Abstract

Thallium autometallography (TIAMG) is a novel method for high-resolution mapping of neuronal activity. With this method, we found that a general depression of neuronal activity occurs in response to optic nerve crush (ONC) within the first 2 weeks postinjury in the contralateral dorsal lateral geniculate nucleus (dLGN) as well as in the contralateral primary visual cortex (V1). Interestingly, the neuronal activity recovered thereafter in both brain regions and reached a plateau in the tenth week postinjury in layers IV and V of V1, monocular area (V1m). Several clusters of highly active neurons in V1m were found 6 weeks after ONC in layers IV and V on the side contralateral to the lesion. We reasoned that these clusters appeared due to a reorganization of the corticocolliucular projections. Employing a combination of biotinylated dextran amine retrograde tract tracing from the superior colliculus (SC) with TIAMG in the same animal, we indeed found that the clusters of neurons with high Tl+ uptake in V1m are spatially in register with those neuronal subpopulations that project to the SC. These data suggest that extensive reorganization plasticity exists in the adult rat visual cortex following ONC.

Introduction

Limited optic nerve crush (ONC) in adult rats induces diffuse axonal injury and has been widely used to study neurodegeneration but also neuroprotection and injury-induced plasticity (Yoles et al. 1992; Sautter and Sabel 1993; Kreutz et al. 1998, 1999; Dieterich et al. 2002). Partial ONC is also considered as an animal model for glaucoma (Schwartz and Yoles 1999), a group of progressive optic neuropathies that are characterized by a gradual loss of retinal ganglion cells (RGCs) and accompanied by a degeneration of the optic nerve. In contrast to axotomy, 3 different cell populations can be discriminated following ONC: 1) degenerating RGC; 2) axotomized RGC that survive for longer periods of time; and, finally, 3) RGC that despite axonal damage can maintain their axonal connection with the superior colliculus (SC) (Bien et al. 1999). Interestingly, this latter population undergoes substantial reorganization plasticity and it was shown that the surviving RGC largely project to the contralateral rostromedial SC (Kreutz et al. 2004) and that following ONC neuronal activity is higher in this region of the SC as compared with others (Macharadze et al. 2009). This regression to a smaller area is different from the expansion of the remaining axonal projections documented in the classical studies on reorganization plasticity of surviving axonal pathways connected to the cortex in adult mammals (Kaas et al. 1983; Milleret and Buser 1984; Kaas et al. 1990).

It is well established that in adulthood retinal and optic nerve lesions are associated with a decline of activity in the primary visual cortex (Kaas et al. 1990; Heinen and Skavenski 1991; Chino et al. 1992; Gilbert and Wiesel 1992; Schmitt et al. 1996; Calford et al. 2000; Brooks et al. 2004). In contrast to development, where monocular enucleation leads to an expansion of the ipsilateral visual projection area in the cortex (Kaas et al. 1990; Heinen and Skavenski 1991; Chino et al. 1992; Gilbert and Wiesel 1992; Calford et al. 2000), no such changes were reported initially in adult animals and humans. Interestingly, however, also unilateral retinal lesions in adult mice induce a shift in ocular dominance (Sawtell et al. 2003). In contrast to developmental plasticity, the shift in ocular dominance is caused by an increase of the cortical responses to the open eye, without a corresponding decrease of the responses to the deprived eye (Sawtell et al. 2003). Moreover, in primates and humans, supportive evidence for reorganization plasticity in the visual system was found after glaucoma (Duncan et al. 2007a, 2007b), optic nerve ischemia (Brooks et al. 2004), macular degeneration (Baker et al. 2005, 2008), and optic neuritis (Toosy et al. 2005; Korsholm et al. 2008; Jenkins et al. 2010). At the cellular level, recovery of activity in the cortex after retinal lesions or enucleation has been shown to correlate with the expression levels of immediate early genes (IEG) like arc, c-fos, and zif268 in cats, mice, monkeys, and rats (Caleo et al. 1999; Arckens et al. 2000; Majdan and Shatz 2006; Hu et al. 2009; Takahata et al. 2009; Van Brussel et al. 2011).

We therefore examined, in this study, how other parts of the visual system than the SC respond to ONC in terms of organization and activity. To this end, we employed thallium autometallography (TIAMG) and biotinylated dextran amine (BDA) tract tracing. TIAMG is a novel method for high-resolution mapping of neuronal activity (Goldschmidt et al. 2004, 2010) that employs the higher uptake rates of K+ ions and Tl+ with increasing neuronal activity (Keynes and Ritchie 1965; Landowne 1975). Compared with the 14C-2-deoxyglucose method (Sokoloff et al. 1977), which is similar in rationale, TIAMG offers the advantage that the tracer can be detected nonradioactively with cellular and subcellular resolution by means of a modified histochemical technique, the Timm staining technique or autometallographic method (Goldschmidt et al. 2004, 2010). Moreover, in contrast to IEG expression, that also provides a read-out of neuronal activity with cellular resolution, TIAMG is not dependent on specific signaling pathways that might differ between different cell types and IEGs.

