Neonatal frontal cortical lesions in rats alter cortical structure and connectivity

BRAIN RESEARCH ELSEVIER

Brain Research 645 (1994) 85-97

Research Report

Neonatal frontal cortical lesions in rats alter cortical structure and connectivity Bryan Kolb a,,, Robbin Gibb a, D e r e k van der Kooy b Department of Psychology, University of Lethbridge, Lethbridge, Alta., Canada T1K 3M4, b Department of Anatomy, University of Toronto, Toronto, Canada (Accepted 18 January 1994)

Abstract

Rats were given frontal cortical lesions at day 1 or 10 of life. Later, as adults, they were either: (1) processed with Golgi-Cox in order to analyze cortical dendritic arborization; (2) given injections of True Blue into the parietal or visual cortex, or (3) given injections of [3H]leucine into the substantia nigra. An additional group of normal rats were given injections of fluorescent dyes into the cortex on day 4 or 10 of life. The main findings were that (1) adult hemispheres with day 10 lesions had greater dendritic arbor than normal hemispheres, (2) adult hemispheres with day 1 lesions had reduced dendritic branching relative to normal hemispheres, (3) adult rats with day 10 lesions had no obvious abnormalities in cortical connections, (4) adult rats with day 1 lesions had abnormal thalamo-cortical, amygdalo-cortical, and nigro-cortical connections, and (5) many of these abnormal connections were present in the brains of 4-day-old normal rats. Since the 'abnormal' connections in the very early frontal operates were present in day 4 animals, it appears that they result from the failure of exuberant connections to retract after the lesions. The increased dendritic growth in day l0 operates does not appear related to qualitative changes in cortical afferents or efferents and may related to increased intrinsic cortical connectivity. Since rats with day 10 lesions have previously been shown to exhibit significant recovery of function, it is possible that the increased dendritic arborization is supporting the functional restitution.

Key words: Cortex; Development; Recovery of function; Frontal cortex; Prefrontal cortex; Parietal cortex; Dendrite

1. Introduction

For over 100 years it has been known that the behavioral effects of cortical injury are frequently different in young and adult animals [9]. This phenomenon gained prominence in the 1940s when Kennard showed that under certain circumstances it appeared that the earlier the cortical injury was incurred in monkeys, the less severe were the behavioral consequences [19,20]. Subsequent investigations looking at the effects of cortical lesions at different ages in various species have confirmed Kennard's general claim that cortical injury in infancy and adulthood may have quite different behavioral effects but the mechanisms

* Corresponding author. Fax: (1) (403) 329-2555. E-mail: [email protected] 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 1 3 8 - 3

underlying the differential effect of cortical injury with age are still poorly understood [1,8]. In order to approach this problem, we have been studying the behavioral effects of bilateral lesions of the prefrontal, motor or parietal cortex of rats at different development ages. As expected, we were able to show that cortical injury at 7-10 days of age was associated with significant behavioral sparing relative to the effects of similar lesions in adulthood. Unexpectedly, however, we also found that cortical injury at 1-5 days was associated with no behavioral sparing and in many cases greater behavioral loss than was experienced by rats with similar removals in adulthood [21, 27,281. Following these behavioral observations we began to look for anatomical correlates of the differential effects of neocortical lesions at 1, 10 or 90 days of age. Our initial experiments showed that behavioral recovery

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after day 10 frontal lesions was correlated with an increase in dendritic arborization in the sensorimotor cortex adjacent to the lesions. Failed recovery after similar day 1 lesions was correlated with a decrease in dendritic arborization [23,25]. In parallel experiments in rats with adult frontal lesions we found a small increase in dendritic branching that was also correlated with modest behavioral recovery over 4 months [24]. This dendritic growth was restricted to the adjacent sensorimotor cortex, however, and was not observed in posterior cortex. Thus, one goal of the current study was to determine if dendritic changes after infant lesions were as restricted as in adult frontal operates or if they might be more widespread. A second goal of this study was to examine the patterns of efferent and afferent connectivity of the neocortex after day 1 or day 10 lesions. We reasoned that if there were widespread changes in dendritic arborization, then this implied an increase in connectivity. Since neonatal motor cortex lesions in rats are associated with changes in corticospinal projections from the remaining cortex, it seemed reasonable to expect that neonatal frontal lesions might also alter cortical connectivity with thalamic, brainstem, or remaining cortical regions. We therefore injected fluorescent dyes into the cortex of adult rats that had sustained frontal lesions on day 1, 10 or 90. Four sets of experimental animals were used in this study. Since different sets of animals were treated differently, the procedures for each set of animals is described as a separate experiment. The results are described in a single section, however, to simplify explanation.

