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The behavioral and dendritic growth effects of focal sensorimotor cortical damage depend on the method of lesion induction
Behavioural Brain Research 133 (2002) 237 /246 www.elsevier.com/locate/bbr
Research report
The behavioral and dendritic growth effects of focal sensorimotor cortical damage depend on the method of lesion induction Ann C. Voorhies a, Theresa A. Jones a,b,c,* a
b
Psychology Department, University of Washington, Seattle, WA 98195, USA Psychology Department, Mezes Hall, Campus Box B3800, The University of Texas, Austin, TX 78712, USA c Institute for Neuroscience Research, The University of Texas, Austin, TX 78712, USA Received 26 June 2001; received in revised form 18 December 2001; accepted 18 December 2001
Abstract Using different models of focal cortical injury in adult rats, the neural structural and behavioral outcomes of unilateral lesions of the forelimb representation of the sensorimotor cortex (SMC) were assessed. Lesions were produced using either electrolytic, aspiration, or combined (‘electroaspiration’) techniques. Measurements of dendritic arborization in layer V of the motor cortex opposite the lesion revealed a growth of pyramidal neuron dendritic processes following electrolytic lesions in comparison to shams. This effect was not found in either the aspiration or electroaspiration lesion groups. Behaviorally, animals in all lesion groups developed a hyper-reliance on the forelimb ipsilateral to the lesion and proportionate disuse of the contralateral (impaired) forelimb for postural support behaviors. In comparison to sham-operated animals, the initial asymmetries in behaviors expressed during movement were similar between lesion groups, but were less enduring following electrolytic lesions than following aspiration and electroaspiration lesions. Furthermore, both aspiration lesion groups had more prevalent adduction of the impaired forelimb than the electrolytic-only lesion rats. Thus, cortical aspiration resulted in more severe and enduring forelimb impairments than the electrolytic lesions, despite similar lesion sizes, as assessed using cortical volume measures. These findings suggest that the aspiration lesion procedures, at least as performed in the present study, exacerbate the behavioral effects of focal cortical injury and limit compensatory plasticity in the contralateral cortex. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cortical injury; Electrolytic lesions; Aspiration lesions; Suction-ablation; Ischemia; Dendritic arborization; Forelimb use; Sensorimotor cortex; Recovery of function
1. Introduction Several previous studies suggest that the neural and behavioral outcomes of neocortical damage depend upon the technique used to induce the lesion. Using adult rats, Napieralski et al. [26] found that large unilateral ischemic lesions produced using thermocoagulation of pial blood vessels overlying the sensorimotor cortex (SMC) versus similarly sized aspiration (suctionremoval) lesions of the SMC resulted in different patterns of impairment and different recovery rates on tests of sensorimotor function. Furthermore, large
* Corresponding author. Tel.: 1-512-475-7763; fax: 1-512-4757765 E-mail address: [email protected] (T.A. Jones).
unilateral thermocoagulatory lesions, but not aspiration lesions, of the SMC in adult rats resulted in crossed corticostriatal axonal sprouting from the motor cortex of the intact hemisphere [27,39]. Additional research has shown that thermocoagulatory and aspiration lesions of the SMC result in different lesion-induced cellular responses in the underlying striatum. Whereas GAP-43 expression in the dorsolateral striatum was not significantly changed following cortical thermocoagulatory lesions, it was significantly decreased after aspiration lesions [38] (see also [32,39]). These lesion-dependent differences were found even when the lesions were of comparable size and were performed in the same laboratory [26,27,38,39]. In more focal lesion models, electrolytic SMC lesions (produced by passing electric current through a platinum electrode as it is moved through the tissue), have been found to result in robust
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dendritic growth and synaptogenesis in the motor cortex opposite the site of cortical damage [16 /18]. However, somewhat larger aspiration lesions have been found not to result in dendritic growth, at least not at comparable time points after the lesions [13,28]. Based on these previous studies, it seemed possible that aspiration lesions either inhibit or fail to promote the compensatory neural plasticity and behavioral recovery that is found after other types of cortical injury. The purpose of the present study was to test the hypothesis that neural growth contralateral to the site of the lesion as well as forelimb behavioral outcomes vary following similarly sized focal unilateral electrolytic versus aspiration lesions of the SMC. Adult male rats were assigned to one of the following groups: (1) electrolytic lesions; (2) aspiration lesions; (3) electroaspiration lesions, used to assess whether differences in electrolytic versus aspiration groups were dependent upon passage of electric current in the former group; and (4) sham controls. These groups were compared for their effects on asymmetrical use of the forelimbs and on the arborization of layer V pyramidal neurons in the cortex opposite the lesion. Day 18 following surgery was chosen as the time point for examination of dendritic arborization based on previous findings indicating that this is a time point of contracortical dendritic growth after unilateral electrolytic SMC lesions [17,18]. To assess the possibility that aspiration lesions may simply result in a slower course of dendritic growth, an additional group of animals were assessed at 30 days after aspiration lesions.
2. Methods 2.1. Subjects Subjects were 36 adult (3 /4 months old) male Long / Evans hooded rats maintained in a vivarium at the University of Washington. Animals were housed in pairs in standard tub-style laboratory cages with wood chip bedding. Rats were kept on 12:12 h light:dark cycle and had food and water ad libitum. Rats were made tame prior to the onset of the experimental procedures by frequent gentle handling. Subjects were randomly assigned to one of the following four groups: electrolytic lesions (n/8), aspiration lesions (n/14), electroaspiration lesions (n/7), and sham surgeries (n/7). All animals were sacrificed at 18 days after surgery with the exception of a subset of animals in the aspiration lesion condition (n/7) and the sham group (n/3), which were used to assess structural changes at 30 days after surgery. Animal use was in accordance with a protocol approved by the Animal Care and Use Committee of the University of Washington.
2.2. Sensorimotor cortex lesions Prior to surgery, all animals were anesthetized with Equithesin (150 mg/kg chloral hydrate and 34 mg/kg pentobarbital). For SMC lesions, following a surgical incision to the scalp, the skull overlying the forelimb representation region of the SMC was removed. Coordinates for skull removal were between 0.5 mm posterior and 1.5 mm anterior to bregma and 3.0 /4.5 mm lateral to midline. The depth of dura was then measured and dura was removed. For electrolytic lesions, an uninsulated 30 gauge platinum wire electrode was lowered to a depth of 1.7 mm below dura. Electric current (1.0 mA) was delivered as the electrode was continuously moved in eight equally spaced transverses through the depth of the exposed SMC for 120 s. Aspiration lesions were created by suction-removal of the exposed gray matter of the SMC, with the aid of a dissecting microscope. All exposed tissue was removed, down to the appearance of white matter. Electroaspiration lesions were created using the same procedure used to make the electrolytic lesions followed immediately by suction-removal of the damaged tissue. As shown in Fig. 1, aspiration lesions and electroaspiration lesions were similar to electrolytic lesions in size and placement. Sham animals received anesthesia and scalp incisions, but the skull was not disturbed, as previous research has indicated that trephination alone may cause behavioral asymmetries [1]. Following surgery, all animals were sutured and given atropine sulfate (0.1 mg/kg) to counteract the respiratory depressive effects of the Equithesin. 2.3. Behavioral analyses Animals were filmed for 2 min durations in an upright, transparent cylinder on the day before surgery and on post-surgical days 2, 7, 16 and 28 (see Fig. 2). This procedure, known as the ‘Cylinder Test’, is used to detect unilateral forelimb impairments and compensatory behaviors, as it encourages vertical-lateral exploratory movements that expose asymmetries in forelimb use [33]. Forelimb use for upright postural support against cylinder walls was observed for all 36 subjects by a rather naive to the experimental conditions, who recorded each instance of left, right and simultaneous bilateral forelimb use for postural support. In the same manner, for each instance of rearing behavior, the forelimb used to initiate upright posture from a quadrupedal position was also recorded. Additionally, the duration of each adduction of the left and right forelimb while the animals were in an otherwise quadrupedal and stationary position in the cylinder was recorded. Limb adduction was defined as retraction of a forelimb for at least 2 s and was observed as a possible indicator of striatal dysfunction [23,34]. These data were later
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Fig. 1. Representative examples of electrolytic, aspiration and electroaspiration lesions. Electrolytic lesions were created via passage of electric current through layers I /V of the forelimb representation region of the sensorimotor cortex (SMC). Aspiration lesions were made by suctionremoval of the cortical matter of the forelimb representation of the SMC, down to the appearance of white matter. Electroaspiration lesions were created using the same technique used to make the electrolytic lesions, followed immediately by suction-removal of the damaged tissue.
by Day interactions. Post-hoc analyses were performed when appropriate using SAS GLM procedure for contrasts. 2.4. Histology and structural analyses
Fig. 2. Cylinder test. Rats were filmed in a transparent cylinder to measure asymmetries in forelimb use for postural support behaviors. Following unilateral SMC lesions, rats have impairments in the forelimb contralateral (arrow) to the lesion and develop a reliance on the ipsilateral forelimb for postural support.