Materials and Methods

Animals and Surgery

Twelve-week-old adult male Wistar rats from the local breeding facilities of the Leibniz Institute for Neurobiology were used for all experiments. All experimental procedures were performed according to the National Institutes of Health guidelines for the care and use of laboratory animals and approved by local authorities. Permission for animal experiments according to German federal law was obtained by the Landesverwaltungsamt Sachsen-Anhalt (2003–2006 42502-2-577 Leibniz Institute for Neurobiology [IfN] Magdeburg; 2007–2010 42502-2-803 and 42502-2-825 Leibniz Institute for Neurobiology [IfN] Magdeburg). ONC was performed as described previously (Sautter and Sabel 1993; Kreutz et al. 1998). Only the left optic nerve was crushed in each animal (see Supplementary Table 1 for the number of animals for each experiment and the group assignment). Animals were kept under a 12:12 h light:dark illumination cycle (lights on at 6 AM).

Thallium Autometallography

TIAMG was performed as described in detail in Goldschmidt et al. (2004, 2010). Rats were implanted with jugular vein catheters and were given 2–3 days to recover from surgery. On the day of the experiment, the catheter was connected under brief halothane anesthesia to a PE tube, and the animals were placed in a novel cage to allow for unrestrained movements of the PE tubes in freely moving animals. Three hours later animals were intravenously injected through the PE tube with 1 mL of a freshly prepared 0.05% TlDDC solution in 0.9% NaCl. Injections were made at ambient light without visual stimulation. The TlDDC solution was prepared by mixing, within the syringe used for injection, 0.5 mL of a 0.1% aqueous thallium (I) acetate (Fluka, Germany) solution with 0.5 mL 0.1% sodium diethyldithiocarbamate trihydrate (Sigma, Germany) dissolved in a 1.8% aqueous NaCl solution.

The TlDDC solution was continuously injected over a period of 4 min. Then, the catheter was cleared with 150 μL 0.9% NaCl. Five minutes after starting the experiment, animals were anesthetized by an intravenous injection of 270–280 μL ketamine (Ketamine-ratiopharm, 50 mg/mL). Since the injected volume corresponds to a dose of 25-mg ketamine per kg body weight, this injection results in an almost immediate (within seconds) onset of anesthesia. Following onset of anesthesia, the animals were transcardially perfused with a sodium sulfide solution (0.325% Na2S in 100 mM phosphate buffer pH 7.4) and a sulfide-glutaraldehyde solution (0.16% Na2S and 3% glutaraldehyde in 100 mM phosphate buffer pH 7.4). Na2S and glutaraldehyde were obtained from Sigma (Deisenhofen/Germany). First, a small amount (6–7 mL) of the sulfide solution was perfused within a time span of about 10 s. This was followed without interrupting the flow by 10 min perfusion with the sulfide-glutaraldehyde solution. Animals were perfused at flow rates of 65 mL/min during the first 3 min of perfusion and 30 mL/min for the remaining time.

After perfusion, brains were removed and cryoprotected for 48 h in 30% sucrose 0.1 M phosphate buffer, pH 7.4. Brains were frozen, and 25 μm thick frontal sections were cut on a Leica cryostat. Sections were air-dried and treated with 0.1 N HCl to remove zinc sulfide. Some sections were treated with DDC for controlled removal of thallium. Sections were stained in a standard gum arabic developer used for autometallography (Danscher 1981; Goldschmidt et al. 2010) for 150 min in the dark. Every tenth section was counterstained with cresyl-violet for better delineation of cytarchitectonically defined areas.

Statistical Analysis of TI+ Uptake Patterns in Primary Visual Cortex and Dorsal Lateral Geniculate Nucleus of the Thalamus

Sections were analyzed by means of a Leica DMR microscope system (Germany). Cortical areas and subcortical structures were identified and later labeled for the illustrations on the basis of their cytoarchitecture using information derived in particular from stereotaxic atlases of the rat (Paxinos and Watson 1986, 1998; Paxinos, Carrive, et al. 1999; Paxinos, Kus, et al. 1999). For the delineation of the cortex, we used the nomenclature of the rat stereotaxic atlas of Paxinos and Watson (1998), based on the work of Swanson (1992), which differs from that of the atlas of Paxinos and Watson (1986), based on the work of Zilles (1985).

Sections of interest were photographed with a Fuji FinePix S2 Pro digital camera mounted on the same microscope system. Photographs were arranged for illustrations using the Adobe Photoshop software for Macintosh. The NIH image software (ImageJ for MacOS X) was used for the analysis of thallium uptake patterns.

