Behavioral and neuroplastic effects of focal endothelin-1 induced sensorimotor cortex lesions

Neuroscience 128 (2004) 473– 486

BEHAVIORAL AND NEUROPLASTIC EFFECTS OF FOCAL ENDOTHELIN-1 INDUCED SENSORIMOTOR CORTEX LESIONS D. L. ADKINS,a A. C. VOORHIESb AND T. A. JONESa,c*

Sharkey et al. (1994) demonstrated that a simple intracerebral microinjection technique for applying ET-1 near the middle cerebral artery (MCA) produced similar patterns of neocortical and striatal damage as that previously reported for MCA occlusions in rats. Application of ET-1 (60 pmol in 3 ␮l saline) near the MCA has been found to reduce cerebral blood flow by 30 –50% in the cortex and striatum, returning to control levels after 16 –22 h in the cortex and 7–10 h in the striatum (Biernaskie et al., 2001). Fuxe et al. (1997) used ET-1 to produce smaller and more localized ischemic lesions by applying it directly onto the surface of the dorsal frontoparietal cortex of rats. This created a dose-dependent reduction in cortical blood flow for the 60 min of observation compared with basal levels and to CSF-injected controls. The ischemic damage extended through all six cortical layers without penetrating the underlying white matter. This study by Fuxe et al. (1997) established the usefulness of ET-1 to induce localized, reproducible focal cortical ischemic lesions. Gilmour et al. (2004) recently assessed behavioral effects of intracortical microinjection of ET-1 into the forelimb representation region of the sensorimotor cortex (SMC) of rats. These animals were compared with rats receiving aspiration or excitotoxic lesions of the SMC. All three lesion methods resulted in enduring (for at least 12 weeks) impairments in skilled forelimb reaching compared with shams, but there were no differences between lesion types. Other studies, however, have indicated that behavioral and neuroplastic responses to focal cortical lesions can depend upon the method of lesion induction. For example, studies comparing unilateral SMC lesions produced either by thermocoagulation of pial blood vessels or by aspiration have revealed that the two lesion methods differ in the severity of the forelimb motor and sensory deficits induced (Napieralski et al., 1998). In addition, aspiration lesions failed to produce the crossed corticostriatal sprouting (Napieralski et al., 1996; Szele et al., 1995; Uryu et al., 2001) and related bilateral synchronous cortical neuronal firing (Carmichael and Chesselet, 2002) that were found after thermocoagulatory lesions of the SMC. Voorhies and Jones (2002) found that, despite being of similar size and placement, aspiration lesions failed to result in the increased contracortical dendritic arborization that was found after electrolytic lesions of the SMC. Thus, it seems important to more thoroughly characterize both the behavioral and neuroplastic effects of focal lesions of the SMC produced using ET-1. The purpose of the present study was to assess the somatic-sensory and motor effects of focal ET-1-induced damage of the SMC as well as cellular and structural

a

Institute for Neuroscience, University of Texas at Austin, Austin, TX 78712, USA b Psychology Department, University of Washington, Seattle, WA 98195, USA c

Psychology Department, University of Texas at Austin, Austin, TX 78712, USA

Abstract—Previous studies have established the usefulness of endothelin-1 (ET-1) for the production of focal cerebral ischemia. The present study assessed the behavioral effects of focal ET-1-induced lesions of the sensorimotor cortex (SMC) in adult rats as well as cellular and structural changes in the contralateral homotopic motor cortex at early (2 days) and later (14 days) post-lesion time points. ET-1 lesions resulted in somatosensory and postural-motor impairments in the contralateral (to the lesion) forelimb as assessed on a battery of sensitive measures of sensorimotor function. The lesions also resulted in the development of a hyper-reliance on the ipsilateral forelimb for postural-support behaviors. In comparison to sham-operated rats, in layer V of the motor cortex opposite the lesions, there were time- and laminardependent increases in the surface density of dendritic processes immunoreactive for microtubule-associated protein 2, in the optical density of N-methyl-D-asparate receptor (NMDA) subunit 1 immunoreactivity, and in the numerical density of cells immunolabeled for Fos, the protein product of the immediate early gene c-fos. These findings corroborate and extend previous findings of the effects of electrolytic lesions of the SMC. It is likely that compensatory forelimb behavioral changes and transcallosal degeneration play important roles in these changes in the cortex opposite the lesion, similar to previously reported effects of electrolytic SMC lesions. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ischemia, motor cortex, behavioral compensation, microtubule-associated protein 2, NMDA receptor subunit 1, Fos.

Endothelin-1 (ET-1) is an endogenous vasospasm-inducing peptide that can be applied near the cerebral arteries (Biernaskie et al., 2001; Robinson et al., 1990; Sharkey et al., 1994; Ward et al., 1998), microinjected into selected brain regions (Fuxe et al., 1992; Gilmour et al., 2004; Hughes et al., 2003) or applied directly onto the cortical surface (Fuxe et al., 1997) to produce ischemic damage. *Correspondence to: T. A. Jones, University of Texas at Austin, Institute for Neuroscience, 1 University Station A8000, Austin, TX 78712, USA. Tel: ⫹1-512-475-7763; fax: ⫹1-512-475-7765. E-mail address: [email protected] (T. A. Jones). Abbreviations: ANOVA, analysis of variance; ET-1, endothelin-1; IR, immunoreactive; MAP2, microtubule-associated protein 2; MCA, middle cerebral artery; NMDA, N-methyl-D-asparate; NMDAR1, N-methylD-asparate receptor subunit 1; PBS, phosphate-buffered saline; SMC, sensorimotor cortex.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.07.019