translated into ipsilateral, contralateral, and bilateral limb use, relative to the side of the lesion. For shamoperated animals, left and right forelimb use were designated ‘ipsilateral’ and ‘contralateral’, respectively. There were no significant differences in any behavioral measures between the aspiration lesion subgroups or between the sham subgroups sacrificed at 18 versus 30 days and these were combined for the behavioral analyses. Data were analyzed using SAS general linear models (GLM) system for 2-way analysis of variance (ANOVA) for the effects of Groups, Days and Group
2.4.1. Golgi /Cox impregnation Animals were perfused intracardially using approximately 300 ml of 0.1 M phosphate buffer followed by light perfusion with approximately 100 ml of 4% paraformaldehyde in the same buffer. The brains were removed, coronally transected at the level of the midbrain, and the cerebrum was bisected at midline. They were then placed in Golgi /Cox solution for 35/60 days of impregnation. The brains were reacted en bloc, resin-embedded and coronally sectioned on a microtome at a section thickness of 200 mm. All slides were coded, to avoid experimenter bias effects. Sections were then assessed for quality of Golgi impregnation, and six brains from the 35 days impregnation duration were removed from the study due to poor impregnation. This included two animals from the electrolytic group, two animals from the aspiration group, and two animals from the electroaspiration group. (These are not reported in the n s above.) 2.4.2. Volume measures Cortical volume of the lesion hemisphere was measured in order to ascertain whether the different lesion types produced similar loss of cortical tissue. The volume of non-necrotic cortical tissue remaining in the SMC region was measured using macrostructural landmarks (rostrally, the midline joining of the corpus callosum and, caudally, the midline joining of the anterior commisure) to delimit the SMC region measured. In brains from the 18 days time point, striatal volume was also measured in the hemispheres ipsilateral and contralateral to the lesion. This measure was expected to be sensitive to direct lesion-induced damage to the striatum but not to striatal atrophy secondary to
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the denervation of corticostriatal projections (which would likely be more sensitively detected at later time points). The ratio of ipsilateral to contralateral striatal volume was calculated to determine if striatum was differentially affected by the different lesion methods. Volume measures were obtained using systematic point counting and the Cavalieri method, as described in detail in Chu and Jones [9]. 2.4.3. Dendritic measures The arborization of basilar dendrites of layer V pyramidal neurons was measured in the forelimb region of the motor cortex contralateral to the damaged hemisphere. This population of dendrites was analyzed because earlier studies have shown that it undergoes robust dendritic growth following electrolytic lesions [18,20]. Individual pyramidal neurons in layer V of the forelimb representation in the motor cortex were randomly chosen for analysis; however neurons were only used if they displayed excellent impregnation of the cell body and dendrites, with no beading of the stain or gaps in the processes of the dendrites. Furthermore, neurons that were overly obscured by astrocytes or capillaries, and neurons that were in the outer approximate ten percent of the section planes, were avoided. Size of the neuron was not a factor in this selection. Using these criteria, 20 neurons per brain were analyzed. Branch point analyses of these dendrites entailed counting the total number of points of bifurcation per topological branch order using a centrifugal ordering system. This counting method designates the point at which each basilar dendrite of a given neuron arises from the cell body as a first order branch point. The point at which each of the first order dendrites bifurcates (or divides) was labeled a second order branch point. Each point of bifurcation of the second order processes was labeled a third order branch point. This was continued to the topologically distal-most dendritic branch point relative to the soma, labeling each point of bifurcation as the next-highest branch order. (Note: branch point analysis differs from branch segment analysis only in that points of bifurcation, rather than branch segments, are counted.) Branch points were totaled to determine the number of branch points per order. These data were then converted to dendritic segment numbers. The number of dendritic segments per each second and higher order is twice the number of branch points. The number of first order segments and first order branch points are the same. Data from individual neurons within brains were pooled to obtain mean branch segment number per order per subject. 2.4.4. Statistical analyses of structural data These data were analyzed using SPSS procedures for contrasts to perform planned comparisons. Compari-
sons between each lesion group and the sham group were used to test the hypothesis that each lesion type resulted in increases in dendritic arborization in the cortex opposite the lesion. Aspiration and electrolyticonly lesion groups were compared to test the hypothesis that these methods of lesion-induction produce different amounts of dendritic growth following damage. Comparison of the electroaspiration and electrolytic lesion groups were used to test the hypothesis that dendritic growth varies depending on the aspiration of the damaged tissue. Aspiration and electroaspiration lesions were compared to determine whether the passage of electric current through cortical tissue at the time of the lesion significantly contributes to lesion-induced dendritic growth. Finally, the day 18 aspiration lesion group was compared with the day 30 aspiration group to test the hypothesis that there are time-dependent dendritic arborization differences in the aspiration lesion animals. There were no significant differences in any of the structural analyses between the sham-operated animals perfused at 18 versus 30 days after surgery, and these subgroups were pooled for these variables.
3. Results 3.1. Structural results Quantification of cortical volume revealed that the lesion types did not result in a differential reduction in cortical volume in the damaged hemisphere. As shown in Fig. 3, all lesion types resulted in a significant reduction in cortical volume relative to shams, but were not significantly different from each other. As shown in Fig. 4, only electrolytic lesions resulted in significant increases in the arborization of layer V
Fig. 3. The volume of the remaining sensorimotor region of the damaged cortex in lesion groups and of one hemisphere of shams was measured using stereological methods. All lesion groups had significantly lower cortical volume relative to shams, but were not significantly different from each other. Data are mean9S.E.M. * P B 0.05 significantly different from shams.
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Fig. 4. Arborization of basilar dendrites of motor cortical layer V pyramidal neurons opposite the lesions. At day 18 after electrolytic lesions, there was significantly increased dendritic arborization relative to shams (A), and this was most evident at topologically higher branch orders (B). The number of dendritic segments in aspiration and electroaspiration lesions were not significantly increased relative to shams and were significantly reduced relative to electrolytic lesions, especially at higher order segments. The days 18 and 30 aspiration lesion groups were not significantly different (Note that previous studies of dendritic growth following electrolytic lesions have reported dendritic branch points, rather than segment number [18,19]. Branch point values are reported in the text.) * P B 0.05 significantly different from shams. Data are means9S.E.M. $ P B 0.05 significantly different from the aspiration lesion group. % P B 0.05 significantly different from the electroaspiration lesion group.
basilar dendrites in the cortex opposite the lesion in comparison to shams. The total number of dendritic segments per neuron was significantly elevated in comparison to the sham group (F1,31 /5.83, P B/0.05; Fig. 4A). There were also significantly greater numbers of dendritic segments in the electrolytic lesion group than in the aspiration lesion group examined at 18 days (F1,31 /12.62, P B/0.001), the aspiration lesion group examined at 30 days (F1,31 /4.98, P B/0.05), and the electroaspiration group (F1,31 /9.68, P B/0.01). There were no other significant differences between groups for the planned comparisons of total dendritic segment number. Animals with aspiration lesions examined at 18 days post-lesion had, on average, the smallest basilar dendritic arbors as measured by segments per neuron, but this was not significantly reduced in comparison to shams (F1,31 /1.21, P /0.279). The mean9/S.E.M. basilar dendritic branch point number was 27.919/3.42 in Shams, 34.729/5.72 in the electrolytic lesion group, 24.719/4.01 and 28.439/5.47 in the 18 and 30 day aspiration groups, respectively, and 25.959/5.16 in the electroaspiration lesion group. As shown in Fig. 4B, the increases in dendritic segments in the cortex opposite electrolytic lesions were primarily found at topologically distal (relative to the soma) branch orders. The number of dendritic segments at each third and higher branch order was significantly increased in comparison to shams and each of the other lesion groups. None of the other lesion groups were significantly different from shams at any branch order. At third and fourth order branches, the day 30 aspiration lesion group tended to have more dendritic branches than the day 18 aspiration lesion
group, but these effects were not significant (F s1,31 / 1.64 and 2.81, P /0.21 and 0.10, for third and fourth orders, respectively). At 18 days after the lesion, there was no evidence of lesion-induced striatal atrophy nor of significant group differences in striatal volume. The mean9/S.E.M. percentage of ipsilateral to contralateral striatal volume was 102.259/4.32 in shams, 96.759/4.42 in the electrolytic lesion group, 94.849/2.9 in the aspiration lesion group and 106.379/2.40 in the electroaspiration group. It is important to note that the 18 day time point is likely to be too early to detect lesion-induced striatal atrophy, as edema may still be present, and/or neuronal debris may not yet have been cleared away. 3.2. Behavioral results 3.2.1. Upright support behaviors As shown in Fig. 5, all lesions resulted in a severe initial asymmetry in forelimb use for upright postural support during exploratory movements. This included disuse of the forelimb contralateral to the lesion and proportionate increased reliance on the forelimb ipsilateral to the lesion (Fig. 5A and B). In addition, there was a moderate reduction in the simultaneous use of both forelimbs in all lesion groups relative to shams (Fig. 5C). However, over days after the lesion, the electrolytic lesion group showed a greater return to more symmetrical limb use than either of the aspiration lesion groups. Two-way ANOVA for asymmetries in ipsilateral support revealed significant effects for Groups (F3,32 /5.15, P B/0.01) and Days (F4,101 /12.96, P B/ 0.0001) and a significant Group by Day interaction
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(F10,101 /2.63, P B/0.01). Two-way ANOVAs for asymmetries in contralateral support also revealed a significant effect for Groups (F3,32 /4.01, P B/0.05), Days (F4,101 /7.67, P B/0.0001) and a Group by Day interaction (F10,101 /2.18, P B/0.05). At 16 days after the lesion, the preferential use of the ipsilateral forelimb in the electrolytic lesion group, although elevated, was no longer significantly different from Shams on post-hoc analyses and the disuse of the contralateral forelimb was moderately reduced. In contrast, on post-hoc analyses, the aspiration and electroaspiration groups showed significantly increased reliance on the ipsilateral fore-
limb (Fig. 5A) and disuse of the contralateral forelimb (Fig. 5B) on day 16 in comparison to Shams. In the aspiration lesion subgroup examined at 28 days, this significant asymmetry continued to be apparent. Although there was a tendency for all lesion groups to show reductions in simultaneous use of both forelimbs and this tendency was maintained in the aspiration lesion group (Fig. 5C), there was no significant Group by Day interaction effect (F10,101 /1.54, P /0.05). There was a significant effect for Days (F4,101 /8.58, P B/0.0001) and a Group effect that approached significance (F3,32 /2.40, P /0.08). 3.2.2. Rearing behaviors Observations of the forelimb used to push off of the floor during rearing also revealed a behavioral asymmetry in lesion groups relative to the sham group (Fig. 6). Animals in all lesion groups initially had a major preference for use of the ipsilateral forelimb for rearing, but by day 16, the electrolytic lesion group had the greatest reduction in this ipsilateral preference. Twoway ANOVAs for asymmetries in ipsilateral forelimb use for rearing revealed a significant effect for Groups (F3,32 /9.34, P B/0.0001), Days (F4,104 /15.10, P B/ 0.0001) and a Group by Day interaction (F10,104 / 2.63, P B/0.01). At days 2 and 7 after surgery, all lesion groups had a significantly increased reliance on their intact forelimb on post-hoc analyses. However, on day 16, the aspiration and electroaspiration lesion groups, but not the electrolytic lesion group, were significantly different from shams. 3.2.3. Forelimb adduction In contrast to the effects found in measures of exploratory movements, the measures of forelimb adduction revealed a much greater initial impairment of the contralateral forelimb following aspiration and electroaspiration lesions than following electrolytic lesions (Fig. 7). On the first post-surgical day of observation, these aspiration lesion groups spent ap-
Fig. 5
Fig. 5. Asymmetries in forelimb use for postural support during upright exploratory movements. The use of the forelimb ipsilateral to the lesion was significantly increased (A) and contralateral (impaired) forelimb use was significantly decreased (B) in the aspiration lesion group relative to shams on the first day of examination following surgery. At day 7 following surgery, all lesion groups were significantly different in both ipsilateral and contralateral forelimb use relative to the sham group. At day 16, the electrolytic lesion group had moderately reduced asymmetries and ipsilateral forelimb use was not significantly different from shams, whereas the aspiration and electroaspiration lesion groups continued to show significant differences in proportionate use of either forelimb in comparison to shams. In addition to the reduction in the use of the impaired forelimb, simultaneous use of both forelimbs (C) tended to be decreased after the lesions. Data are mean percentages of ipsilateral, contralateral and simultaneous bilateral limb use9S.E.M. * P B 0.05 significantly different from shams.