2. Materials and methods

AGE PN1

PN4

PN10

PN120

PN127

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7

8 Fig. 1. Schematic illustration of the design of the experiments. Different groups of rats received different treatments at postnatal day 1, 4, 10, 120, or 127 (PN1, PN4, PN10, PN120, PN127). Groups I - 3 received frontal cortical lesions at PNI or PN10 and then received fluorescent dye injections on PNI20. They were sacrificed on PN127. Rats in groups 3 and 4 received dye injections on PN4 or PN10 and were sacrificed on PN127. Rats in groups 6 and 7 received frontal ablations on PN1 or PN10, were sacrificed on PN127, and the brains stained with Golgi-Cox. Rats in group 8 received serial lesions in which one frontal cortex was removed on PN1 and the other frontal cortex was removed on PN10. These animals were either processed with Golgi-Cox or Cresyl violet in adulthood.

2.1. Experiment 1 In this experiment rats were given lesions on postnatal day 1 (PN1) or 10 (PN10) and sacrificed on day 127 (Fig. 1, groups 6-8). Their brains were processed for Golgi-Cox.

2.1.1. Subjects The observations were made on 28 male Long-Evans rats derived from the Charles River Long Evans strain. There were 10 control rats, five rats with P N I lesions, five rats with PN10 lesions, and eight rats with a serial lesions in which the right frontal cortex was removed at PN1 and the left frontal cortex was removed at PN10. The animals were weaned at 22 days of age, sexed, and group housed in stainless steel hanging cages until adulthood. They were maintained on ad lib food and water with a 12:12 h l i g h t / d a r k cycle.

2.1.2. Surgical and anatomical procedures The neonatal rats were anesthetized by cooling them in a Thermatron cooling chamber set at - 5°C. They were cooled for about 15 rain by which time their rectal temperature was in the range of 18-20°C and they were immobile. Frontal decortication was achieved by removing the frontal bone by cutting it with iris scissors, and then

removing the cortex anterior to the bregma by aspiration. The wound then was sutured with silk thread. The rats were then warmed up in cupped hands until they began to squirm, and then they were allowed to warm up slowly to about 35°C under a heat lamp before being returned to the mother. T h e control rats were anesthetized in the same m a n n e r and the skin was incised and sutured. The brains of five of the serial lesion rats were processed for Nissl staining and the remainder were processed for Golgi-Cox staining. The rats were anesthetized with sodium pentobarbital and perfused intracardially with 0.9% saline. The rats processed for Nissl were then perfused with 10% formaldehyde, placed in 30% sucrose formalin until they sunk, cut frozen at 4 0 / z m and every tenth section was m o u n t e d on slides and stained with Cresyl violet. T h e brains processed for Golgi-Cox were removed after saline perfusion and immersed whole in 50 ml of Golgi-Cox solution [12]. T h e solution was changed after 2 days and the brains left in Golgi-Cox solution for an additional 12 days. T h e brains were either embedded in celloidin (PNI group) and cut at 120 /xm using the procedure of Glaser and van der Loos [12] or they (PN10 group, serial group) were placed in a 30% sucrose solution for 2 days and cut frozen at 120 p.m and developed using a procedure described by Kolb and McLimans [29].