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changes in the motor cortex opposite these lesions. The SMC region was chosen to permit comparison with previous findings of lesion-induced forelimb behavioral and contra-cortical dendritic changes after electrolytic lesions of similar size and placement. Adult rats with unilateral electrolytic lesions of the SMC were found to have impairments in the use of the forelimb contralateral to the lesion and developed a compensatory hyper-reliance on the ipsilateral (non-impaired) forelimb (Jones and Schallert, 1992a). Additionally, these lesions were found to produce timedependent alterations in the motor cortex contralateral and homotopic to the lesion, including increases in layer V dendritic arborization (Jones et al., 1996; Jones and Schallert, 1992a), increases in the number of synapses per neuron and changes in synaptic ultrastructure (Jones, 1999; Jones et al., 1996, 1999). Increases in dendrites immunoreactive (IR) for the cytoskeletal protein, microtubule-associated protein 2 (MAP2), have also been found at time points corresponding to the dendritic growth (Bury and Jones, 2002). The dendritic growth has been linked to both the degenerative effects of the lesion and to increased reliance on the unimpaired forelimb (e.g. Adkins et al., 2002; Bury et al., 2000). Therefore, an important goal of the present study was to determine whether ET-1 induced ischemic SMC lesions produce forelimb somaticsensory and motor asymmetries as well as dendritic changes in the cortex opposite the lesion. The behavioral effects of the lesions were characterized using a battery of sensitive measures of sensorimotor impairments. Dendritic structural plasticity in the motor cortex opposite the lesions was assessed using quantitative measures of dendrites immunostained for MAP2. We also sought to extend previous findings by examining cellular changes in the cortex associated with altered neural activity. Quantitative immunocytochemistry was used to assess motor cortical changes in Fos, the protein product of the immediate early gene, c-fos, and the N-methyl-D-asparate (NMDA) receptor subtype, NMDAR1. Examination of Fos was chosen as a general marker of changes in neural activity (e.g. Robertson, 1992) and because increased c-fos has been linked to several forms of learning (e.g. Castro-Alamancos et al., 1992; Kaczmarek and Chaudhuri, 1997; Kleim et al., 1996; Rose, 1991). NMDAR1 was examined because its upregulation following brain damage has been related to increased excitability in tissue surrounding and connected to the site of a lesion and it is likely to play a role in functional reorganization (e.g. Buchkremer-Ratzmann et al., 1998; Reinecke et al., 1999; Schiene et al., 1996). Immunocytochemistry data were obtained at early (day 2) and later (day 14) time points after ET-1 lesions and compared with sham-operated animals.

EXPERIMENTAL PROCEDURES Subjects Twenty-nine adult (3– 4 months) male Long-Evans hooded rats were housed in pairs, received food and water ad libitum and were kept on a 12-h light/dark cycle. Animal use was in accordance with

protocols approved by the Animal Care and Use Committees of the University of Washington and the University of Texas, were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and all efforts were made to minimize the number of animals used and their suffering. Rats underwent ET-1 lesions and then were randomly assigned to be killed at 2 days (n⫽8) or 14 days (n⫽8) postsurgery. Sham-operated animals killed at 2 (n⫽6) and 14 days (n⫽4) were combined into one group after statistical analyses indicated no significant behavioral or anatomical differences between the two time points. In an additional three animals (two lesion and one sham), immunocytochemistry for ET-1 was used to verify its cortical diffusion.

Surgical procedure Rats were anesthetized with Equithesin (150 mg/kg chloral hydrate and 34 mg/kg pentobarbital). The side of the lesion was randomly assigned to one rat per caged pair and the cage mate received a lesion in the opposite hemisphere so that there were equal numbers of left and right lesions. The skull overlying the forelimb representation region of the SMC was removed between 0.5 mm posterior and 1.5 mm anterior to bregma and 3.0 – 4.5 mm lateral to midline. Three penetrations of dura mater were made using a 25 gauge needle. ET-1 (Peninsula Laboratory, San Carlos, CA, USA; 120 pmol, 0.2 ␮g/␮l delivered in 1.5 ␮l sterile saline) was then delivered via microsyringe onto the dura mater, 0.5 ␮l at a time with a 2 min interval between applications. After the third application, the skull was left undisturbed for 5 min prior to suturing the scalp. This dosage of ET-1 was chosen because it was found to produce very localized ischemic lesions through all six cortical layers while sparing the underlying white matter (Fuxe et al., 1997). Sham-operated rats received all procedures up to, but not including, skull removal. Removal of skull was not performed in sham-operated animals because it has been found to produce behavioral and neurochemical asymmetries (Adams et al., 1994). Atropine sulfate (0.1 mg/kg) was used post-operatively to counteract the respiratory depressive effects of Equithesin.

Behavioral tests A battery of behavioral tests previously found to be highly sensitive to sensorimotor function (e.g. Schallert et al., 2000) was administered before surgery (day 0), and on day 1 after the lesion. In animals killed at the later time point (day 14), most tests were also administered on days 3, 7 and 13 after surgery. For shamoperated animals, one forelimb was randomly designated as “contralateral” at the time of surgery. Forelimb postural-motor asymmetries. The Schallert Cylinder test was used to examine asymmetries in forelimb use for postural support behaviors (e.g. Schallert et al., 2000). This test encourages upright support against the cylinder walls which sensitively reveals forelimb asymmetries. Rats were videotaped for 2 min in the cylinder and then, during slow-motion video play back, instances of the sole use of the ipsilateral (to the lesion) or contralateral forelimb or the simultaneous bilateral use of both forelimbs for upright support were recorded. The ipsilateral asymmetry score was computed using the formula: % (ipsilateral forelimb support⫹1/2 bilateral forelimb support)/(ipsilateral⫹contralateral⫹bilateral forelimb support). Forelimb adduction. Instances of the adduction of either forelimb were also recorded during the slow-motion viewing of rats in the cylinder. Limb adduction was defined as retraction of a forelimb for at least 2 s while rats were stationary, in a horizontal position resting on the remaining paws (Lindner et al., 2003; Schallert and Lindner, 1990). The number of ipsilateral and contralateral adductions per 2 min of observation was recorded.