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Fig. 6. Ipsilateral forelimb use for rearing behaviors. Preferred use of the ipsilateral (intact) forelimb to push off the floor during rearing was significantly increased in all lesion groups relative to shams during the first week following surgery. At day 16, the electrolytic lesion group was no longer significantly different from the sham group, although both aspiration and electroaspiration groups continued to show significant levels of disuse of their impaired forelimb for rearing behaviors at this time point. At day 28, ipsilateral forelimb use for rearing was not significantly different in the aspiration group relative to the sham group. Data are mean percentages of ipsilateral limb use9S.E.M. * P B 0.05 significantly different from shams.
proximately 22% of the 2-min observation period with the impaired forelimb adducted (held in a retracted position) versus approximately 11% in the electrolyticonly lesion group and 4% in the sham group. Although the time spent with the impaired forelimb adducted was partially reduced on subsequent days in the aspiration groups, the duration of adduction was maintained at higher levels than shams or electrolytic lesion animals throughout the course of observation. A 2-way ANOVA resulted in a significant effect for Groups (F3,32 /6.98, P B/0.001) and Days (F3,72 /3.87, P B/0.05), but there
Fig. 7. Duration of contralateral forelimb adduction. Throughout the course of observation, the aspiration and electroaspiration lesion groups showed significantly more adduction of the contralateral (impaired) forelimb than the sham and the electrolytic lesion groups. Data are means9S.E.M. * P B 0.05 significantly different from shams. $ P B 0.05 significantly different from aspiration lesions. % P B 0.05 significantly different from electroaspiration lesions.
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was no significant Group by Day interaction effect (F7,72 /1.05, P /0.05). On post-hoc analyses of group effects, the aspiration and electroaspiration lesions resulted in a significantly increased duration of limb adduction in comparison to both electrolytic lesion (F1,32 /6.88, P B/0.05 and F1,32 /5.87, P B/0.05, respectively) and sham (F1,32 /18.20, P B/0.001 and F1,32 /14.37, P B/0.001, respectively) groups. Although rats with electrolytic lesions tended to show greater durations of contralateral forelimb adduction during the first week after the lesion than did sham-operated animals, limb adduction was not significantly increased in this group in comparison to shams (F1,32 /2.03, P / 0.05). There were no significant changes in adduction of the ipsilateral (non-impaired) forelimb following the lesions in comparison to shams.
4. Discussion In summary, despite similarly sized lesions and initial similarities in some types of behavioral impairments, animals with aspiration lesions, with or without preceding electrolytic damage, evidenced much more limited behavioral recovery and contracortical structural plasticity than animals with electrolytic lesions. The electrolytic lesions resulted in increased dendritic arborization of layer V pyramidal neurons in the motor cortex contralateral to the site of damage at 18 days after the lesions in comparison to shams. The magnitude and pattern of the electrolytic lesion-induced dendritic growth, with greatest increases at topologically higher orders, was similar to that reported in previous studies [18,20,22]. In contrast, both aspiration and electroaspiration lesions failed to result in increases in dendritic arborization at this time point. Aspiration lesions also failed to evidence dendritic growth at 30 days in comparison to shams. Behavioral results showed that, despite similar initial asymmetries in forelimb use for upright postural support and rearing behaviors, the aspiration and electroaspiration lesion groups failed to show evidence of functional recovery in these behavioral measures during the course of observation, whereas animals with electrolytic lesions showed a partial return to more symmetrical behavior. Furthermore, both types of aspiration lesions resulted in a more prevalent adduction of the impaired forelimb. Together, these behavioral results suggest that the aspiration lesions resulted in a more severe and chronic postural-motor impairment of the forelimb opposite the lesion. The dendritic arborization results are consistent with previous findings of robust dendritic growth in layer V opposite the site of electrolytic lesions [17,18,20,22,33], as well as the failure to find dendritic growth at comparable time points following aspiration lesions [13,28]. The induction of neural structural and physio-
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logical changes in the contralateral cortex is not specific to electrolytic lesions. Focal ischemic lesions of the SMC produced using the endothelin-1 (a vasoconstrictor) method [14] have recently been found to result in major increases in microtubule-associated protein-2 (MAP-2), a marker of dendritic growth and plasticity, as well as increased immunoreactivity for NMDA (NR1) receptor protein in layers II/III and V of the motor cortex opposite the lesion [40]. Unilateral neocortical infarctions also result in increased synaptophysin expression in the contralateral cortex between 14 and 60 days after the infarct [36,37]. Furthermore, ischemic lesions produced using either middle cerebral artery occlusion or photothrombosis result in a major enhancement of the excitability of the contralateral homotopic cortex [41]. This includes enduring (at least 28 days) elevations in neuronal calcium currents [4], enhanced paired-pulse excitation [31], reduced paired-pulse inhibition [5] increased NMDA receptor binding [29] and decreased GABAA receptor binding [30]. It is important to note that contracortical dendritic growth is not found when the electrolytic lesions are made to be much smaller than those used in the present study, either as a result of experimental error in the creation of the lesions [17] or as a result of using an unmoving platinum electrode to produce the lesions [13]. The prolonged adduction of the impaired forelimb found in the aspiration lesion groups is a type of forelimb impairment that has previously been associated with striatal damage [23,34]. It has long been hypothesized that the impairments following cortical lesions are due, in part, to disruption of the striatum and other subcortical structures [10 /12,19,35]. The failure to find lesion-induced differences in striatal volume at 18 days suggests that it is unlikely that the greater behavioral impairments following aspiration lesions are simply due to greater direct injury to the underlying striatum. However, this does not rule out the possibility of lesion-dependent differences in the severity of secondary degenerative effects in the striatum, a possibility that is unlikely to be detected using the volume measures of the present study. Atrophy resulting from lesion-induced degeneration is likely to take more than a few weeks to be sensitively detected using volume measurements. Thus, it remains possible that the more enduring severe impairments found following aspiration lesions are related to greater disruption of striatal and/or other subcortical structures. The failure to find differences in cortical volume loss between lesion groups also, of course, does not indicate equivalency in the extent of secondary injury within the peri-lesion cortex because diffuse degenerative effects of the lesions may not necessarily be detected with volume measures. It remains possible that examination of later time points would be revealing of dendritic growth effects in the cortex opposite aspiration lesions. Although not
significant, the number of dendritic branch segments in the aspiration lesion group that survived to 30 days after surgery tended to be elevated relative to the aspiration lesion group examined at 18 days. It has been previously found that dendritic and synaptic changes in layer V of the motor cortex are time-dependent following electrolytic damage. Maximal increases in dendritic arborization contralateral to the site of unilateral lesions are found 18 days following injury [18], whereas increases in the ratio of synapses to neurons are not found until day 30 [17], which coincides with a partial pruning of dendritic arbors [17,18,20,22]. Contracortical increases in the dendritic cytoskeletal protein, MAP-2, have also been found to be time-dependent after both electrolytic lesions [25] and endothelin-1 induced ischemic lesions [40] of the sensorimotor cortex. Thus, it is conceivable that examination at an even later time point would reveal greater dendritic arborization changes in the cortex opposite aspiration lesions. Although electrolytic lesions have previously been found to result in enduring asymmetries in some types of limb use for upright postural support behaviors [33], aspiration lesions clearly resulted in more prolonged time course of severe behavioral impairments in the present study. It is also possible that the aspiration lesions result in structural changes that are not revealed in measures of dendritic branch number, such as changes in spine density. Forelimb behavioral manipulations and transcallosal denervation have each been found to result in localized spine addition in the motor cortex which is not coupled to increases in dendritic arborization [2]. The similarity in the behavioral and structural effects of aspiration and electroaspiration lesions indicates that the passage of electric current is unlikely to be a key contributor to these lesion-dependent effects. One alternative possibility is that the presence of damaged tissue at the site of cortical injury plays an important role in behavioral and structural outcomes following cortical lesions. That is, it is possible that the slower loss of damaged cells following electrolytic lesions, and the glial response to this loss, contributes to compensatory plasticity in regions of both the damaged and intact hemispheres. Boyeson et al. [3] have found that SMC lesion procedures that do not remove tissue (percussive injury and cortical undercuts) result in transient increases in the thresholds of microstimulation-induced movements in adjacent SMC regions. This effect was not found after aspiration lesions, consistent with the possibility that removal of the damage tissue alters the nature of the ipsilateral cortical response to the lesion. However, there are two lines of research that fail to support the hypothesis that the contralateral cortical effects are directly attributable to the presence of the damaged tissue. The first is that it is possible to reproduce growth of motor cortical layer V dendritic arbors with the combination of transections of the