For cell counting, the region of interest (ROI)—for example, the monocular part of the primary visual cortex V1m (occipital cortex area 1, monocular part Oc1M, according to Zilles 1985), V1b, V2b, and V2l—was photographed at ×10 magnification. The colored photomicrographs were converted to gray scale images using unweighted conversions in Adobe Photoshop (Goldschmidt et al. 2010) or ImageJ. In these images, all the cells above a certain gray value were counted semiautomatically using the ImageJ (Version 1.39J) built-in function “Analyze Particles.” Gray value thresholds, which had to be adjusted due to different staining intensities of sections across animals, were defined in the following way: From a rostrocaudal series of sections through the visual cortex in a certain animal, the most central section was chosen for adjusting the threshold. ROI in dorsal lateral geniculate nucleus (dLGN) were scanned from Bregma-4 to Bregma-5, covering almost the entire DLG except the very rostral and caudal parts. The threshold was adjusted in the way that gray values in the entire somata of at least 80% of layer 5 pyramidal neurons on the side ipsilateral to the optic nerve lesion were above threshold. This threshold was used for the entire series of sections. In order to avoid the counting of blood vessels or staining artifacts like nonspecific silver grains or nonspecific staining of section edges, the size and shape of the counted structures were restricted. Only the structures above 200 and below 24 000, contingent pixels in size were counted. This corresponded to the structures covering areas of approximately 5–60 μm2. The shape was additionally restricted by using a circularity factor of 0.3–1 with “1” meaning perfectly circular. This excludes highly noncircular structures (circularity < 0.3), for instance, the endothelial linings of blood vessels, from being counted. In essence, cells with staining intensities as high as or higher than clearly stained ipsilateral layer V pyramidal neurons were counted. The cells were pooled for each animal, and statistical analysis was performed with a two-way analysis of variance (ANOVA) and subsequent t-test.

To confirm the statistical analysis of the thallium signal and to visualize the clustered cells, the visual cortex was cut out manually of the gray scale photomicrographs for each slice by using the “Straighten”-PlugIn of ImageJ. These partial images were then transformed to a rectangular shape and then reconstructed as a 3D Stack. The gray values of this stack were inverted, so that a high thallium signal results in high gray value intensity. A projection over all slices visualized the average thallium signal in the visual cortex in rostrocaudal direction. Similarly, the dLGN was photographed at ×10 magnification, converted, and the mean of the gray value on the ipsi- and contralateral side was calculated.

Tract Tracing with BDAs

Animals were anaesthetized briefly with 2-Bromo-2-chloro-1,1,1-trifluoroethane and then intraperitoneally injected with ketamine (25 mg/kg). The cranial skin was incised and partly removed, and the muscles were displaced so that a large opening could be drilled into the skull over the right SC. In each animal, 50 nL of 10% BDA (1:1 mixture of 10.000 Da and 3.000 Da MW; lysine-fixable; Molecular Probes, Eugene, Oregon), dissolved in distilled water containing 0.1% sodium acid and 1% dimethyl sulfoxide, were injected by pressure via a fine glass pipette (tip diameter 20 μm) and an oil hydraulic nanoliter delivery system (WPI, Germany) into the superficial layers of the rostromedial SC. For the injections, we used coordinates from the rat stereotaxic atlas (Paxinos and Watson 1986, 1998): 3.0-mm dorsoventral, 0.6-mm mediolateral, and 6.3-mm posterior from bregma. Injections were made 5 weeks post-crush. Thereafter, the animals were allowed to recover and survive for 7 days. Then, the animals were reanesthetized and perfused transcardially with 50 mL of 0.1 M phosphate-buffered saline (PBS), followed by 200 mL of 4% paraformaldehyde (PFA) in PBS. The brains were removed, postfixed overnight in 4% PFA, and then cryoprotected by soaking them in 30% sucrose in PBS for 48 h at 4 °C. The brains were then cut in a cryostat into 50-μm thick frontal sections. In order to visualize the retrograde transport of BDA, the sections were stained using the avidin–biotin–peroxidase method (ABC kit, Vector Laboratories) and diaminobenzidine as the chromogen (Budinger et al. 2000). In order to correlate the cytoarchitecture of brain areas with the BDA labeling, every tenth section was counterstained for cell bodies with cresyl-violet (Nissl stain, intensive staining of complete somata); all other sections were counterstained with methylgreen for nuclei (moderate staining which allows an easier visualization of the BDA labeling than Nissl). For the analysis of the BDA transport, sections were again examined light microscopically, and ROIs were digitally photographed (LEICA/FUJI). The images were arranged and labeled for figures using Adobe Photoshop software. The diameters of the injection sites were calculated from direct measurements in the frontal plane and the thickness (i.e., 50 μm) of consecutive sections, covering the injection sites, by means of the “Neurolucida” system and software (MicroBrightField Europe; Supplementary Table 2). The numbers of retrogradely labeled somata were counted in the ROIs using Neurolucida (Fig. 8).

Figure 8.

Images and quantitative assessment of the number of BDA-labeled cells in the visual cortex 6 weeks after ONC in comparison to sham-control animals. Images were taken from the V2M, V1m, V1b, and V2L region of the contralateral side with respect to the lesion in control (A, C, E, G) and crushed animals (B, D, F, H) after injection of BDA into the rostromedial SC (mean diameter of injection sites 825 μm and 815 μm, respectively, see Supplementary Table 2). The scale bar is 100 μm. (I) Quantitative assessment of the labeled pyramidal cells of the ipsilateral projection of sham-control animals versus animals 6 weeks after ONC of the contralateral eye. The data were collected for each brain region separately and averaged over each group. Average values of the control group (±1 standard deviation) were plotted against the average values of the crushed group for each brain region separately. N = 5 in each group. Despite the fact that the data points are all under the bisector of angle, illustrating the lower number of cortical projection neurons in rats 6 weeks after ONC, the differences are not statistically significant.

Figure 8.