D. L. Adkins et al. / Neuroscience 128 (2004) 473– 486 Vibrissae-stimulated forelimb placing. Asymmetries in forelimb-placing behaviors were assessed using the Placing test and the Extinction Placing test (Barth et al., 1990a) on postoperative day 1. For the Placing test, animals were held by the torso with their forelimbs hanging freely. They were then moved slowly and laterally toward the edge of the countertop until the vibrissae of one side made contact with the edge. Typically intact animals will quickly place the forelimb onto the edge of a counter top when the ipsilateral vibrissae are brushed against the table edge. In contrast, rats with unilateral electrolytic SMC lesions have impairments in the placing response with the limb contralateral to the lesion (Barth et al., 1990a; Hoane et al., 2000). This deficit is even more pronounced with the Extinction Placing test. In the Extinction Placing test, as the vibrissae of one side are brushed against the edge of the counter top, the contralateral vibrissae are gently stimulated by the experimenter, thus providing competing vibrissal stimulation. Animals were tested on 10 trials of each forelimb, performed in balanced order for each of the two placing tests. Placing asymmetries are presented as the percentage of contralateral and ipsilateral forelimb placements per trial. One animal was excluded from the placing analyses because it struggled during the tests, producing unreliable data. Coordinated forelimb placement during locomotion. The Footfault test was used to measure coordinated forelimb placement during locomotor movements (Barth et al., 1990b). Animals were placed on an elevated grid platform (33 cm ⫻30 cm; grid openings: 12.25, 8.4 and 6.25 cm2) and were videotaped for 2 min as they wandered the platform. Rats moved across the platform placing their paws on the rungs of the grid openings. Errors (“footfaults”) were measured as slips with either forelimb through the grid openings. Following unilateral SMC lesions, the forelimb opposite the lesion is frequently incorrectly placed and slips through the grid opening (e.g. Aronowski et al., 1996; Jones et al., 1999; Schmanke et al., 1996). Using slow-motion video play-back, the total number of steps and the number of footfaults were assessed. Data were analyzed as the percentage of ipsilateral and contralateral errors per forelimb step. Somatosensory asymmetry. The Bilateral Tactile Stimulation test (also known as the “Sticky Tape test”) was used to measure forelimb somatosensory asymmetries (Schallert et al., 1983; Schallert and Whishaw, 1984). This test measures responsiveness to tactile stimulation applied to the distal forelimbs. Asymmetries on this test have been found to be independent of postural-motor asymmetries and may be largely insensitive to practice and other experience effects (Rose et al., 1987; Schallert et al., 1983). Animals were tested in their home cage, with their cage mates temporarily removed. For each trial, small adhesive backed circular patches (Avery multipurpose round removable labels; 1.2 cm diameter; Avery Dennison, Brea, CA, USA) were applied to the radial aspect of the wrists of both forelimbs. The rat was returned to the homecage and the order (left versus right) in which the animal contacted the stimuli was recorded. Four to five trials were administered per each time-point. Animals with forelimb somatosensory impairments show a bias toward contacting the stimulus on the ipsilateral (non-impaired) limb before the contralateral stimulus (e.g. Barth et al., 1990a; Schallert and Whishaw, 1984). Data are presented as the percentage of trials (four or five) in which the animal contacted the ipsilateral stimulus first.

Immunocytochemistry tissue processing Rats were deeply anesthetized with sodium pentobarbital (100 mg/kg) and were perfused intracardially with 200 ml of 0.1 M phosphate buffer and 400 ml of 4% paraformaldehyde in the same buffer. Coronal sections (50 ␮m) were obtained throughout the frontoparietal cortex using a vibratome. Six rostral to caudal sets of tissue sections were collected into 0.01 M phosphate-buffered

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saline (PBS). For rats killed 2 and 14 days post-surgery, adjacent sets of sections were used for MAP2, Fos and NMDR1 immunocytochemistry. One set was stained with Toluidine Blue and used for lesion verification. Lesion reconstructions were based on the extent of remaining tissue and were drawn on to schematics of cerebral coronal sections adapted from Paxinos and Watson (1986). Rats used for ET-1 immunostaining were perfused within 45 min after surgery. For immunocytochemistry processing, a free-floating section method was used. Sections were placed in 0.3% H2O2 in 0.01 M PBS at room temperature for 30 min to exhaust endogenous peroxidase activity. Sections were then rinsed in PBS and placed for 2 h at room temperature in a solution to block non-specific protein binding. This blocking solution consisted of 0.4% Triton-X and 0.1% bovine serum albumin in PBS with either 2% horse serum (MAP2) or goat serum (Fos, NMDAR1 and ET-1). Following rinses in PBS, adjacent sections were incubated at 4 °C for 72 h in one of the following: anti-MAP2a⫹b (1:500; Sigma, St. Louis, MO, USA; clone AP20), anti-NMDAR1 (1:500; Chemicon, Temecula, CA, USA), anti-Fos (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-ET-1 (1:500; Peninsula Laboratories, Inc., San Carolos, CA, USA). Following 1 h incubation in secondary antibody (1:200 horse anti-mouse for MAP2 staining, goat anti-rabbit for NMDR1, Fos and ET-1 staining), sections were rinsed and incubated for 1 h in peroxidase-linked avidin– biotin complex (ABC kit; Vector Laboratories, Burlingame, CA, USA). Immunoreactivity was visualized using 3–3= diaminobenzidine with nickel ammonium sulfate intensification. Tissue from all groups was included in each batch of immunocytochemical processing. To verify the specificity of antibody labeling, each batch of immunocytochemical processing included tissue sections processed without primary antibody (noprimary controls). Quantitative analyses were performed in layers II/III and V of the motor cortex (the lateral agranular region of the SMC) contralateral and homotopic to the lesions or within the randomly assigned hemisphere of sham-operated rats. Because of histological processing errors, some animals were excluded from immunocytochemical analyses (n⫽1, day 2 lesion group excluded from all analyses, n⫽2, day 14 lesion group excluded from the layer 2/3 MAP2 analysis, and n⫽1 sham operate excluded from the layer 2/3 Fos analysis).