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corpus callosum and forced use of the opposite forelimb [6]. This indicates the necessity of callosal denervation and behavioral demand, but not of slow-growing cortical damage, to produce neuronal growth. Obviously, all lesions in the present study produced these two necessary conditions. Although corpus callosum transections result in a variable amount of electrodetract damage to the medial agranular and/or cingulate cortex, there were no differences in the magnitude of dendritic growth that could be linked to the extent of cortical damage [6]. Secondly, although other researchers have failed to find crossed-corticostriatal sprouting following aspiration lesions [27,39]. McNeill et al. have found both significant crossed-corticostriatal growth [8] as well as increases in several plasticity related proteins [7,24] in the motor cortex opposite unilateral aspiration lesions in young adult (250 /300 g) animals. In addition to pointing to potential methodological differences in aspiration lesion procedures, this work suggests that factors other than the removal of tissue at the lesion site are likely to be involved in the differences in contracortical effects. Another possible explanation for these lesion-dependent differences is that the aspiration procedure, at least as it is performed in many laboratories, results in greater disruption of underlying brain structures, including the underlying white matter and the striatum. If so, it is possible that this could result in the more severe behavioral asymmetries observed, and possibly in alterations of mechanisms that either promote or inhibit neuronal adaptation to the damage. It has been found, for example, that lack of neural sprouting in adult rats is linked to the presence of the inhibitory factor, Nogo-A, a myelin-associated inhibitor of neurite growth [15]. Kartje et al. [21] have found that, whereas aspiration lesions in adult rats failed to result in crossed corticostriatal sprouting originating from the cortex opposite the lesion, blocking Nogo-A following the lesions did result in an increase in crossed corticostriatal fibers and other crossed cortical projections. Consistent with the possibility that aspiration lesions result in a greater inhibition of structural plasticity, growth associated protein-43 (GAP-43), a marker of axonal sprouting, has been found to be decreased in the underlying striatum after large unilateral aspiration, but not after ischemic cortical lesions [38,39]. Also potentially related is the finding that fetal cortical grafts placed within the territory of ischemic cortical infarcts of adult rats developed zinc-positive (likely glutamatergic) terminals within the transplanted tissue. This was not found when the grafts were placed in the territory of aspiration lesions [42]. In addition to providing evidence that electrolytic lesions and aspiration lesions produce different structural and behavioral outcomes, and are therefore not interchangeable models of cortical injury, the findings of
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this study further support the idea that lesion-specific factors may contribute to the outcomes of cortical damage. Chesselet et al. have found marked differences in the effects of ischemic versus aspiration lesions when pre-, peri-, and post-operative conditions were identical between lesion groups [26,27,38,39]. Together with the present results, these findings indicate that some of the ‘‘normal’’ sequelae of compensatory neuroplasticity seen after electrolytic and ischemic injury are either absent or considerably less prevalent after aspiration lesions. At this point, it is necessary to continue this comparison of lesion models in order to examine structural and behavioral outcomes at later time points, as well as to address the possibility of differences in the cellular responses to electrolytic and ischemic versus aspiration lesions in both peri-lesion and remote regions. The presence of different growth-promoting and inhibitory substances and substrates may be revealing, not only of the differences in these lesion types, but also of the conditions that promote adaptive plasticity after ischemic injury. The present finding that aspiration and electrolytic lesions are not interchangeable models of cortical injury is also illustrative of the limits in generalizing between the commonly used models of neural and behavioral adaptation to brain damage.
Acknowledgements We are very grateful to Drs Timothy Schallert and Otto Witte for comments on this study, to DeAnna Adkins and Scott Bury for technical assistance, to Samuel Pedigo and Shannon Carr for assistance in preliminary data collection and analysis, to Kevin McCullough for taming and caring for the animals and to Todd Martin for assistance in assembling the manuscript. Supported by NIH Grant MH56361.
References [1] Adams FS, Schwarting RK, Huston JP. Behavioral and neurochemical asymmetries following unilateral trephination of the rat skull: is this control operation always appropriate? Physiol Behav 1994;55:947 /52. [2] Adkins DL, Bury SD, Jones TA. Laminar-dependent dendritic spine alterations in the motor cortex of adult rats following callosal denervation and forelimb behavioral change. Neurobiol Learn Mem, 2002 (in press). [3] Boyeson MG, Feeney DM, Dail WG. Cortical microstimulation thresholds adjacent to sensorimotor cortex injury. J Neurotrauma 1991;8:205 /17. [4] Bruel C, Neumann-Haefelin T, Witte OW. Enhancement of whole cell calcium currents following transient MCAO. Brain Res 2000;884:129 /38. [5] Buchkremmer-Ratzmann I, Witte WO. Extended brain disinhibition following small photothrombotic lesions in rat frontal cortex. Neuroreport 1997;8:519 /22.
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[6] Bury SD, Adkins DL, Ishida JT, Kotzer CM, Eichhorn AC, Jones TA. Denervation facilitates neuronal growth in the motor cortex of rats in the presence of behavioral demand. Neurosci Lett 2000;287:85 /8. [7] Cheng H-W, Jiang T, Mori N, McNeill TH. Upregulation of stathmin (p19) gene expression in adult rat brain during injuryinduced synapse formation. Neuroreport 1997;8:3691 /5. [8] Cheng HW, Tong J, McNeill TH. Lesion-induced axon sprouting in the deafferented striatum of adult rat. Neurosci Lett 1998;242:69 /72. [9] Chu CJ, Jones TA. Experience-dependent structural plasticity in cortex heterotopic to focal sensorimotor cortical damage. Exp Neurol 2000;165:1 /12. [10] Deuel RK, Collins RC. The functional anatomy of frontal lobe neglect in the monkey: behavioral and quantitative 2-deoxyglucose studies. Ann Neurol 1984;15:521 /9. [11] Deuel RK, Collins RC. Recovery from unilateral neglect. Exp Neurol 1983;81:733 /48. [12] Feeney DM. Diaschisis: new data rekindle interest in an old theory. Neurol Neurosurg 1988;32:2 /7. [13] Forgie ML, Gibb R, Kolb B. Unilateral lesions of the forelimb area of rat motor cortex: lack of evidence for use-dependent neural growth in the undamaged hemisphere. Brain Res 1996;710:249 /59. [14] Fuxe K, Bjelke B, Andbjer B, Grahn H, Rimondini R, Agnati LF. Endothelin-1 induced lesions of the fronto-parietal cortex of the rat. A possible model of focal cortical ischemia. Neuroreport 1997;8:2623 /9. [15] Huber AB, Schwab ME. Nogo-A, a potent inhibitor of neurite outgrowth and regeneration. Biol Chem 2000;381:407 /19. [16] Jones TA, Chu CJ, Grande LA, Gregory AD. Motor skills training enhances lesion-induced structural plasticity in the motor cortex of adult rats. J Neurosci 1999;19:10153 /63. [17] Jones TA, Kleim JA, Greenough WT. Synaptogenesis and dendritic growth in the cortex opposite unilateral sensorimotor cortex damage in adult rats: a quantitative electron microscopic examination. Brain Res 1996;733:142 /8. [18] Jones TA, Schallert T. Overgrowth and pruning of dendrites in adult rats recovering from neocortical damage. Brain Res 1992;581:156 /60. [19] Jones TA, Schallert T. Subcortical deterioration after cortical damage: effects of diazepam and relation to recovery of function. Behav Brain Res 1992;51:1 /13. [20] Jones TA, Schallert T. Use-dependent growth of pyramidal neurons after neocortical damage. J Neurosci 1994;14:2140 /52. [21] Kartje GL, Schulz MK, Lopez-Yunez A, Schnell L, Schwab ME. Corticostriatal plasticity is restricted by myelin-associated neurite growth inhibitors in the adult rat. Ann Neurol 1999;45:778 /86. [22] Kozlowski DA, Schallert T. Relationship between dendritic pruning and behavioral recovery following sensorimotor cortex lesions. Behav Brain Res 1998;97:89 /98. [23] Lindner MD. Development of behavioral outcome measures for preclinical Parkinson’s research. In: Emerich DF, Dean RL III, Sanberg PR, editors. Central Nervous System Diseases: Innovative Models of CNS Diseases from Molecule to Therapy. Totowa, NJ: Humana Press, 2000:153 /69. [24] McNeill TH, Mori N, Cheng H-W. Differential regulation of the growth-associated proteins, GAP-43 and SCG-10, in response to unilateral cortical ablation in adult rats. Neuroscience 1999;90:1349 /60. [25] Monahan TM, Adkins DL, Jones TA. Alterations in microtubuleassociated protein 2 in the cortex contralateral and homotopic to focal neocortical damage. Soc Neurosci Abstr 2000;26:73.20.
