the posterior–anterior axis is displayed in a manner such that the Horsley–Clark coordinates of the sight view and the graph correlate with each other. Along the posterior–anterior axis, we were able to determine the spatial coordinates of the shifting border of the deprived zone. Next to the permanently deafferented center of the LPZ (C; P7.0 to P3.0) and the transiently deprived border of the LPZ (B; P3.0 to A3.0), the far peripheral cortex (P; A3–A12) appeared as consisting of 2 parts: a part adjacent to the LPZ border (A3–A9) in which time-dependent changes in activity occurred especially at 14 days and 8 months and a second remote portion (A9–A12) in which no obvious changes in c-fos expression were observed independent of postlesion survival time, as statistically assessed (see asterisks).

Figure 7.

Spatiotemporal evolution of the deprived zone in area 17: Below, a medial sight view of the cat brain illustrates the retinotopic organization of the visual field in area 17 (adapted from Tusa et al. [1978]: Copyright [1978, J. Comp. Neurol.]; “Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley and Sons, Inc.”). The size of the deprived zone is shown in yellow for the 14 days retinal lesion cat (14d RL), in green for the 1 month (1m) RL, in red for the 3 months (3m) RL, and in blue for the 8 months (8m) RL. The figure gives a clear visualization of the time-dependent shrinkage of the inactivated cortex along the dorsoventral axis as well as the posterior–anterior axis. In the right lower inset, this reorganization is shown in the visual field. In the line graph, the activity gradient along the posterior–anterior axis is displayed in a manner such that the Horsley–Clark coordinates of the sight view and the graph correlate with each other. Along the posterior–anterior axis, we were able to determine the spatial coordinates of the shifting border of the deprived zone. Next to the permanently deafferented center of the LPZ (C; P7.0 to P3.0) and the transiently deprived border of the LPZ (B; P3.0 to A3.0), the far peripheral cortex (P; A3–A12) appeared as consisting of 2 parts: a part adjacent to the LPZ border (A3–A9) in which time-dependent changes in activity occurred especially at 14 days and 8 months and a second remote portion (A9–A12) in which no obvious changes in c-fos expression were observed independent of postlesion survival time, as statistically assessed (see asterisks).

In the upper line graph of Figure 7, the activity gradient along the P–A axis is displayed, which we have obtained through real-time PCR for c-fos. This graph clearly shows that after an initial decrease in area 17, the cortical c-fos activity increased again in ensuing months. A wave of normal c-fos activity progressively moved into the LPZ. Indeed, at 14 days, this plateau of normal c-fos expression was only discernible anterior to A9 (yellow line). The c-fos activity level in the part of peripheral area 17 adjacent to the border of the LPZ (A3–A9) was significantly lower than normal (P < 0.05). The 1-month experimental animals displayed a normal expression level even in this region of area 17 closest to the border of the LPZ (green line). Separate statistical analysis of these 2 subdivisions in peripheral area 17 underscored these observations. This wave of recovered gene expression levels even moved into the LPZ, since 3 months postlesion, the c-fos expression in the border zone of the LPZ was no longer significantly different from normal values (red line). At 8 months, low c-fos activity reached from the center of the LPZ up to A9 (all P < 0.05). Only the outermost region of area 17 displayed c-fos levels indistinguishable from normal subjects. Consequently, far peripheral area 17 consisted of 2 subdivisions with one adjacent to the LPZ border displaying a dynamic c-fos expression, and one most remote region never differing from the normal situation concerning c-fos expression.