Immunocytochemistry measures of the motor cortex opposite the lesion MAP2 immunoreactive (IR) surface density. The cycloid grid intersection method (Baddeley et al., 1986) was used to measure the surface density (Sv) of MAP-2 IR processes (AdkinsMuir and Jones, 2003). MAP2-IR somata were excluded from these measurements. A set of test lines (cycloid arcs) arrayed in a complex, staggered manner was superimposed on light microscopic images. The vertical axis of the cycloid arc test grid was aligned parallel to the orientation of cortical columns, defined as the orientation of the apical dendritic shafts of pyramidal neurons (i.e. apical shaft orientation was used to define local vertical windows; Baddeley et al., 1986). The number of intersections between IR dendritic processes and cycloid arcs was counted. The surface density was then calculated using the formula: Sv⫽2(I/L), where I is the total number of intersections and L is the total test line length (2500 ␮m) within the sample area. Within layers II/III and V, 12 samples per layer (three to four samples, 250 ␮m apart per each of three to four coronal sections) were chosen using a systematic random sampling method and were visualized using a 100⫻ oil immersion objective (⫻1680 final magnification). Fos IR cell density. The density of Fos-IR cells was determined using the optical disector method (e.g. Harding et al., 1994; Korbo et al., 1990; Sterio, 1984). Fos positive cells were visualized

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using a 100⫻ oil immersion objective (⫻1680 final magnification) and cells were counted from 12 samples per layer (three to four samples, 250 ␮m apart per each of three to four coronal sections). An unbiased counting frame was superimposed over each sample and then Fos-IR cells that appeared in the top focal plain were marked and not counted whereas cells that appeared focusing through the depth of the section were counted. Cell density (Nv) was then calculated with the following equation: Nv⫽⌺Q⫺/ ⌺v(frame), where ⌺Q⫺ is the total number of Fos positive cells counted for each brain and ⌺v(frame) is the total sample volume (3.9⫻106␮m3). NMDAR1 optical density. Light microscopic images from three to four coronal sections per brain were captured using a high-resolution digital camera (⫻84 final magnification). Images were captured using standardized microscope and camera settings and then opened in Adobe Photoshop. Using cytoarchitectural landmarks, layers II/III and V and the corpus callosum (background control area) were outlined and Adobe Photoshop Histogram feature was used to determine the mean luminosity for each region. The corpus callosum was used as a background control area. Optical density was then calculated as the inverse of the luminosity. Layer II/III and V data are presented as a ratio of the background optical density.

Statistical analyses Behavioral data were analyzed using SPSS general linear models for two-way analysis of variance (ANOVA) for the effects of Groups, Days and Group by Day interactions with Subjects treated as a random factor to produce the appropriate error terms. SPSS procedure for contrasts was used for post hoc tests when needed to further analyze group differences. The day 1 behavioral data of the early and late time point groups were combined because there were no significant differences between the two lesion groups or between the two sham groups on this day. Immunocytochemistry data were analyzed using SPSS procedure for the effects of Group (Sham, post-lesion day 2, and post-lesion day 14). The a priori ␣ level was 0.05 for all comparisons.

RESULTS Lesion reconstruction Fig. 1A and B show schematic reconstructions and representative photomicrographs of unilateral ET-1 lesions as assessed 2 and 14 days after the lesion. All lesions resulted in major damage in the SMC. Most lesions produced at least superficial damage to the underlying white matter (n⫽6 day 2 group; n⫽6 day 14 group) and four produced superficial damage to underlying striatum (n⫽3 day 2 group; n⫽1 day 14 group). Reconstruction of the extent of the lesions revealed a range of lesion sizes and lesion placement which could be found in both the early and late time point groups, although the early time point lesions tended to extend more rostrally than those in the later time point. (However, as previously mentioned, the behavioral data of the two lesion groups obtained on day 1 did not differ significantly.) Fig. 1C shows immunolabeling for ET-1 at 30 min after the onset of its application, which indicates a diffusion into the depth of the cortex underlying the craniotomy. Behavioral results Forelimb postural-motor asymmetries. As shown in Fig. 2, the unilateral lesions resulted in an increase in the