[26] Napieralski JA, Banks RJA, Chesselet M-F. Motor and somatosensory deficits following uni- and bilateral lesions of the cortex induced by aspiration or thermocoagulation in the adult rat. Exp Neurol 1998;154:80 /8. [27] Napieralski JA, Butler AK, Chesselet M-F. Anatomical and functional evidence for lesion-specific sprouting of corticostriatal input in the adult rat. J Comp Neurol 1996;373:484 /97. [28] Prusky G, Whishaw IQ. Morphology of identified corticospinal cells in the rat following motor cortex injury: absence of usedependent change. Brain Res 1996;714:1 /8. [29] Que M, Schiene K, Witte OW, Zilles K. Widespread upregulation of N-methyl-D-aspartate receptors after focal photothrombotic lesion in rat brain. Neurosci Lett 1999;273:77 /80. [30] Que M, Witte OW, Neumann-Haefelin T, Schiene K, Schroeter M, Zilles K. Changes in GABA(A) and GABA(B) receptor binding following cortical photothrombosis: a quantitative receptor autoradiographic study. Neuroscience 1999;93:1233 /40. [31] Reinecke S, Lutzenburg M, Hagemann G, Bruehl C, NeumannHaefelin T, Witte OW. Electrophysiological transcortical diaschisis after middle cerebral artery occlusion (MCAO) in rats. Neurosci Lett 1999;261:85 /8. [32] Salin P, Chesselet M-F. Paradoxical increases in striatal neuropeptide gene expression following ischemic lesions of the cerebral cortex. Proc Natl Acad Sci USA 1992;89:9954 /8. [33] Schallert T, Kozlowski DA, Humm JL, Cocke RR. Use-dependent structural events in recovery of function. Adv Neurol 1997;73:229 /38. [34] Shallert T, Lindner MD. Rescuing neurons from trans-synaptic degeneration after brain damage: helpful, harmful, or neutral in recovery of function? Can J Psychol 1990;44:276 /92. [35] Schallert T, Whishaw IQ. Bilateral cutaneous stimulation of the somatosensory system in hemi-decorticate rats. Behav Neurosci 1984;98:518 /40. [36] Stroemer RP, Kent TA, Hulsebosch CE. Enhanced neocortical neural sprouting, synaptogenesis, and behavioral recovery with D-amphetamine therapy after neocortical infarction in rats. Stroke 1998;11:2381 /93. [37] Stroemer RP, Kent TA, Hulsebosch CE. Neocortical neural sprouting, synaptogenesis, and behavioral recovery after neocortical infarction in rats. Stroke 1995;26:2135 /44. [38] Szele FG, Alexander C, Chesselet M-F. Expression of molecules associated with neuronal plasticity in the striatum after aspiration and thermocoagulatory lesions of the cerebral cortex in adult rats. J Neurosci 1995;15:4429 /48. [39] Uryu K, MacKenzie L, Chesselet M. Ultrastructural evidence for differential axonal sprouting in the striatum after thermocoagulatory and aspiration lesions of the cerebral cortex in adult rats. Neuroscience 2001;105:307 /16. [40] Voorhies AC, Adkins DL, Jones TA. Cortical structural plasticity and behavioral deficits following endothelin-1 induced ischemic lesions of the sensorimotor cortex. Soc Neurosci Abstr 2001;27:190.7. [41] Witte OW, Bidmon HJ, Schiene K, Redecker C, Hagemann G. Functional differentiation of multiple perilesional zones after focal cerebral ischemia. J Cereb Blood Flow Metab 2000;20:1149 /65. [42] Zeng J, Mattsson B, Schulz MK, Johansson BB, Sorensen JC. Expression of zinc-positive cells and terminals in fetal neocortical homografts to adult rat depends on lesion type and rearing conditions. Exp Neurol 2000;164:176 /83.
Research report
The behavioral and dendritic growth effects of focal sensorimotor cortical damage depend on the method of lesion induction Ann C. Voorhies a, Theresa A. Jones a,b,c,* a
b
Psychology Department, University of Washington, Seattle, WA 98195, USA Psychology Department, Mezes Hall, Campus Box B3800, The University of Texas, Austin, TX 78712, USA c Institute for Neuroscience Research, The University of Texas, Austin, TX 78712, USA Received 26 June 2001; received in revised form 18 December 2001; accepted 18 December 2001
Abstract Using different models of focal cortical injury in adult rats, the neural structural and behavioral outcomes of unilateral lesions of the forelimb representation of the sensorimotor cortex (SMC) were assessed. Lesions were produced using either electrolytic, aspiration, or combined (‘electroaspiration’) techniques. Measurements of dendritic arborization in layer V of the motor cortex opposite the lesion revealed a growth of pyramidal neuron dendritic processes following electrolytic lesions in comparison to shams. This effect was not found in either the aspiration or electroaspiration lesion groups. Behaviorally, animals in all lesion groups developed a hyper-reliance on the forelimb ipsilateral to the lesion and proportionate disuse of the contralateral (impaired) forelimb for postural support behaviors. In comparison to sham-operated animals, the initial asymmetries in behaviors expressed during movement were similar between lesion groups, but were less enduring following electrolytic lesions than following aspiration and electroaspiration lesions. Furthermore, both aspiration lesion groups had more prevalent adduction of the impaired forelimb than the electrolytic-only lesion rats. Thus, cortical aspiration resulted in more severe and enduring forelimb impairments than the electrolytic lesions, despite similar lesion sizes, as assessed using cortical volume measures. These findings suggest that the aspiration lesion procedures, at least as performed in the present study, exacerbate the behavioral effects of focal cortical injury and limit compensatory plasticity in the contralateral cortex. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cortical injury; Electrolytic lesions; Aspiration lesions; Suction-ablation; Ischemia; Dendritic arborization; Forelimb use; Sensorimotor cortex; Recovery of function
1. Introduction Several previous studies suggest that the neural and behavioral outcomes of neocortical damage depend upon the technique used to induce the lesion. Using adult rats, Napieralski et al. [26] found that large unilateral ischemic lesions produced using thermocoagulation of pial blood vessels overlying the sensorimotor cortex (SMC) versus similarly sized aspiration (suctionremoval) lesions of the SMC resulted in different patterns of impairment and different recovery rates on tests of sensorimotor function. Furthermore, large
* Corresponding author. Tel.: 1-512-475-7763; fax: 1-512-4757765 E-mail address: [email protected] (T.A. Jones).
unilateral thermocoagulatory lesions, but not aspiration lesions, of the SMC in adult rats resulted in crossed corticostriatal axonal sprouting from the motor cortex of the intact hemisphere [27,39]. Additional research has shown that thermocoagulatory and aspiration lesions of the SMC result in different lesion-induced cellular responses in the underlying striatum. Whereas GAP-43 expression in the dorsolateral striatum was not significantly changed following cortical thermocoagulatory lesions, it was significantly decreased after aspiration lesions [38] (see also [32,39]). These lesion-dependent differences were found even when the lesions were of comparable size and were performed in the same laboratory [26,27,38,39]. In more focal lesion models, electrolytic SMC lesions (produced by passing electric current through a platinum electrode as it is moved through the tissue), have been found to result in robust
0166-4328/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 2 ) 0 0 0 2 9 - 3
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dendritic growth and synaptogenesis in the motor cortex opposite the site of cortical damage [16 /18]. However, somewhat larger aspiration lesions have been found not to result in dendritic growth, at least not at comparable time points after the lesions [13,28]. Based on these previous studies, it seemed possible that aspiration lesions either inhibit or fail to promote the compensatory neural plasticity and behavioral recovery that is found after other types of cortical injury. The purpose of the present study was to test the hypothesis that neural growth contralateral to the site of the lesion as well as forelimb behavioral outcomes vary following similarly sized focal unilateral electrolytic versus aspiration lesions of the SMC. Adult male rats were assigned to one of the following groups: (1) electrolytic lesions; (2) aspiration lesions; (3) electroaspiration lesions, used to assess whether differences in electrolytic versus aspiration groups were dependent upon passage of electric current in the former group; and (4) sham controls. These groups were compared for their effects on asymmetrical use of the forelimbs and on the arborization of layer V pyramidal neurons in the cortex opposite the lesion. Day 18 following surgery was chosen as the time point for examination of dendritic arborization based on previous findings indicating that this is a time point of contracortical dendritic growth after unilateral electrolytic SMC lesions [17,18]. To assess the possibility that aspiration lesions may simply result in a slower course of dendritic growth, an additional group of animals were assessed at 30 days after aspiration lesions.