Images and quantitative assessment of the number of BDA-labeled cells in the visual cortex 6 weeks after ONC in comparison to sham-control animals. Images were taken from the V2M, V1m, V1b, and V2L region of the contralateral side with respect to the lesion in control (A, C, E, G) and crushed animals (B, D, F, H) after injection of BDA into the rostromedial SC (mean diameter of injection sites 825 μm and 815 μm, respectively, see Supplementary Table 2). The scale bar is 100 μm. (I) Quantitative assessment of the labeled pyramidal cells of the ipsilateral projection of sham-control animals versus animals 6 weeks after ONC of the contralateral eye. The data were collected for each brain region separately and averaged over each group. Average values of the control group (±1 standard deviation) were plotted against the average values of the crushed group for each brain region separately. N = 5 in each group. Despite the fact that the data points are all under the bisector of angle, illustrating the lower number of cortical projection neurons in rats 6 weeks after ONC, the differences are not statistically significant.

Combination of Tract Tracing and TIAMG

For combining tract tracing and TIAMG in the same animal, the standard protocol for BDA tracing described above was modified. Five days after BDA injection, jugular vein catheters were implanted. Two days later, TlDDC was intravenously injected as described above. The animals were transcardially perfused with the sulfide/glutaraldehyde fixative for TIAMG. The brains were removed and cryoprotected as described above. Cryostat-sections were made alternating in thickness between 25 and 50 μm. Fifty-micrometer sections were processed for BDA staining with appropriate counterstaining (see above), and 25 μm sections were used for TIAMG.

Results

ONC Initially Decreased TI+ Uptake in the Primary Visual Cortex

Utilizing TIAMG to study reorganization plasticity in the brain following ONC, we found that TI+ uptake patterns in the cortex of untreated control animals were in general similar to the 14C-deoxyglucose uptake patterns in nonstimulated animals reported in the literature (Fig. 1; Sokoloff et al. 1977; Cooper 2002). Interestingly, however, the thallium uptake in the primary visual cortex (V1), as identified with a Nissl stain by a cell-dense layer IV, appears to be higher than in secondary areas (Fig. 1).

Figure 1.

(A) TI+ uptake pattern in the visual cortex of control rats. (BG) Different cortical areas differ in Tl+-uptake patterns and in cell density in layer IV as demonstrated in the lower panels (EG/layer IV is marked by lines). Shown are the details from a section neighboring that of the upper panel. The section was Nissl-stained after TIAMG. The cell density is highest in layer IV of the monocular (V1m) and binocular part (V1b) of the primary visual cortex and lower in the medial secondary visual cortex (V2M) (EG). In addition, nonpyramidal cells or interneurons (white arrows) in layer IV are stained differently in the different areas (EG). The differences in topography, metabolism, and cell density in Nissl-stained sections can be used for delineating V1m from the neighboring areas of the visual cortex. The scale bar is 1 mm in the overview (A), 100 μm in upper panel (BD), and 50 μm in the lower panel (EG). Tl+ uptake in the lateral part of V1 was higher than in the medial part. Topographically, this lateral part corresponds to the V1b. These lateral-to-medial differences were also present in optic nerve-crushed animals (see Fig. 2). We used these criteria—Nissl staining, differences in Tl+ uptake and topographical relationships—for delineating the V1m.

Figure 1.

(A) TI+ uptake pattern in the visual cortex of control rats. (BG) Different cortical areas differ in Tl+-uptake patterns and in cell density in layer IV as demonstrated in the lower panels (EG/layer IV is marked by lines). Shown are the details from a section neighboring that of the upper panel. The section was Nissl-stained after TIAMG. The cell density is highest in layer IV of the monocular (V1m) and binocular part (V1b) of the primary visual cortex and lower in the medial secondary visual cortex (V2M) (EG). In addition, nonpyramidal cells or interneurons (white arrows) in layer IV are stained differently in the different areas (EG). The differences in topography, metabolism, and cell density in Nissl-stained sections can be used for delineating V1m from the neighboring areas of the visual cortex. The scale bar is 1 mm in the overview (A), 100 μm in upper panel (BD), and 50 μm in the lower panel (EG). Tl+ uptake in the lateral part of V1 was higher than in the medial part. Topographically, this lateral part corresponds to the V1b. These lateral-to-medial differences were also present in optic nerve-crushed animals (see Fig. 2). We used these criteria—Nissl staining, differences in Tl+ uptake and topographical relationships—for delineating the V1m.

It is well established that cellular activity declines after lesions of the retina or the optic nerve in the visual system of primates as well as in the rodent visual system (Schmitt et al. 1996; Brooks et al. 2004; Macharadze et al. 2009). However, previous studies with the 14C-deoxyglucose uptake method could not distinguish between glial and neuronal cells, and since neurons are greatly outnumbered by glial cells, it is therefore unclear whether the changes reported previously reflect altered neuronal activity. We indeed found utilizing TIAMG a general decline of signal intensity in neurons of the primary visual cortex (Figs 2 and 3; two-way ANOVA factor time df4,24, P < 0.0012) and the dLGN (Figs 4 and 5) of the adult rat 2 weeks after ONC that resembled in time course and extent those observed previously in the SC (Macharadze et al. 2009). In animals subjected to ONC, a quantitative assessment of Tl+ uptake revealed a significantly lower number of stained cells in the V1m (Student t-test; P < 0.01) and to a lesser extent V2l (Student t-test; P < 0.05) region on the contralateral side as compared with the ipsilateral side with respect to optic nerve lesion (Figs 2 and 3; Supplementary Fig. 1). This difference was particularly pronounced in layer V. Interestingly, the difference in the number of stained cells gradually decreased over postinjury time and was no longer statistically significant after 6 weeks (Figs 2 and 3). No such changes were found in the V1b and V2m region (Supplementary Fig. 1). However, Tl+ uptake was initially reduced in the contralateral dLGN as compared with the ipsilateral side after ONC, and this difference was also no longer visible after 6 weeks and a complete recovery of activity was found like in V1m (Figs 4 and 5).