Discussion

IEGs as Markers of Neuronal Activity

The IEGs zif268 and c-fos can be rapidly and transiently induced by synaptic stimulation. Light stimulation specifically boosts IEG expression in mammalian visual cortex after a period of darkness (Worley et al. 1991; Arckens, Van der Gucht, et al. 2000; Correa-Lacárcel et al. 2000; Van der Gucht et al. 2002). Both IEGs are popular molecular tools for mapping functional brain activity (Sagar et al. 1988; Worley et al. 1991; Hoffman et al. 1993; Kaczmarek and Chaudhuri 1997; Herdegen and Leah 1998; Lehner et al. 2004). Especially, c-fos is well correlated with neuronal activity, whereas zif268 is more related to long-term potentiation-dependent processes (Heynen and Bear 2001; Soares et al. 2005; Aydin-Abidin et al. 2008). Several studies have demonstrated that c-fos expression is strongly linked to functional activity because electrophysiology (Hajós et al. 1999; Herry et al. 2007; Librizzi et al. 2007; Yamazaki et al. 2008) as well as fMRI (Aoki et al. 2004; Lawrence et al. 2004, 2007; Herry et al. 2007) experiments show similar activation patterns as c-fos. Our investigations further support this notion that IEGs can be considered as secondary indicators of activity. Indeed, the light-induced zif268 and c-fos observations in retinally lesioned animals corroborate all previous electrophysiological findings. Our 10° retinal lesions clearly caused a permanently deprived center in the primary visual cortex, characterized by a persistent low IEG expression. Kaas et al. (1990) also concluded, based on electrophysiological experiments that an LPZ originally larger than 5 visual degrees can not reorganize completely, leaving the most centrally situated neurons permanently unresponsive to visual stimuli. Yet, we observed a time-dependent IEG expression recovery in the outer rim of a given LPZ. As a result, within 3 months, the border of the LPZ, as detected with zif268 and c-fos, moves 2.5–6 mm along the D–V and the P–A axes of the brain, respectively, with the largest shifts occurring in the first month of the reorganization period. These observations correspond to the electrophysiological findings of Darian-Smith and Gilbert (1995) and Kaas et al. (1990), who described a topographical reorganization over 3–5 and 4–8 mm of visual cortex, respectively. Only recently, 2-photon imaging in mouse visual cortex also reported the recovery of visual responsiveness after a monocular retinal lesion 1–2 months postlesion (Keck et al. 2008). Smirnakis et al. (2005) challenged the occurrence of such a cortical reorganization in the monkey based on fMRI data. Yet, recent fMRI work in visual cortex of patients with macular degeneration or stroke did deliver solid evidence for shrinkage of the initially unresponsive cortical scotoma in humans (Baker et al. 2005, 2008; Dilks et al. 2007; Masuda et al. 2008).

Activity Changes outside the LPZ

Another important aspect of cortical reorganization that emerged from this study is the clear activity difference between far peripheral area 17 of normal and retinal lesion animals. This difference implies that retinal lesions placed in the area centralis affect neuronal activity well beyond central area 17. Cross-modal plasticity is another good example of how extensive the influence of sensorial manipulation can be. Apparently, the influence of a visual manipulation can reach far outside the directly sensory-deprived cortical zone, even into another sensorial system (Rauschecker 1996; Yaka et al. 1999). In 2000, Qu et al. discovered that the concentrations of the neuromodulators noradrenalin and dopamine were significantly increased in far peripheral area 17 of cats with retinal lesions. Moreover, microdialysis studies for the neurotransmitters γ-aminobutyric acid (GABA) and glutamate also revealed that over a distance of several millimeters, the cortex surrounding the LPZ is aberrant for the main inhibitory and excitatory cortical neurotransmitter. Massie, Cnops, Jacobs, et al. (2003) and Massie, Cnops, Smolders, et al. (2003) found increased concentrations for glutamate and GABA in far peripheral area 17 after a survival time of 1–2 months pointing to a positive net change in the activity balance in this part of area 17. The real-time data that we have obtained showed that at first the c-fos expression in peripheral area 17 was significantly lower than in normal cortex. In time, however, the c-fos quantity augmented, pointing to a “normalization” of the cortical activity around 1–3 months postlesion. Consequently, the timing of the recovery of the IEG expression fits the earlier microdialysis data for GABA and glutamate (Massie, Cnops, Jacobs, et al. 2003; Massie, Cnops, Smolders, et al. 2003).

The reason why the far peripheral regions of area 17, spared from a direct deafferentation-induced impact of the retinal lesions, also undergo a significant decrease in activity shortly after the induction of central retinal lesions (14d, 1m) might be a reduced lateral excitation via the horizontal fiber system originating inside the silenced LPZ. In normal circumstances, a neuronal cell in the visual cortex receives ascending input arising from the retina and lateral input from the surrounding cells. Central retinal lesions do not only abolish the ascending input to the LPZ but also the lateral input from the LPZ to the surrounding peripheral area 17. As a consequence, the adjoining peripheral part of area 17 will be decreased in neuronal activity because it is not stimulated by the horizontal connections coming from the LPZ. At the same time, the most remote peripheral part will exhibit a normal activity level because it gets normal upward as well as normal lateral input from the adjacent peripheral part. Therefore, the cortical activity is dependent on the distance from the LPZ. A similar reasoning may hold for the descending inputs from higher-order visual areas. Because higher order areas have been shown to also display an LPZ detectable with IEGs (Arckens, Van der Gucht, et al. 2000), also top-down inputs into area 17 may change with distance from the LPZ and may be responsible for the observed activity gradient in area 17.

Evidence for Functionally Meaningful Reorganization in Area 17

At the area centralis (P4), the D–V axis exhibited a maximal reorganization of 2.5 mm along the horizontal meridian (Figs 1A–C and 2). In contrast, this was around 6 mm for the P–A axis, which represents the vertical meridian (Figs 1A,B,D, 3, and 6). In visual degrees, the restoration of function is about equal in both dimensions (approximately 3.5°). The difference in distance of reorganization along the horizontal meridian and along the vertical meridian by a factor of about 2 might have its anatomical correlate in the anisotropic organization of horizontal connection networks along the 2 visuotopic axes. Indeed, Gilbert and Wiesel (1982) described that clustered axonal fields have an asymmetric organization, extending for greater distances along one cortical axis than along the orthogonal axis. Furthermore, single fiber tracings after injections of biocytine in layer III of cat area 17 revealed a difference in the extent of horizontal axonal fields on average by a factor of 1.72 in favor of the P–A axis (Kisvarday and Eysel 1992). These observations imply that the activity changes should not be expressed in millimeters but rather in degrees of the visual field to understand the equal restoration of activity in all dimensions. This underlines the functional relevance of the reorganization because it then respects visual angle as well as anatomical organization rather than absolute cortical distance.