2. Methods 2.1. Subjects Subjects were 36 adult (3 /4 months old) male Long / Evans hooded rats maintained in a vivarium at the University of Washington. Animals were housed in pairs in standard tub-style laboratory cages with wood chip bedding. Rats were kept on 12:12 h light:dark cycle and had food and water ad libitum. Rats were made tame prior to the onset of the experimental procedures by frequent gentle handling. Subjects were randomly assigned to one of the following four groups: electrolytic lesions (n/8), aspiration lesions (n/14), electroaspiration lesions (n/7), and sham surgeries (n/7). All animals were sacrificed at 18 days after surgery with the exception of a subset of animals in the aspiration lesion condition (n/7) and the sham group (n/3), which were used to assess structural changes at 30 days after surgery. Animal use was in accordance with a protocol approved by the Animal Care and Use Committee of the University of Washington.
2.2. Sensorimotor cortex lesions Prior to surgery, all animals were anesthetized with Equithesin (150 mg/kg chloral hydrate and 34 mg/kg pentobarbital). For SMC lesions, following a surgical incision to the scalp, the skull overlying the forelimb representation region of the SMC was removed. Coordinates for skull removal were between 0.5 mm posterior and 1.5 mm anterior to bregma and 3.0 /4.5 mm lateral to midline. The depth of dura was then measured and dura was removed. For electrolytic lesions, an uninsulated 30 gauge platinum wire electrode was lowered to a depth of 1.7 mm below dura. Electric current (1.0 mA) was delivered as the electrode was continuously moved in eight equally spaced transverses through the depth of the exposed SMC for 120 s. Aspiration lesions were created by suction-removal of the exposed gray matter of the SMC, with the aid of a dissecting microscope. All exposed tissue was removed, down to the appearance of white matter. Electroaspiration lesions were created using the same procedure used to make the electrolytic lesions followed immediately by suction-removal of the damaged tissue. As shown in Fig. 1, aspiration lesions and electroaspiration lesions were similar to electrolytic lesions in size and placement. Sham animals received anesthesia and scalp incisions, but the skull was not disturbed, as previous research has indicated that trephination alone may cause behavioral asymmetries [1]. Following surgery, all animals were sutured and given atropine sulfate (0.1 mg/kg) to counteract the respiratory depressive effects of the Equithesin. 2.3. Behavioral analyses Animals were filmed for 2 min durations in an upright, transparent cylinder on the day before surgery and on post-surgical days 2, 7, 16 and 28 (see Fig. 2). This procedure, known as the ‘Cylinder Test’, is used to detect unilateral forelimb impairments and compensatory behaviors, as it encourages vertical-lateral exploratory movements that expose asymmetries in forelimb use [33]. Forelimb use for upright postural support against cylinder walls was observed for all 36 subjects by a rather naive to the experimental conditions, who recorded each instance of left, right and simultaneous bilateral forelimb use for postural support. In the same manner, for each instance of rearing behavior, the forelimb used to initiate upright posture from a quadrupedal position was also recorded. Additionally, the duration of each adduction of the left and right forelimb while the animals were in an otherwise quadrupedal and stationary position in the cylinder was recorded. Limb adduction was defined as retraction of a forelimb for at least 2 s and was observed as a possible indicator of striatal dysfunction [23,34]. These data were later
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Fig. 1. Representative examples of electrolytic, aspiration and electroaspiration lesions. Electrolytic lesions were created via passage of electric current through layers I /V of the forelimb representation region of the sensorimotor cortex (SMC). Aspiration lesions were made by suctionremoval of the cortical matter of the forelimb representation of the SMC, down to the appearance of white matter. Electroaspiration lesions were created using the same technique used to make the electrolytic lesions, followed immediately by suction-removal of the damaged tissue.
by Day interactions. Post-hoc analyses were performed when appropriate using SAS GLM procedure for contrasts. 2.4. Histology and structural analyses
Fig. 2. Cylinder test. Rats were filmed in a transparent cylinder to measure asymmetries in forelimb use for postural support behaviors. Following unilateral SMC lesions, rats have impairments in the forelimb contralateral (arrow) to the lesion and develop a reliance on the ipsilateral forelimb for postural support.
translated into ipsilateral, contralateral, and bilateral limb use, relative to the side of the lesion. For shamoperated animals, left and right forelimb use were designated ‘ipsilateral’ and ‘contralateral’, respectively. There were no significant differences in any behavioral measures between the aspiration lesion subgroups or between the sham subgroups sacrificed at 18 versus 30 days and these were combined for the behavioral analyses. Data were analyzed using SAS general linear models (GLM) system for 2-way analysis of variance (ANOVA) for the effects of Groups, Days and Group
2.4.1. Golgi /Cox impregnation Animals were perfused intracardially using approximately 300 ml of 0.1 M phosphate buffer followed by light perfusion with approximately 100 ml of 4% paraformaldehyde in the same buffer. The brains were removed, coronally transected at the level of the midbrain, and the cerebrum was bisected at midline. They were then placed in Golgi /Cox solution for 35/60 days of impregnation. The brains were reacted en bloc, resin-embedded and coronally sectioned on a microtome at a section thickness of 200 mm. All slides were coded, to avoid experimenter bias effects. Sections were then assessed for quality of Golgi impregnation, and six brains from the 35 days impregnation duration were removed from the study due to poor impregnation. This included two animals from the electrolytic group, two animals from the aspiration group, and two animals from the electroaspiration group. (These are not reported in the n s above.) 2.4.2. Volume measures Cortical volume of the lesion hemisphere was measured in order to ascertain whether the different lesion types produced similar loss of cortical tissue. The volume of non-necrotic cortical tissue remaining in the SMC region was measured using macrostructural landmarks (rostrally, the midline joining of the corpus callosum and, caudally, the midline joining of the anterior commisure) to delimit the SMC region measured. In brains from the 18 days time point, striatal volume was also measured in the hemispheres ipsilateral and contralateral to the lesion. This measure was expected to be sensitive to direct lesion-induced damage to the striatum but not to striatal atrophy secondary to
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the denervation of corticostriatal projections (which would likely be more sensitively detected at later time points). The ratio of ipsilateral to contralateral striatal volume was calculated to determine if striatum was differentially affected by the different lesion methods. Volume measures were obtained using systematic point counting and the Cavalieri method, as described in detail in Chu and Jones [9]. 2.4.3. Dendritic measures The arborization of basilar dendrites of layer V pyramidal neurons was measured in the forelimb region of the motor cortex contralateral to the damaged hemisphere. This population of dendrites was analyzed because earlier studies have shown that it undergoes robust dendritic growth following electrolytic lesions [18,20]. Individual pyramidal neurons in layer V of the forelimb representation in the motor cortex were randomly chosen for analysis; however neurons were only used if they displayed excellent impregnation of the cell body and dendrites, with no beading of the stain or gaps in the processes of the dendrites. Furthermore, neurons that were overly obscured by astrocytes or capillaries, and neurons that were in the outer approximate ten percent of the section planes, were avoided. Size of the neuron was not a factor in this selection. Using these criteria, 20 neurons per brain were analyzed. Branch point analyses of these dendrites entailed counting the total number of points of bifurcation per topological branch order using a centrifugal ordering system. This counting method designates the point at which each basilar dendrite of a given neuron arises from the cell body as a first order branch point. The point at which each of the first order dendrites bifurcates (or divides) was labeled a second order branch point. Each point of bifurcation of the second order processes was labeled a third order branch point. This was continued to the topologically distal-most dendritic branch point relative to the soma, labeling each point of bifurcation as the next-highest branch order. (Note: branch point analysis differs from branch segment analysis only in that points of bifurcation, rather than branch segments, are counted.) Branch points were totaled to determine the number of branch points per order. These data were then converted to dendritic segment numbers. The number of dendritic segments per each second and higher order is twice the number of branch points. The number of first order segments and first order branch points are the same. Data from individual neurons within brains were pooled to obtain mean branch segment number per order per subject. 2.4.4. Statistical analyses of structural data These data were analyzed using SPSS procedures for contrasts to perform planned comparisons. Compari-
sons between each lesion group and the sham group were used to test the hypothesis that each lesion type resulted in increases in dendritic arborization in the cortex opposite the lesion. Aspiration and electrolyticonly lesion groups were compared to test the hypothesis that these methods of lesion-induction produce different amounts of dendritic growth following damage. Comparison of the electroaspiration and electrolytic lesion groups were used to test the hypothesis that dendritic growth varies depending on the aspiration of the damaged tissue. Aspiration and electroaspiration lesions were compared to determine whether the passage of electric current through cortical tissue at the time of the lesion significantly contributes to lesion-induced dendritic growth. Finally, the day 18 aspiration lesion group was compared with the day 30 aspiration group to test the hypothesis that there are time-dependent dendritic arborization differences in the aspiration lesion animals. There were no significant differences in any of the structural analyses between the sham-operated animals perfused at 18 versus 30 days after surgery, and these subgroups were pooled for these variables.
3. Results 3.1. Structural results Quantification of cortical volume revealed that the lesion types did not result in a differential reduction in cortical volume in the damaged hemisphere. As shown in Fig. 3, all lesion types resulted in a significant reduction in cortical volume relative to shams, but were not significantly different from each other. As shown in Fig. 4, only electrolytic lesions resulted in significant increases in the arborization of layer V
Fig. 3. The volume of the remaining sensorimotor region of the damaged cortex in lesion groups and of one hemisphere of shams was measured using stereological methods. All lesion groups had significantly lower cortical volume relative to shams, but were not significantly different from each other. Data are mean9S.E.M. * P B 0.05 significantly different from shams.