Figure 2.

(AF) TI+ uptake patterns in V1m 2 weeks after ONC. Images in the left row (A, C, E) show the visual cortex contralateral to the crushed nerve, those in the right row (B, D, F) show the visual cortex ipsilateral to the lesion. Overviews are shown in the top row (A and B), details from layer IV and layer V of V1m are shown in the middle row (C and D). The bottom row depicts in red cells in layers IV and V that were counted after thresholding using the ImageJ software. Most of the cells above threshold were present in these layers but for statistical analysis cells above threshold in all cortical layers were counted. Note the markedly lower density of labeled neurons contralateral to the crushed nerve (E) as compared with ipsilateral (F). The scale bar is 500 μm in A and B and 50 μm in CF.

Figure 2.

(AF) TI+ uptake patterns in V1m 2 weeks after ONC. Images in the left row (A, C, E) show the visual cortex contralateral to the crushed nerve, those in the right row (B, D, F) show the visual cortex ipsilateral to the lesion. Overviews are shown in the top row (A and B), details from layer IV and layer V of V1m are shown in the middle row (C and D). The bottom row depicts in red cells in layers IV and V that were counted after thresholding using the ImageJ software. Most of the cells above threshold were present in these layers but for statistical analysis cells above threshold in all cortical layers were counted. Note the markedly lower density of labeled neurons contralateral to the crushed nerve (E) as compared with ipsilateral (F). The scale bar is 500 μm in A and B and 50 μm in CF.

Figure 3.

Quantitative assessment of Tl+ stained cells in the monocular part of the primary visual cortex (V1m) in the ipsilateral and contralateral cortex control, 2, 6, and 10 weeks after ONC. The data are normalized with respect to the numbers on the ipsilesional side, which was set to 1 in each group at the indicated time points. Cells in all cortical layers were counted. Controls: n: 80 sections from 6 animals; 2 weeks: n: 74 sections from 6 animals; 6 weeks: n: 72 sections from 6 animals; 10 weeks: n: 58 sections from 6 animals. Error bars show standard error of the mean. **P < 0.01 with Student t-test.

Figure 3.

Quantitative assessment of Tl+ stained cells in the monocular part of the primary visual cortex (V1m) in the ipsilateral and contralateral cortex control, 2, 6, and 10 weeks after ONC. The data are normalized with respect to the numbers on the ipsilesional side, which was set to 1 in each group at the indicated time points. Cells in all cortical layers were counted. Controls: n: 80 sections from 6 animals; 2 weeks: n: 74 sections from 6 animals; 6 weeks: n: 72 sections from 6 animals; 10 weeks: n: 58 sections from 6 animals. Error bars show standard error of the mean. **P < 0.01 with Student t-test.

Figure 4.

TI+ uptake in the dLGN 2 (A) and 6 weeks (B) after ONC. Pseudocolored images of TI+ uptake patterns are shown in a rostral to caudal series of coronal sections through the dLGN. Note that the difference between the ipsi- and contralateral side does vanish 6 weeks after ONC.

Figure 4.

TI+ uptake in the dLGN 2 (A) and 6 weeks (B) after ONC. Pseudocolored images of TI+ uptake patterns are shown in a rostral to caudal series of coronal sections through the dLGN. Note that the difference between the ipsi- and contralateral side does vanish 6 weeks after ONC.

Figure 5.

Quantitative assessment of Tl+ stained cell in dLGN on the ipsilateral and contralateral side 2 and 6 weeks after ONC. Bar graphs indicate the total number of cells counted in all animals in the 2 groups on the contra- and ipsilesional side, respectively. The data are normalized with respect to the numbers on the ipsilesional side that were set to 1 (see Fig. 2). Analyses were done with the ImageJ program, as described above. Two weeks: n: 27 sections from 6 animals; 6 weeks: n: 17 sections from 6 animals. *P < 0.05 with Student t-test.

Figure 5.

Quantitative assessment of Tl+ stained cell in dLGN on the ipsilateral and contralateral side 2 and 6 weeks after ONC. Bar graphs indicate the total number of cells counted in all animals in the 2 groups on the contra- and ipsilesional side, respectively. The data are normalized with respect to the numbers on the ipsilesional side that were set to 1 (see Fig. 2). Analyses were done with the ImageJ program, as described above. Two weeks: n: 27 sections from 6 animals; 6 weeks: n: 17 sections from 6 animals. *P < 0.05 with Student t-test.