Possible Reorganization Mechanisms throughout Area 17

The restoration of activity in area 17 had largely been completed on a short-term basis, meaning within 3 months, rendering the unresponsive zone strongly reduced in size. Between 3 and 8 months, there was no evidence for a further change in the dimensions of the permanently deprived center of the LPZ. In the first 3 months, this cortical reorganization may be carried by strengthening of the synaptic efficiency through the unmasking of existing connections, for example, the long range horizontal connections (Gilbert and Wiesel 1992; Chino, Smith, et al. 1995; Calford et al. 2003; Giannikopoulos and Eysel 2006). Yet, at 8 months, almost the entire area 17 again displayed lower c-fos expression levels compared with earlier time points. At the long-term, there may be a need for other reorganization mechanisms in comparison to early time points. At 8 months, mechanisms may apply in which c-fos does not play a relevant role anymore. Indeed, axonal outgrowth and branching inside the LPZ have been described at this time point following lesioning (Darian-Smith and Gilbert 1994, 1995; Donoghue 1995) and might bring about additional newly acquired receptive fields as recording probability increases inside the LPZ between 3 months and 1 year (Giannikopoulos and Eysel 2006). Other researchers also reported evidence that plasticity-mediated reorganization is not based on one mechanism but rather on the cooperation of various time-dependent mechanisms (Obata et al. 1999; Arckens 2006). Homeostatic plasticity has been described to occur in a late phase after Hebbian plasticity to counteract the destabilizing effects of Hebbian plasticity (Surmeier and Foehring 2004; Turrigiano and Nelson 2004; Pérez-Otaño and Ehlers 2005; Rabinowitch and Segev 2006). Our findings suggest that area 17 requires a new activity level many months after the lesions. This new, lower, and more economical activity balance between the 3 different regions within area 17 might be achieved through homeostatic plasticity whereby neurons seek to maintain a certain amount of total drive.

It is now clear that even at the long-term, there are still major modifications going on in area 17 due to the retinal lesions, in correspondence with earlier descriptions of gene and protein expression differences at these survival times (Obata et al. 1999, Arckens et al 2003). In fact, long-term alterations after the induction of retinal lesions are quite reasonable because it is commonly known that the topographical reorganization causes an overrepresentation of the perilesion area (Kaas et al. 1990; Gilbert and Wiesel 1992, Chino 1995; Gilbert et al. 1996). This filling-in process can lead to reactivation of visual responsiveness in the visual cortex but certainly not to the reinstatement of a normal representation of the complete visual field in the visual cortex. It is more a compensation mechanism through which as many as possible cells are again usefully recruited. Indeed, it has been reported that experience-dependent brain plasticity includes compensatory mechanisms (Rauschecker 1995; Temple et al. 2003; Metz et al. 2005). Because reorganization implies the development of new strategies, it is essential to have long-term changes to maintain these newly developed characteristics.

As in patients with macular degeneration, animals with central retinal lesions may implement adaptive strategies involving the use of the intact peripheral retina in place of the central scotoma for eccentric fixation. In humans, the development of a preferred retinal location can take many months and based on our observations could rely on activity changes in far peripheral parts of the visual cortex (Crossland et al. 2005; Baker et al. 2008). It is also possible that in animals focal retinal deficits could result in a rereferencing of the oculomotor system in the months following the lesions (Heinen and Skavenski 1992).

In conclusion, we characterized significant, time-dependent adjustments in the expression of the activity marker genes c-fos and zif268 in the center and the border regions of the LPZ in accordance with prior electrophysiological measurements of changes in neuronal activity in the primary visual cortex of retinally lesioned animals. In addition, significant parallel changes were also observed in far peripheral area 17. Furthermore, the restoration of IEG expression in the border zone in the first months postlesion appeared over equal dimensions in visual degrees along the P–A axis and the D–V axis of the brain indicative for a proportional topographic map reorganization. We propose that lasting changes in activity throughout area 17 are fundamental in implementing and maintaining newly acquired network properties that help in compensating the visual deficit.

Funding

Scientific Research-Flanders (FWO-Vlaanderen); Research Council of the K.U.Leuven (OT 05/33); Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) to T.-T.H.

We gratefully thank Ria Van Laer for technical assistance and Tim Theuwissen and Julie Puttemans for image processing. Conflict of Interest: None declared.

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