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Fig. 4. Arborization of basilar dendrites of motor cortical layer V pyramidal neurons opposite the lesions. At day 18 after electrolytic lesions, there was significantly increased dendritic arborization relative to shams (A), and this was most evident at topologically higher branch orders (B). The number of dendritic segments in aspiration and electroaspiration lesions were not significantly increased relative to shams and were significantly reduced relative to electrolytic lesions, especially at higher order segments. The days 18 and 30 aspiration lesion groups were not significantly different (Note that previous studies of dendritic growth following electrolytic lesions have reported dendritic branch points, rather than segment number [18,19]. Branch point values are reported in the text.) * P B 0.05 significantly different from shams. Data are means9S.E.M. $ P B 0.05 significantly different from the aspiration lesion group. % P B 0.05 significantly different from the electroaspiration lesion group.
basilar dendrites in the cortex opposite the lesion in comparison to shams. The total number of dendritic segments per neuron was significantly elevated in comparison to the sham group (F1,31 /5.83, P B/0.05; Fig. 4A). There were also significantly greater numbers of dendritic segments in the electrolytic lesion group than in the aspiration lesion group examined at 18 days (F1,31 /12.62, P B/0.001), the aspiration lesion group examined at 30 days (F1,31 /4.98, P B/0.05), and the electroaspiration group (F1,31 /9.68, P B/0.01). There were no other significant differences between groups for the planned comparisons of total dendritic segment number. Animals with aspiration lesions examined at 18 days post-lesion had, on average, the smallest basilar dendritic arbors as measured by segments per neuron, but this was not significantly reduced in comparison to shams (F1,31 /1.21, P /0.279). The mean9/S.E.M. basilar dendritic branch point number was 27.919/3.42 in Shams, 34.729/5.72 in the electrolytic lesion group, 24.719/4.01 and 28.439/5.47 in the 18 and 30 day aspiration groups, respectively, and 25.959/5.16 in the electroaspiration lesion group. As shown in Fig. 4B, the increases in dendritic segments in the cortex opposite electrolytic lesions were primarily found at topologically distal (relative to the soma) branch orders. The number of dendritic segments at each third and higher branch order was significantly increased in comparison to shams and each of the other lesion groups. None of the other lesion groups were significantly different from shams at any branch order. At third and fourth order branches, the day 30 aspiration lesion group tended to have more dendritic branches than the day 18 aspiration lesion
group, but these effects were not significant (F s1,31 / 1.64 and 2.81, P /0.21 and 0.10, for third and fourth orders, respectively). At 18 days after the lesion, there was no evidence of lesion-induced striatal atrophy nor of significant group differences in striatal volume. The mean9/S.E.M. percentage of ipsilateral to contralateral striatal volume was 102.259/4.32 in shams, 96.759/4.42 in the electrolytic lesion group, 94.849/2.9 in the aspiration lesion group and 106.379/2.40 in the electroaspiration group. It is important to note that the 18 day time point is likely to be too early to detect lesion-induced striatal atrophy, as edema may still be present, and/or neuronal debris may not yet have been cleared away. 3.2. Behavioral results 3.2.1. Upright support behaviors As shown in Fig. 5, all lesions resulted in a severe initial asymmetry in forelimb use for upright postural support during exploratory movements. This included disuse of the forelimb contralateral to the lesion and proportionate increased reliance on the forelimb ipsilateral to the lesion (Fig. 5A and B). In addition, there was a moderate reduction in the simultaneous use of both forelimbs in all lesion groups relative to shams (Fig. 5C). However, over days after the lesion, the electrolytic lesion group showed a greater return to more symmetrical limb use than either of the aspiration lesion groups. Two-way ANOVA for asymmetries in ipsilateral support revealed significant effects for Groups (F3,32 /5.15, P B/0.01) and Days (F4,101 /12.96, P B/ 0.0001) and a significant Group by Day interaction
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(F10,101 /2.63, P B/0.01). Two-way ANOVAs for asymmetries in contralateral support also revealed a significant effect for Groups (F3,32 /4.01, P B/0.05), Days (F4,101 /7.67, P B/0.0001) and a Group by Day interaction (F10,101 /2.18, P B/0.05). At 16 days after the lesion, the preferential use of the ipsilateral forelimb in the electrolytic lesion group, although elevated, was no longer significantly different from Shams on post-hoc analyses and the disuse of the contralateral forelimb was moderately reduced. In contrast, on post-hoc analyses, the aspiration and electroaspiration groups showed significantly increased reliance on the ipsilateral fore-
limb (Fig. 5A) and disuse of the contralateral forelimb (Fig. 5B) on day 16 in comparison to Shams. In the aspiration lesion subgroup examined at 28 days, this significant asymmetry continued to be apparent. Although there was a tendency for all lesion groups to show reductions in simultaneous use of both forelimbs and this tendency was maintained in the aspiration lesion group (Fig. 5C), there was no significant Group by Day interaction effect (F10,101 /1.54, P /0.05). There was a significant effect for Days (F4,101 /8.58, P B/0.0001) and a Group effect that approached significance (F3,32 /2.40, P /0.08). 3.2.2. Rearing behaviors Observations of the forelimb used to push off of the floor during rearing also revealed a behavioral asymmetry in lesion groups relative to the sham group (Fig. 6). Animals in all lesion groups initially had a major preference for use of the ipsilateral forelimb for rearing, but by day 16, the electrolytic lesion group had the greatest reduction in this ipsilateral preference. Twoway ANOVAs for asymmetries in ipsilateral forelimb use for rearing revealed a significant effect for Groups (F3,32 /9.34, P B/0.0001), Days (F4,104 /15.10, P B/ 0.0001) and a Group by Day interaction (F10,104 / 2.63, P B/0.01). At days 2 and 7 after surgery, all lesion groups had a significantly increased reliance on their intact forelimb on post-hoc analyses. However, on day 16, the aspiration and electroaspiration lesion groups, but not the electrolytic lesion group, were significantly different from shams. 3.2.3. Forelimb adduction In contrast to the effects found in measures of exploratory movements, the measures of forelimb adduction revealed a much greater initial impairment of the contralateral forelimb following aspiration and electroaspiration lesions than following electrolytic lesions (Fig. 7). On the first post-surgical day of observation, these aspiration lesion groups spent ap-
Fig. 5
Fig. 5. Asymmetries in forelimb use for postural support during upright exploratory movements. The use of the forelimb ipsilateral to the lesion was significantly increased (A) and contralateral (impaired) forelimb use was significantly decreased (B) in the aspiration lesion group relative to shams on the first day of examination following surgery. At day 7 following surgery, all lesion groups were significantly different in both ipsilateral and contralateral forelimb use relative to the sham group. At day 16, the electrolytic lesion group had moderately reduced asymmetries and ipsilateral forelimb use was not significantly different from shams, whereas the aspiration and electroaspiration lesion groups continued to show significant differences in proportionate use of either forelimb in comparison to shams. In addition to the reduction in the use of the impaired forelimb, simultaneous use of both forelimbs (C) tended to be decreased after the lesions. Data are mean percentages of ipsilateral, contralateral and simultaneous bilateral limb use9S.E.M. * P B 0.05 significantly different from shams.
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Fig. 6. Ipsilateral forelimb use for rearing behaviors. Preferred use of the ipsilateral (intact) forelimb to push off the floor during rearing was significantly increased in all lesion groups relative to shams during the first week following surgery. At day 16, the electrolytic lesion group was no longer significantly different from the sham group, although both aspiration and electroaspiration groups continued to show significant levels of disuse of their impaired forelimb for rearing behaviors at this time point. At day 28, ipsilateral forelimb use for rearing was not significantly different in the aspiration group relative to the sham group. Data are mean percentages of ipsilateral limb use9S.E.M. * P B 0.05 significantly different from shams.
proximately 22% of the 2-min observation period with the impaired forelimb adducted (held in a retracted position) versus approximately 11% in the electrolyticonly lesion group and 4% in the sham group. Although the time spent with the impaired forelimb adducted was partially reduced on subsequent days in the aspiration groups, the duration of adduction was maintained at higher levels than shams or electrolytic lesion animals throughout the course of observation. A 2-way ANOVA resulted in a significant effect for Groups (F3,32 /6.98, P B/0.001) and Days (F3,72 /3.87, P B/0.05), but there
Fig. 7. Duration of contralateral forelimb adduction. Throughout the course of observation, the aspiration and electroaspiration lesion groups showed significantly more adduction of the contralateral (impaired) forelimb than the sham and the electrolytic lesion groups. Data are means9S.E.M. * P B 0.05 significantly different from shams. $ P B 0.05 significantly different from aspiration lesions. % P B 0.05 significantly different from electroaspiration lesions.
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was no significant Group by Day interaction effect (F7,72 /1.05, P /0.05). On post-hoc analyses of group effects, the aspiration and electroaspiration lesions resulted in a significantly increased duration of limb adduction in comparison to both electrolytic lesion (F1,32 /6.88, P B/0.05 and F1,32 /5.87, P B/0.05, respectively) and sham (F1,32 /18.20, P B/0.001 and F1,32 /14.37, P B/0.001, respectively) groups. Although rats with electrolytic lesions tended to show greater durations of contralateral forelimb adduction during the first week after the lesion than did sham-operated animals, limb adduction was not significantly increased in this group in comparison to shams (F1,32 /2.03, P / 0.05). There were no significant changes in adduction of the ipsilateral (non-impaired) forelimb following the lesions in comparison to shams.