To obtain a complete data set of the entire primary visual cortex, we performed a 3D reconstruction of the area. For a better visualization, the TI+ signal was coded as height to achieve a surface plot of the area. Figure 6 shows representative examples of pseudocolored surface plots of the averaged signal over all slices of the visual cortex in one animal without ONC for control, one at 2 weeks, another at 6 weeks, and a fourth at 10 weeks after nerve crush. The profile showed a clearly higher signal in the ipsilateral side of the V1m region, which is the center part of the depicted area at 2 weeks after ONC (Fig. 6D). At 6 weeks and at 10 weeks after ONC, the signal was on the same level in the V1m region in both sides (Fig. 6E–H). In accord, 6 weeks after crush also the automated counting of cells in V1m did not reveal any significant differences between the ipsi- and contralateral side with respect to the number of cells reaching Tl+ uptake above the threshold (Fig. 2).

Figure 6.

Surface plots of the projection and profiles of the visual cortex of control animals (A, B), 2 weeks (C, D), 6 weeks (E, F), and 10 weeks (G, H) after ONC, contralateral to the crush on the left and ipsilateral on the right. The upper row shows surface plots of the average intensity over all slices in the ROI as pseudo colors (white: high thallium signal, blue: low signal). The lower row denotes the profile over the averaged intensities. At 2 weeks after ONC, the signal in the Oc1M region (the center part of the ROI) at the contralateral side is lower compared with the ipsilateral side, whereas after 6 weeks, the signal at the contralateral is restored. The signal of the control animal (without ONC/A) shows no prominent difference between both hemispheres. Most interestingly, after 6 weeks, high TI+ uptake appears to be clustered in columns in the contralateral side in layer 5 (see black arrows in E).

Figure 6.

Surface plots of the projection and profiles of the visual cortex of control animals (A, B), 2 weeks (C, D), 6 weeks (E, F), and 10 weeks (G, H) after ONC, contralateral to the crush on the left and ipsilateral on the right. The upper row shows surface plots of the average intensity over all slices in the ROI as pseudo colors (white: high thallium signal, blue: low signal). The lower row denotes the profile over the averaged intensities. At 2 weeks after ONC, the signal in the Oc1M region (the center part of the ROI) at the contralateral side is lower compared with the ipsilateral side, whereas after 6 weeks, the signal at the contralateral is restored. The signal of the control animal (without ONC/A) shows no prominent difference between both hemispheres. Most interestingly, after 6 weeks, high TI+ uptake appears to be clustered in columns in the contralateral side in layer 5 (see black arrows in E).

Pyramidal Layer V Cells with High Tl+ Uptake after ONC Are Clustered and Project to the SC

Differences were present, however, with respect to the spatial pattern of Tl+ uptake. Six weeks following ONC, the signal appears to be clustered in columns on the contralateral side of layer V (Fig. 6E, black arrows) as compared with control animals not subjected to ONC the signal showed no prominent difference between both hemispheres (Fig. 6A,B). We indeed observed on the contralateral side with respect to the lesion small groups of layer V pyramidal cells with substantially higher Tl+ uptake that were clustered in columns (Fig. 7B), while no such effect was seen in the ipsilateral side (Fig. 7A).

Figure 7.

TI+ uptake patterns in V1m 6 weeks after ONC. V1m ipsilateral to the crushed nerve is shown in A, V1m contralateral to the lesion is shown below in B. Compared with 2 weeks after crush (see Fig. 2), the density of neurons with high TI+ uptake has increased contralateral to the lesion (B). However, in layer V neurons with high TI+ uptake are clustered (black arrows) separated by gaps with only few cells labeled (red arrows). The scale bar in A and B is 100 μm.

Figure 7.

TI+ uptake patterns in V1m 6 weeks after ONC. V1m ipsilateral to the crushed nerve is shown in A, V1m contralateral to the lesion is shown below in B. Compared with 2 weeks after crush (see Fig. 2), the density of neurons with high TI+ uptake has increased contralateral to the lesion (B). However, in layer V neurons with high TI+ uptake are clustered (black arrows) separated by gaps with only few cells labeled (red arrows). The scale bar in A and B is 100 μm.

In a previous study (Kreutz et al. 2004), we found that in contrast to sham-operated animals, where only RGC from the lower temporal quadrant of the retina project to the superficial layers of the rostromedial SC, in animals 6 weeks after partial ONC significantly more axons from all regions of the retina with a marginally higher number of RGC in the ventrotemporal retina terminate in the rostromedial SC. To address the question whether the reorganization in the SC and cortex might be linked to each other, we injected the retrograde tracer BDA in the superficial layers of the rostromedial SC and analyzed the labeling pattern in the visual cortex. The injection sites in the SC were quantified in terms of the covered layers and the mean diameter size (Supplementary Fig. 2 and Table 2). Only animals with an injection site covering the upper layers of the rostromedial SC were analyzed. We found retrograde BDA labeling in the ipsilateral cortex and staining of cells in regions of the medial (V2M) and lateral (V2L) secondary visual (V2) as well as monocular (V1m) and binocular part (V1b) of the primary visual cortex (V1) largely in accordance to previously published data (Lund 1966, 1969; Harvey and Worthington 1990 and Fig. 8). Although slightly more cells were labeled under control conditions, a subsequent quantitative assessment revealed that this difference between crushed animals 6 weeks after ONC as compared with noncrushed controls is statistically not significant (Fig. 8).