asymmetry score on the Schallert Cylinder test, indicating preferential use of the ipsilateral (to the lesion) forelimb for upright postural support. This asymmetry was evident throughout the days of observation. Sham-operated animals did not show this pattern of asymmetrical forelimb use. Repeated measures ANOVA revealed a significant Group by Day interaction effect (F4,54⫽8.86, P⬍0.00001). There were also significant effects of Group (F1,24⫽27.29, P⬍0.0001) and of Day (F4,54⫽5.04, P⬍0.002). Post hoc analyses indicated that the lesion group had a significantly greater asymmetry on each post-lesion day of observation. The increased asymmetry score in the lesion group reflects an increase in sole support using the ipsilateral forelimb and a proportionate decrease in both sole support with the contralateral limb and in simultaneous bilateral forelimb support behaviors. Pooled over days post-lesion, the mean⫾S.E.M. percentage of ipsilateral, contralateral and simultaneous bilateral support observations was 52.06⫾2.48, 20.35⫾1.54 and 27.58⫾1.27, respectively in the lesion group versus 34.17⫾1.87, 31.56⫾1.91 and 34.27⫾0.99, respectively in the sham group. There were significant Group, Day and Group by Day interaction effects for each of these variables (P’s⬍0.05). Forelimb adduction. Unilateral SMC lesions resulted in a major, but transient, increase in the adduction of the contralateral forelimb compared with shams (see Fig. 3). In a two-way ANOVA for the number of contralateral adductions per 2 min in the cylinder, there were significant effects of Group (F1,24⫽11.69, P⬍0.01), Day (F4,54⫽7.55, P⬍0.001) and Group by Day interaction (F4,54⫽5.42, P⬍0.001). In post hoc analyses, contralateral limb adduction was significantly increased relative to shams on days 1 and 3 after the lesion, but not on the later test days. There were no significant differences in the instances of adduction of the ipsilateral forelimb between lesion and sham groups. Vibrissae-stimulated forelimb placing. SMC lesions produced significant reductions in vibrissae-stimulated placing of the contralateral forelimb compared with sham animals, as measured on the first day after the lesion (see Fig. 4). This reduction was particularly evident in the Extinction-Placing test, when competing stimuli were applied to the ipsilateral vibrissae. One-way ANOVAs indicated that the percentage of limb placements (out of 10 trials) made with the contralateral forelimb was significantly reduced in rats with lesions compared with sham-operated animals for both the Placing (F1,23⫽13.09, P⬍0.001) and Extinction Placing (F1,23⫽29.51, P⬍0.0001) tests. There were no significant group differences in the percentage of ipsilateral placing responses in either measure. Coordinated forelimb placement during locomotion. On the Footfault test, ET-1 induced lesions increased the percentage of contralateral errors (footfaults) per step compared with those made with one limb in shamoperated rats. The greatest error rate was found on the first day after the lesion and there was a partial return to control levels after this (see Fig. 5). In two-way ANOVA of contralateral footfaults per step, there were significant effects

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Fig. 1. (A) Schematic representations of the largest lesion (black outlines) and a typical lesion (gray) of each post-lesion time point. (B) Representative photomicrographs of smallest, typical and large ET-1 induced lesions of the SMC as viewed in Nissl-stained coronal sections. (C) Immunolabeling for ET-1 in the cortex underlying the craniectomy 30 min after its application (left), in the contralateral cortex of the same coronal section (middle) and in the cortex of a sham-operated rat. An area of intense immunolabeling (outlined in arrowheads) was seen in the cortex underlying the ET-1 application. Scale bars⫽1 mm.

of Group (F1,24⫽25.63, P⬍0.00001), Day (F4,54⫽10.41, P⬍0.00001) and Group by Day interaction (F4,54⫽3.44, P⬍0.01). In post hoc contrasts, rats with lesions made significantly more contralateral errors per step on days 1 and 6 post-lesion compared with shams.

Somatosensory asymmetry. Following ET-1 lesions, animals demonstrated a significant ipsilateral bias on the Bilateral Tactile Stimulation test compared with sham-operated animals, as indicated by the increase in the percentage of trials in which the ipsilateral stimulus was

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Fig. 2. Forelimb postural-motor asymmetries. (A) The Schallert Cylinder test was used to examine asymmetries in forelimb use for upright postural support. The arrows indicate the forelimb ipsilateral to the lesion. (B) Unilateral ET-1 lesions resulted in significantly increased reliance on the ipsilateral forelimb for postural support compared with sham-operated animals and this persisted throughout the days of observation. The asymmetry score was calculated using the formula: (ipsilateral⫹1/2 bilateral forelimb support observations)/total number of support observations. Data are means⫾S.E.M. * P⬍0.05, ** P⬍0.01, ‡ P⬍0.0001 significantly different from sham.

contacted before the contralateral stimulus (see Fig. 6). There were significant effects of Group (F1,24⫽10.40, P⬍0.01), Day (F3,44⫽2.92, P⬍0.04) and interaction of Group by Day (F3,44⫽6.12, P⬍0.001). This ipsilateral bias was significantly different from the sham group on days 1 and 13 of post-lesion testing. Immunocytochemistry results Changes in layer II/III contralateral to the lesion. In layer II/III, there were robust and time-dependent increases in measures of Fos and NMDAR1, but not in MAP2 IR processes in the motor cortex opposite the lesions. As shown in Fig. 7, the lesions resulted in increased density of Fos IR cells in layer II/III on day 2 compared with shams (F1,21⫽6.58, P⬍0.01) and compared with post-

Fig. 3. Contralateral forelimb adduction. (A) Limb adduction was defined as retraction of the forelimb (arrow) for at least 2 s while rats were in a stationary horizontal position. (B) Early after unilateral ET-1 induced SMC lesions, animals adducted the contralateral (impaired) forelimb significantly more often than sham-operated animals. Data are mean adductions per 2 min⫾S.E.M. * P⬍0.05, ** P⬍0.001 significantly different from shams.