4. Discussion In summary, despite similarly sized lesions and initial similarities in some types of behavioral impairments, animals with aspiration lesions, with or without preceding electrolytic damage, evidenced much more limited behavioral recovery and contracortical structural plasticity than animals with electrolytic lesions. The electrolytic lesions resulted in increased dendritic arborization of layer V pyramidal neurons in the motor cortex contralateral to the site of damage at 18 days after the lesions in comparison to shams. The magnitude and pattern of the electrolytic lesion-induced dendritic growth, with greatest increases at topologically higher orders, was similar to that reported in previous studies [18,20,22]. In contrast, both aspiration and electroaspiration lesions failed to result in increases in dendritic arborization at this time point. Aspiration lesions also failed to evidence dendritic growth at 30 days in comparison to shams. Behavioral results showed that, despite similar initial asymmetries in forelimb use for upright postural support and rearing behaviors, the aspiration and electroaspiration lesion groups failed to show evidence of functional recovery in these behavioral measures during the course of observation, whereas animals with electrolytic lesions showed a partial return to more symmetrical behavior. Furthermore, both types of aspiration lesions resulted in a more prevalent adduction of the impaired forelimb. Together, these behavioral results suggest that the aspiration lesions resulted in a more severe and chronic postural-motor impairment of the forelimb opposite the lesion. The dendritic arborization results are consistent with previous findings of robust dendritic growth in layer V opposite the site of electrolytic lesions [17,18,20,22,33], as well as the failure to find dendritic growth at comparable time points following aspiration lesions [13,28]. The induction of neural structural and physio-
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logical changes in the contralateral cortex is not specific to electrolytic lesions. Focal ischemic lesions of the SMC produced using the endothelin-1 (a vasoconstrictor) method [14] have recently been found to result in major increases in microtubule-associated protein-2 (MAP-2), a marker of dendritic growth and plasticity, as well as increased immunoreactivity for NMDA (NR1) receptor protein in layers II/III and V of the motor cortex opposite the lesion [40]. Unilateral neocortical infarctions also result in increased synaptophysin expression in the contralateral cortex between 14 and 60 days after the infarct [36,37]. Furthermore, ischemic lesions produced using either middle cerebral artery occlusion or photothrombosis result in a major enhancement of the excitability of the contralateral homotopic cortex [41]. This includes enduring (at least 28 days) elevations in neuronal calcium currents [4], enhanced paired-pulse excitation [31], reduced paired-pulse inhibition [5] increased NMDA receptor binding [29] and decreased GABAA receptor binding [30]. It is important to note that contracortical dendritic growth is not found when the electrolytic lesions are made to be much smaller than those used in the present study, either as a result of experimental error in the creation of the lesions [17] or as a result of using an unmoving platinum electrode to produce the lesions [13]. The prolonged adduction of the impaired forelimb found in the aspiration lesion groups is a type of forelimb impairment that has previously been associated with striatal damage [23,34]. It has long been hypothesized that the impairments following cortical lesions are due, in part, to disruption of the striatum and other subcortical structures [10 /12,19,35]. The failure to find lesion-induced differences in striatal volume at 18 days suggests that it is unlikely that the greater behavioral impairments following aspiration lesions are simply due to greater direct injury to the underlying striatum. However, this does not rule out the possibility of lesion-dependent differences in the severity of secondary degenerative effects in the striatum, a possibility that is unlikely to be detected using the volume measures of the present study. Atrophy resulting from lesion-induced degeneration is likely to take more than a few weeks to be sensitively detected using volume measurements. Thus, it remains possible that the more enduring severe impairments found following aspiration lesions are related to greater disruption of striatal and/or other subcortical structures. The failure to find differences in cortical volume loss between lesion groups also, of course, does not indicate equivalency in the extent of secondary injury within the peri-lesion cortex because diffuse degenerative effects of the lesions may not necessarily be detected with volume measures. It remains possible that examination of later time points would be revealing of dendritic growth effects in the cortex opposite aspiration lesions. Although not
significant, the number of dendritic branch segments in the aspiration lesion group that survived to 30 days after surgery tended to be elevated relative to the aspiration lesion group examined at 18 days. It has been previously found that dendritic and synaptic changes in layer V of the motor cortex are time-dependent following electrolytic damage. Maximal increases in dendritic arborization contralateral to the site of unilateral lesions are found 18 days following injury [18], whereas increases in the ratio of synapses to neurons are not found until day 30 [17], which coincides with a partial pruning of dendritic arbors [17,18,20,22]. Contracortical increases in the dendritic cytoskeletal protein, MAP-2, have also been found to be time-dependent after both electrolytic lesions [25] and endothelin-1 induced ischemic lesions [40] of the sensorimotor cortex. Thus, it is conceivable that examination at an even later time point would reveal greater dendritic arborization changes in the cortex opposite aspiration lesions. Although electrolytic lesions have previously been found to result in enduring asymmetries in some types of limb use for upright postural support behaviors [33], aspiration lesions clearly resulted in more prolonged time course of severe behavioral impairments in the present study. It is also possible that the aspiration lesions result in structural changes that are not revealed in measures of dendritic branch number, such as changes in spine density. Forelimb behavioral manipulations and transcallosal denervation have each been found to result in localized spine addition in the motor cortex which is not coupled to increases in dendritic arborization [2]. The similarity in the behavioral and structural effects of aspiration and electroaspiration lesions indicates that the passage of electric current is unlikely to be a key contributor to these lesion-dependent effects. One alternative possibility is that the presence of damaged tissue at the site of cortical injury plays an important role in behavioral and structural outcomes following cortical lesions. That is, it is possible that the slower loss of damaged cells following electrolytic lesions, and the glial response to this loss, contributes to compensatory plasticity in regions of both the damaged and intact hemispheres. Boyeson et al. [3] have found that SMC lesion procedures that do not remove tissue (percussive injury and cortical undercuts) result in transient increases in the thresholds of microstimulation-induced movements in adjacent SMC regions. This effect was not found after aspiration lesions, consistent with the possibility that removal of the damage tissue alters the nature of the ipsilateral cortical response to the lesion. However, there are two lines of research that fail to support the hypothesis that the contralateral cortical effects are directly attributable to the presence of the damaged tissue. The first is that it is possible to reproduce growth of motor cortical layer V dendritic arbors with the combination of transections of the
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corpus callosum and forced use of the opposite forelimb [6]. This indicates the necessity of callosal denervation and behavioral demand, but not of slow-growing cortical damage, to produce neuronal growth. Obviously, all lesions in the present study produced these two necessary conditions. Although corpus callosum transections result in a variable amount of electrodetract damage to the medial agranular and/or cingulate cortex, there were no differences in the magnitude of dendritic growth that could be linked to the extent of cortical damage [6]. Secondly, although other researchers have failed to find crossed-corticostriatal sprouting following aspiration lesions [27,39]. McNeill et al. have found both significant crossed-corticostriatal growth [8] as well as increases in several plasticity related proteins [7,24] in the motor cortex opposite unilateral aspiration lesions in young adult (250 /300 g) animals. In addition to pointing to potential methodological differences in aspiration lesion procedures, this work suggests that factors other than the removal of tissue at the lesion site are likely to be involved in the differences in contracortical effects. Another possible explanation for these lesion-dependent differences is that the aspiration procedure, at least as it is performed in many laboratories, results in greater disruption of underlying brain structures, including the underlying white matter and the striatum. If so, it is possible that this could result in the more severe behavioral asymmetries observed, and possibly in alterations of mechanisms that either promote or inhibit neuronal adaptation to the damage. It has been found, for example, that lack of neural sprouting in adult rats is linked to the presence of the inhibitory factor, Nogo-A, a myelin-associated inhibitor of neurite growth [15]. Kartje et al. [21] have found that, whereas aspiration lesions in adult rats failed to result in crossed corticostriatal sprouting originating from the cortex opposite the lesion, blocking Nogo-A following the lesions did result in an increase in crossed corticostriatal fibers and other crossed cortical projections. Consistent with the possibility that aspiration lesions result in a greater inhibition of structural plasticity, growth associated protein-43 (GAP-43), a marker of axonal sprouting, has been found to be decreased in the underlying striatum after large unilateral aspiration, but not after ischemic cortical lesions [38,39]. Also potentially related is the finding that fetal cortical grafts placed within the territory of ischemic cortical infarcts of adult rats developed zinc-positive (likely glutamatergic) terminals within the transplanted tissue. This was not found when the grafts were placed in the territory of aspiration lesions [42]. In addition to providing evidence that electrolytic lesions and aspiration lesions produce different structural and behavioral outcomes, and are therefore not interchangeable models of cortical injury, the findings of
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this study further support the idea that lesion-specific factors may contribute to the outcomes of cortical damage. Chesselet et al. have found marked differences in the effects of ischemic versus aspiration lesions when pre-, peri-, and post-operative conditions were identical between lesion groups [26,27,38,39]. Together with the present results, these findings indicate that some of the ‘‘normal’’ sequelae of compensatory neuroplasticity seen after electrolytic and ischemic injury are either absent or considerably less prevalent after aspiration lesions. At this point, it is necessary to continue this comparison of lesion models in order to examine structural and behavioral outcomes at later time points, as well as to address the possibility of differences in the cellular responses to electrolytic and ischemic versus aspiration lesions in both peri-lesion and remote regions. The presence of different growth-promoting and inhibitory substances and substrates may be revealing, not only of the differences in these lesion types, but also of the conditions that promote adaptive plasticity after ischemic injury. The present finding that aspiration and electrolytic lesions are not interchangeable models of cortical injury is also illustrative of the limits in generalizing between the commonly used models of neural and behavioral adaptation to brain damage.
Acknowledgements We are very grateful to Drs Timothy Schallert and Otto Witte for comments on this study, to DeAnna Adkins and Scott Bury for technical assistance, to Samuel Pedigo and Shannon Carr for assistance in preliminary data collection and analysis, to Kevin McCullough for taming and caring for the animals and to Todd Martin for assistance in assembling the manuscript. Supported by NIH Grant MH56361.
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