We next started to investigate whether the columns of enhanced neuronal activity contain projection neurons that terminate in the rostromedial SC. To this end, a method was developed to combine TlAMG and retrograde BDA tract tracing and that allowed to detect the TlAMG and the BDA signal in adjacent sections (Fig. 9). Indeed, analysis of adjacent sections from animals 6 weeks after ONC showed a clear overlap of the BDA and the Tl+ signal (Fig. 9). Thus, clusters of Tl+-stained neurons were spatially coregistered with clusters of BDA positive neurons in V1m contralateral to the optic nerve lesion side and the vast majority of cells connected to the SC exhibited high Tl+ uptake, whereas only very few cells with low to moderate thallium uptake were retrogradely labeled (Fig. 9).

Figure 9.

Clusters of BDA-labeled neurons are in register with clusters of neurons with high Tl+ uptake. Images from neighboring sections stained for either BDA or Tl+, respectively, were aligned using boundaries of cortical gray matter and underlying white matter as landmarks (outlined in black). After alignment, images were converted to gray-level images. BDA images were then colorized in red, Tl+ images in blue. Both images were fused with 50% transparency of the BDA images. Two examples are shown at 2 different levels in the same animal. Black arrows point to clusters of stained neurons in BDA and in fused images. Asterisks indicate clusters of neurons with high Tl+ uptake. Details from fused images are shown in the lowermost panels. Note that neurons stained for BDA (red arrows) and for Tl+ (blue arrows) are intermingled and coregister. Scale bars are 500 μm in the overviews and 100 μm in the details.

Figure 9.

Clusters of BDA-labeled neurons are in register with clusters of neurons with high Tl+ uptake. Images from neighboring sections stained for either BDA or Tl+, respectively, were aligned using boundaries of cortical gray matter and underlying white matter as landmarks (outlined in black). After alignment, images were converted to gray-level images. BDA images were then colorized in red, Tl+ images in blue. Both images were fused with 50% transparency of the BDA images. Two examples are shown at 2 different levels in the same animal. Black arrows point to clusters of stained neurons in BDA and in fused images. Asterisks indicate clusters of neurons with high Tl+ uptake. Details from fused images are shown in the lowermost panels. Note that neurons stained for BDA (red arrows) and for Tl+ (blue arrows) are intermingled and coregister. Scale bars are 500 μm in the overviews and 100 μm in the details.

Discussion

The effect of early visual deprivation on ocular dominance in the developing primary visual cortex belongs to the best-characterized forms of experience-dependent plasticity. Only few studies, however, have addressed the impact of enucleation or retinal and optic nerve damage in the adult rodent visual system. A consistent finding of these studies is a lesion-induced drop of neuronal activity as evidenced by the reduced expression of the IEGs arc/arg3.1, c-fos, and zif268 (see for instance Caleo et al. 1999; Van der Gucht et al. 2007; van Brussel et al. 2009, 2011). Experimental glaucoma in primates, which resembles ONC in certain aspects, induces neuronal degeneration of the LGN that is accompanied by activity changes in the visual cortex (Vickers et al. 1997; Crawford et al. 2000, 2001; Weber et al. 2000; Yucel et al. 2001). Interestingly, some reports suggest a remarkable degree of postlesion plasticity in the adult mouse primary visual cortex (Satwell et al. 2003; Keck et al. 2008; Van Brussel et al. 2011). After an initial loss of neuronal activity, a recovery takes place that is accompanied by an activity-dependent establishment of new cortical circuits (Keck et al. 2008) and that can include cross-modal innervation of the lesioned area (Van Brussel et al. 2011).

In the present study, TlAMG was used for high-resolution mapping of neuronal activity in the adult rat visual system after partial ONC. In order to keep the experimental conditions as simple as possible, Tl+ was injected at ambient light and no visual stimulus was applied. In 14C-2-deoxyglucose studies performed in rodents without visual stimulation (Cooper 2002), it was found that the primary visual cortex had a higher metabolic rate than the secondary visual areas and that, within the primary area, metabolism was higher in the binocular than in the monocular field. We confirmed these findings with TlAMG and used these differences in metabolism in addition to the differences in cytoarchitecture as a criterion for delineating the monocular part of V1 from neighboring areas. In this monocular part, contralateral to the crushed optic nerve, the changes in Tl+ uptake were most pronounced in V1m. Two weeks after ONC, the number of stained cells was significantly lower than on the ipsilateral side. The differences between the contra- and ipsilateral side decreased postinjury within the following weeks. At 6 weeks, no significant differences in the number of stained neurons between the ipsi- and contralateral side were found, clearly indicating that the neuronal activity in V1m recovered. A similar time course of recovery of neuronal activity was seen in the dLGN. In addition, we have analyzed V1b, V2M, and V2L and found a statistically significant decline in V2L. In mouse, V1b is a binocular region, whereas V2M and V2L are partly monocularly driven (van Brussel et al. 2009). Basal activity in V2M was extremely low, and it was therefore unlikely to detect a further drop in Tl+ uptake after ONC. The lack of significant changes in V1b and the relatively minor changes in the dLGN might also reflect the fact that there is no strict correlation between silencing retinal input and spontaneous activity in the dLGN and the input to cortex in awake mice (Linden et al. 2009).