lesion day 14 (F1,21⫽12.86, P⬍0.001). The optical density of NMDAR1 immunoreactivity was significantly elevated in layer II/III at day 14 compared with shams (F1,23⫽12.15, P⬍0.002) and approached significance compared with post-lesion day 2 (F1,23⫽4.07, P⫽0.055), as shown in Fig. 8. However, in contrast to the effects found in layer V (described below), there were no significant post-lesion changes in the surface density of MAP2 IR processes in layer II/III of the motor cortex at post-lesion day 2 (F1,20⫽2.46, P⬎0.05) or post-lesion day 14 (F1,20⫽1.25, P⬎0.05) compared with shams (Fig. 9). Changes in layer V contralateral to the lesion. In layer V of the motor cortex opposite the ET-1 lesions, there were significant increases in the measures of MAP2 IR dendrites and a time-dependent increase in NMDAR1 compared with sham-operated rats. As shown in Fig. 9, the surface density of dendritic processes IR for MAP2 was significantly increased at both 2 (F1,22⫽9.96, P⬍0.02) and 14 days post-lesion (F1,22⫽5.06, P⬍0.02) compared with shams. There were no significant differences in layer V MAP2 IR process surface density between the two postlesion time points (F1,22⫽.27, P⬎0.05). There was also a

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Fig. 4. Forelimb placing responses to vibrissal stimulation. (A) Arrows indicate the forelimb just before and after the initiation of a placing response to stimulation of the vibrissae with a counter edge. This is an example of the Extinction Placing test in which competing stimulation is made to the opposite vibrissae with the experimenter’s finger (asterisks). The Placing test is performed without the competing stimulation. (B) ET-1 lesions resulted in major reductions in placing responses with the contralateral forelimb in the Placing test and this effect was even more pronounced in the ExtinctionPlacing test. Data are means⫾S.E.M. ‡ P⬍0.001 significantly different from sham.

significant increase in the optical density of NMDAR1 immunoreactivity (F1,23⫽8.47, P⬍0.01) in layer V in animals at 14, but not at 2 days compared with shams (Fig. 8). As shown in Fig. 7, Fos-IR cell density tended to be increased at day 2 in layer V, but this was not significant compared with shams (F1,22⫽2.49, P⫽0.13). However, the decrease at day 14 was significant compared with the earlier time point (F1,22⫽6.40, P⬍0.01).

DISCUSSION In summary, unilateral ET-1-induced lesions of the SMC resulted in substantial postural-motor and somatosensory impairments of the forelimb contralateral to the site of the lesion as well as a corresponding increase in reliance on the ipsilateral forelimb. Some of these behavioral asymmetries persisted during the 14 days of observation. ET-1 lesions also produced time- and laminar-dependent cellular changes in layer II/III and V of

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Fig. 5. Coordinated forelimb placement during locomotion. (A) In the Footfault test, rats were videotaped while moving across a grid floor and errors were recorded when a forepaw slipped through a grid opening (arrow). (B) Animals with unilateral ET-1 lesions had an initial major impairment in placements of the contralateral forelimb in comparison with shams and this partially improved over time. Data are means⫾S.E.M. % (contralateral forelimb errors/contralateral forelimb steps). ** P⬍0.001; ‡ P⬍0.0001.

the motor cortex opposite the lesion. Increases in the surface density of dendrites immunolabeled for the cytoskeletal protein, MAP2, were most evident in layer V at both 2 and 14 days post-lesion compared with shamoperated animals. Layer V dendritic changes were coincident with increased optical density of NMDAR1 at 14 days post-lesion. Although layer II/III of the SMC opposite the lesion did not show an increase in the surface density of MAP2 positive processes, there was a robust increase in the numerical density of cells immunolabeled for Fos protein at 2 days post-lesion and in NMDAR1 optical density at 14 days post-lesion compared with sham-operated rats in this layer. As discussed below, these changes in the motor cortex opposite the lesion may be due to the influences of both the central neurodegenerative and the forelimb behavioral effects of the lesion.

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position has been found after unilateral striatonigral degeneration (Schallert and Lindner, 1990). Thus, it is possible that this behavior was a result of temporary disruption of striatal activity, e.g. related to deafferentation of corticostriatal afferents (e.g. Hoane et al., 1998, 2000; Jones and Schallert, 1992b). Finally, these contralateral forelimb impairments were coupled with a greater use of the forelimb ipsilateral to the lesion. Rats developed a hyper-reliance upon the unimpaired forelimb, as measured in the Schallert Cylinder test and this was evident throughout the 14 days of observations post-lesion. Although direct comparisons with other lesion techniques were not conducted in this study, the pattern of forelimb asymmetries induced by the unilateral ET-1 lesions of the SMC appear similar to those found after comparably sized and placed electrolytic lesions (e.g. Barth et al., 1990a,b; Hernandez and Schallert, 1988; Jones et al., 1999; Jones and Schallert, 1992a,b, 1994; Voorhies and Jones, 2002). These lesioninduced changes in forelimb behavior are likely to contribute, in part, to the cellular and structural changes found in the contralateral motor cortex. Laminar-dependent changes in MAP2 IR dendrites in the cortex opposite the lesion

Fig. 6. Somatosensory asymmetry. (A) The Bilateral Tactile Stimulation test measures responsiveness to somatosensory stimulation that is applied simultaneously to both forelimbs. Sequential photographs (left to right) show a rat contacting and removing a stimulus (arrows) from one forelimb. (B) Rats with unilateral ET-1 lesions typically contacted the ipsilateral forelimb’s stimulus before the contralateral stimulus and this was significantly different compared with sham-operated animals on days 1 and 13 post-lesion. Data are the means⫾S.E.M. % of trials in which the ipsilateral stimulus was contacted first. * P⬍0.01.