We used an observer-independent simple semiautomated method for counting neurons. This is a robust approach of detecting differences but, since all cells in V1m are counted, small, but nevertheless significant, differences in Tl+ uptake that affect only certain cell types or certain parts of V1m may not be detected. To confirm the results, we analyzed the spatial distribution of the gray value intensity in V1, which corresponds to the Tl+ uptake in all cells and employed a 3D reconstruction of V1m. The statistical analysis in both cases confirmed the same differences between the contra- and ipsilateral side as counting the cells. Importantly, we observed that 6 weeks after the lesion layer V pyramidal cells in the contralateral V1m appear clustered, indicating that the recovery of neuronal activity may not be homogenous.

The reorganization in activity patterns of the visual cortex observed in this study is different from several examples of reorganization processes after peripheral nerve injury or deprivation that suggest that the basic principle is to allow neighboring regions to expand into territory normally occupied by input from the deprived or lesioned sensory pathway (Florence and Kaas 1995; Bao et al. 2002). Similar results were found after small retinal lesions in adult mice (Keck et al. 2008). This is in stark contrast to the situation after partial ONC. Here, small clusters of neurons show a recovery of neuronal activity, and these clusters are dispersed over the entire V1m region. What could be the reason for this difference? A major aspect that might account for these contradictory findings is the lesion paradigm itself. In most cases where neighboring axons fill in the adjacent territory in the cortex either only a small percentage of the projection was removed or the entire pathway was deprived during development, which frequently leads to an expansion of another sensory pathway. Partial ONC was performed in adult rats and leads to a degeneration of the vast majority of axons (Bien et al. 1999; Kreutz et al. 2004). In rats, virtually all RGC project to the contralateral SC (Lund 1965; Dreher et al. 1985), while only a subset of layer V pyramidal neurons of the primary visual cortex project to the ipsilateral SC and a substantial proportion of layer V neurons project to the contralateral visual cortex (Lund 1966, 1969; Harvey and Worthington 1990; Larkman and Mason 1990; Kasper et al. 1994). We reasoned that the recovery of neuronal activity after unilateral ONC in column-like clusters in V1m could be particularly pronounced in pyramidal neurons of the colliculocortical projection. We therefore developed a protocol for combining, in the same animal, TlAMG and neuronal tract tracing with BDAs. Using this technique, we found that the termination sites of the corticocollicular projection colocalize with clusters of neurons with high Tl+ uptake. This raises the intriguing possibilities that the regression of the retinocollicular projection to the rostromedial SC following partial ONC might be responsible for the formation of small clusters of active neurons in the primary visual cortex. The reorganization of the retinocollicular projection is already visible in the first weeks following ONC and correlates with the fasciculation of the remaining RGC axons that are still in continuity with the SC in the optic nerve (Kreutz et al. 2004). Previous work on enucleation in adulthood suggests that corticocollicular fibers are subjected to plastic processes (García del Caño et al. 2002). Although adult enucleation did not alter the terminal pattern in SC with regard to its rostrocaudal and lateromedial dimensions but a redistribution toward the most superficial strata of the SC was reported (García del Caño et al. 2002), suggesting that corticocollicular axons retain some capacity for postlesional plastic remodeling. This also suggests that a retrograde signal might be instructive for the formation of the clusters. The nature of this signal is not clear but a retrograde transport of neurotrophins would be a plausible candidate. Interestingly, however, after monocular enucleation in adult mice a differential laminar and temporal reactivation profile was observed in the visual cortex that required activity of nonvisual cortex (Van Brussel et al. 2011) raising the possibility that preexisting connections between the visual and other cortices can affect the recovery of neuronal activity. Thus, it is also possible that cross-modal plasticity could underlie the altered activity pattern observed after partial ONC and that these clusters of enhanced neuronal activity reflect the neuronal population that originally projects to the rostromedial SC, which might serve to realign sensory maps in the SC for multimodal integration. At present it is difficult to distinguish between these 2 possibilities.

In summary, our data suggest that extensive reorganization occurs at the level of processing in V1m and future studies might aim at elucidating how this reorganization relates to the retinotopic organization of the cortex after ONC. The regression of the retinocollicular projection to a smaller area of the SC can either indicate a loss of retinocollicular topography or, alternatively, a compressed but topographically organized representation of the visual field in the rostromedial SC as described after early postnatal lesions in hamster (Finlay et al. 1979). An essential unanswered question in this regard is therefore whether a representation of the entire visual field in a smaller subcortical area like the rostromedial SC, whose activity is driven by a subset of cortical neurons in V1m, is more favorable under these conditions.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/

Funding

Bundesministerium für Bildung und Forschung; Deutsche Forschungsgemeinschaft; the State Saxony-Anhalt; Deutsches Zentrum für Neurodegenerative Erkrankungen; Deutsche Akademische Austauschdienst; Otto-von-Guericke University Magdeburg (to T.M.).

The authors thank Monika Marunde and Kathrin Gruss for professional technical assistance throughout the project. Conflict of Interest : None declared.

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