ET-1 lesion-induced changes in behavior Lesion-induced changes in forelimb behavior were revealed using a test battery that is highly sensitive to forelimb sensory and motor asymmetries (e.g. Schallert et al., 2000; Schmanke et al., 1996). Forelimb somatic-sensory asymmetries were evidenced by an increased ipsilateral somatosensory response bias on the Bilateral Tactile Stimulation test, a measure which has been found not to be sensitive to postural motor asymmetries or practice effects (Rose et al., 1987; Schallert et al., 1983). Postural-motor impairments included contralateral impairments in forelimb placing responses to vibrissal stimulation and in the coordinated placement of the forelimb during locomotion, as revealed by the Footfault test. Transient increases in the adduction of the contralateral forelimb were also seen. The tendency for rats to hold one forelimb in an adducted

Unilateral ET-1-induced SMC lesions increased the surface density of MAP2 IR processes in layer V of the contralateral motor cortex at both post-lesion time points examined. MAP2 is a cytoskeletal protein localized in neuronal cell bodies and dendrites that mediates microtubule stabilization and dendritic cytoskeletal growth (reviewed in Itoh et al., 1997; Sánchez et al., 2000). MAP2 alterations accompany increased arborization and morphological plasticity of dendrites and appear to be a key component of activity-dependent plasticity in developing and adult animals (Diez-Guerra and Avila, 1993, 1995; Kaech et al., 1996; Martinez et al., 1997; Quinlan and Halpain, 1996a; Wilson and Keith, 1998; Woolf et al., 1994). In layer V of the motor cortex opposite similarly sized and placed electrolytic lesions of the SMC, increases in the dendritic arborization of Golgi-Cox-impregnated pyramidal neurons (Jones and Schallert, 1992a), in the membrane surface area and volume per neuron of dendrites visualized using electron microscopy (Jones et al., 1996) and in the surface density of MAP2 IR dendritic processes (Bury and Jones, 2002) have been found at time points between 14 and 30 days after the lesion compared with shams. More subtle, but significant, increases in MAP2 IR processes were also found in layer II/III at 20 days after electrolytic lesions of the SMC (Bury and Jones, 2002) whereas, in the present study, increases in MAP2 were nonsignificant in Layer II/III, as assessed at 2 and 14 days. This difference in the statistical significance of the layer II/III changes may be due to the earlier time-point examined in the present study or could reflect differences in the lesions. Nevertheless, Bury and Jones (2002) found much more robust lesioninduced increases in MAP2 in layer V than in layer II/III, which is in general agreement with the laminar-dependent pattern of dendritic change in the present study.

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Fig. 7. Fos IR cell density. (A) Representative photomicrographs of Fos IR cells in motor cortical layers II/III and V of a sham-operated rat and in the hemisphere opposite the lesion at post-lesion days 2 and 14. Scale bar⫽50 ␮m. (B) A significant increase in the numerical density of Fos IR cells was found in layer II/III, but not layer V at day 2 when compared with shams. Both layers had significantly increased Fos IR cell density at day 2 relative to day 14 following ET-1 lesions. Data are means⫾S.E.M. * P ⬍0.05 vs. sham, † P⬍0.05 vs. Day 2.

Lesions of the SMC produce degeneration of crossedcorticocortical afferents as well as increased reliance on the ipsilateral forelimb, either of which might induce the dendritic changes observed in the present study. We have previously independently examined these effects by transecting the corpus callosum and by using limb restricting vests to peripherally force animals to rely upon only one forelimb. Transections primarily increased dendritic spines in layer V whereas forced forelimb use primarily increased dendritic spines in layer II/III, as assessed 18 days after surgery (Adkins et al., 2002). The combination of the two manipulations robustly increased layer V (but not layer II/III) dendritic arborization and spine density compared with either independent manipulation (Adkins et al., 2002; Bury et al., 2000). These results suggest that the present MAP2 changes may be a combined result of the enhanced reliance on the forelimb ipsilateral to the lesion and the degeneration of transcallosal fibers. As we have previously hypothesized, the growth-promoting effects of moderate degeneration may facilitate forelimb behavior-

driven changes in dendrites in the motor cortex. The pattern of MAP2 changes found in the present study is also consistent with the laminar-dependent pattern of denervation- and behaviorally induced dendritic change. It should be noted that the MAP2 antibody used in this study labels the high-molecular weight forms of MAP2 (a⫹b). It would be valuable to investigate whether there are also changes in the surface density of the low-molecular weight forms of MAP2 that, in contrast to MAP2a and MAP2b, are present in the CNS at high levels in development and decrease in adulthood (Sánchez et al., 2000). There may be lesion- and/or behaviorally induced changes in the proportion of these MAP2 isoforms which the use of a single antibody has not revealed. Changes in Fos and NMDR1 in the cortex opposite the lesion Despite the lack of significant MAP2 increases in layer II/III, there were robust changes in Fos and NMDR1 im-

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Fig. 8. NMDAR1 optical density. (A) Representative photomicrographs of NMDA receptor subtype 1 (R1) immunoreactivity in layers II/III and V from sham-operated, post-lesion day 2, and post-lesion day 14 animals. Images are from the motor cortex contralateral to the lesion. Scale bar⫽50 ␮m. (B) In layer II/III and V, there was a significant increase in the optical density of NMDAR1 14 days after ET-1 lesions compared with shams. Data are the means⫾S.E.M. optical density in each layer/background (white matter) optical density. * P⬍0.05.

munoreactivity, clearly supporting a participation of layer II/III in post-lesion neural plasticity. Several studies have found c-fos or Fos increases in areas adjacent or connected to a site of injury, especially at time points early after the injury (e.g. Dragunow and Robertson, 1988; Johansson et al., 2000; Walton et al., 1999). Many other studies have found that behavioral experience can induce c-fos (e.g. Castro-Alamancos et al., 1992; Kaczmarek and Chaudhuri, 1997; Rose, 1991). Particularly relevant to the present finding is that motor skill learning, but not simple motor activity, transiently increased the percentage of Fos positive cells in layer II/III of the motor cortex (Kleim et al., 1996). Thus, in the present study, the increased density of Fos positive cells may have been a result of lesion-induced denervation, the motor learning required for the animals to rely on the nonimpaired forelimb, or a combination of these factors. The induction of Fos may have then led to upstream neurochemical events (such as increases in neurotrophic factors; e.g. Buytaert et al., 2001) which contribute to increased MAP2 and NMDAR1, as well as dendritic growth and synaptogenesis.

Fourteen days after the lesions, there was significantly greater optical density of NMDAR1 immunoreactivity in the contralateral motor cortex in both layers II/III and V. As with the changes in Fos and dendrites, there is evidence for both denervation- (Gazzaley et al., 1997; Ulas et al., 1990) and behaviorally induced (Rose, 2000) changes in NMDA receptors. NMDA receptor changes have also been linked to increased excitability in the contralateral and peri-lesion cortices and may be related to functional reorganization after brain damage (e.g. Buchkremer-Ratzmann et al., 1998; Que et al., 1999; Reinecke et al., 1999; Schiene et al., 1996). It seems likely that the increase in NMDAR1 protein found at 14 days post-lesion in the present study is linked to such changes in neural excitability (see Nudo et al., 2001; Witte and Stoll, 1997). There is also considerable evidence for a close association between NMDA receptor activation and changes in MAP2 (Halpain and Greengard, 1990; Montoro et al., 1993; Quinlan and Halpain, 1996a,b; Sánchez et al., 1997). Because increased NMDA receptor protein followed MAP2 increases, it is

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Fig. 9. MAP2 surface density. (A) Representative photomicrographs of MAP2 IR processes in layer II/III and layer V from sham, post-lesion day 2, and post-lesion day 14 animals. Images are from the motor cortex contralateral to the lesion. The surface density of dendritic processes immunolabeled with MAP2 was measured using the cycloid grid intersection method. Scale bar⫽50 ␮m. (B) In layer II/III, there were no significant differences between animals with ET-1 lesions compared with sham-operated animals. There was a significant increase in the surface density of MAP2 IR processes in layer V at both 2 and 14 days following ET-1 lesions compared with shams. Data are means⫾S.E.M. * P⬍0.05.

possible that both proteins are increased in response to changes in cortical activity. It also seems likely that the increase in NMDA receptor protein is related to the increase in asymmetric (presumed excitatory) synapses that has been found in layer V of the contralateral motor cortex 25–30 days after SMC lesions (Jones et al., 1999; Luke et al., 2003). Use of ET-1 for the induction of focal ischemia At the concentrations used in this study, ET-1 is an extremely potent vasoconstriction-inducing peptide that can be used to produce small and highly localized ischemic lesions with a minimally invasive surgical procedure. It is likely that the lesions produced by ET-1 are primarily a

result of its induction of vasoconstriction. Co-administration of a vasodilator with ET-1 has been found to block the production of striatal lesions (Fuxe et al., 1992). ET-1 application to neuron-enriched or glia-enriched cultures fails to induce cell toxicity (Lustig et al., 1992; Nikolov et al., 1993) and fails to modify hypoxic damage resulting from sodium cyanide (Nikolov et al., 1993). However, ET-1 does have direct effects on glia and neurons, as demonstrated in culture and slice preparations (reviewed in Rubanyi and Polokoff, 1994). ET-1 application in cultured astrocytes results in cellular proliferation (MacCumber et al., 1990) and hypertrophy (Egnaczyk et al., 2003), alters the spread of intracellular Ca2⫹ waves and reduces gap junction permeability (Blomstrand et al., 1999; Giaume et al., 1992;

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Venance et al., 1997), increases glutamate efflux (Sasaki et al., 1997), induces the expression of neurotrophic factors (Egnaczyk et al., 2003; Koyama et al., 2003; Ladenheim et al., 1993) and increases the production of 2-arachidonylglycerol (Walter and Stella, 2003). ET-1 has also been found to increase intracellular calcium in neural cell lines, cultured neurons and hippocampal slices (Koizumi et al., 1994; MacCumber et al., 1990; Reiser and Donie, 1990; Yue et al., 1990). Although ET-1 appears not to be directly toxic to neurons and glia, the possibility that these non-vascular effects of ET-1 may modify degenerative and/or neuroplastic responses to the ischemic damage has not been ruled out. Summary and conclusions In summary, unilateral SMC lesions induced by ET-1 produced significant sensory and motor deficits of the contralateral forelimb as well as an increased reliance on the ipsilateral forelimb. In the motor cortex contralateral and homotopic to the lesion, there were laminar- and timedependent changes in Fos, NMDAR1 and MAP2. These changes may be a result of both lesion-induced degenerative events and forelimb behavioral changes. The results are consistent with previous studies using electrolytic lesions of the SMC in indicating major dendritic structural plasticity in layer V of the cortex opposite the lesion, but they extend these findings with evidence of changes in neural activity and plasticity in layer II/III, as evidenced by the major increases in measures of Fos at 2 days and in NMDAR1 at 14 days post-lesion in this layer. Finally, this study provides further support for the usefulness of ET-1 as a means of producing very localized ischemic damage of cortical subregions. Acknowledgments—The authors would like to thank Kevin McCullough for animal care, Drs. Ilene Bernstein and Ellen Covey for advice on antibodies, Scott Bury and Linslee Luke for discussion and comments on this study, and Nicole Donlan and Monica Maldonado for help in stereological quantification. This study was supported by National Institutes of Health grants MH56361 and MH64586.

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(Accepted 4 July 2004) (Available online 8 September 2004)