B. Kolb et al. /Brain Research 645 (1994) 85-97 (The operated and control brains in each experiment were processed using the same procedures.) In order to be included in the data analysis the dendritic trees of pyramidal cells had to fulfill the following criteria: (1) the cell had to be well impregnated and not obscured with stain precipitations, blood vessels or heavy clusters of dendrites from other cells; (2) the cell had to lie in the middle of the section thickness so that the apical and basilar dendrites were clearly visible in the plane of section. The cells were analyzed by drawing the cells via camera lucida and then each branch segment was counted and summarized by branch order using the procedure of Coleman and Riesen [5]. Branch order was determined for the basilar dendrites such that branches originating at the cell body were first order; after one bifurcation, second order; and so on. Branch order was determined for the apical dendrites such that branches originating from the primary apical dendrite were first order and so on. Cells were chosen by locating the areas in question and then by drawing each cell in that section that met the criterion listed above. Ten layer I I / I I I pyramidal cells per hemisphere were drawn from each of Zilles' [40] areas Par 1 (parietal cortex), FP (motor forelimb cortex), TE2 (temporal visual cortex), Oc 1, and Oc2L (primary and secondary visual cortex). Statistical analyses were performed by averaging across the 10 cells per area per hemisphere. Since there were no differences between the two hemispheres of either the control animals or operated animals, each hemisphere of all animals was considered as a separate data point. 2.2. Experiment 2 T h e rats in this experiment were given frontal lesions or control procedures on PN1, PN10 or postnatal day 90 (PN90). They were given injections of True Blue into either the cortex or striatum on PN120 (Fig. 1, groups 1-3).

2.2.1. Subjects Forty-four Long-Evans rats derived from Charles River breeding stock were divided into five groups: normal control (n = 20), unilateral PN1 frontal lesion (n = 5), bilateral PN1 frontal lesion (n = 12), bilateral PN10 frontal lesion (n = 10), and bilateral PN90 (n = 2). There were approximately equal n u m b e r s of males and females in each group and since there were no obvious sex differences the data were pooled. The animals were weaned at 22 days of age, sexed, and group housed in stainless steel hanging cages until adulthood. They were maintained on ad lib food and water with a 12:12 h l i g h t / d a r k cycle.

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For the striatal injections the syringe was lowered 4 m m below the pial surface. The animals were allowed to survive for 7 days before being anesthetized and intracardially perfused with 0.9% saline followed by 10% formalin. The brains were removed and placed in 30% sucrose formalin until they sank (usually 48 h) and were cut frozen at 40 tzm. Every ninth and tenth sections were m o u n t e d to make two complete sets of sections through the entire brain. O n e section was kept for fluorescence microscopy and the other was stained with Cresyl violet.

2.3. Experiment 3 The rats in this experiment were given frontal lesions on PN1 and then in adulthood they were given [3H]leucine injections into the substantia nigra.

2.3.1. Subjects Two Long-Evans rats, who were littermates of those described in Expt. 2, were used. They were operated on PN1 and treated the same as those in Expt. 2.

2.3.2. [ 3H]Leucine injections Anesthetized adult rats with frontal lesions on day 1 were microinjected into the substantia nigra over 5 min with 50 /zCi of [3H]leucine in 0.1 Izl of distilled water. After a 24 h survival these rats were sacrificed by anesthetic overdose and transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline at 4°C. Brains were frozen and cut into 32 ~ m sections and collected in ice-cold phosphate-buffered saline before immunostaining. Nearby sections were saved and m o u n t e d for immunohistochemistry or autoradiography. Sections for autoradiographic demonstration of the [3H]leucine anterogradely transported from the substantia nigra to the striatum and cortex were coated with Kodak NTB2 photographic emulsion and stored at 4°C for 8 weeks. Slides were then developed and counterstained, and the distribution of radiolabeling noted.

2.2.2. Surgical and anatomical procedures 2.2.2.1. Cortical lesions. The neonatal rats were anesthetized and received frontal lesions using the same procedures as in Expt. 1. T h e adult animals were anesthetized with sodium pentobarbital (65 m g / k g for males and 45 m g / k g for females) and placed 'in a stereotaxic apparatus. The bone was removed from the bregma forward and the anterior to the bregma was removed by aspiration. Following hemostasis the wound was closed and sutured.

2.2.2.2. Retrograde dye injections. The adult animals were anesthetized with sodium pentobarbital (65 m g / k g for males and 45 m g / k g for females) and placed in a stereotaxic apparatus. Animals were either given one or more 0.2 /~1 injections of a 5% True Blue solution into the cortex or a 0.2 /zl injection into the striatum. For the cortical injections the syringe was lowered into place 1 m m below the pial surface and the injection made in two 0.1 /zl stages, separated by 5 min, after which the syringe was left in place for 10 min before removal. The parietal injections were m a d e at the level of the bregma and lateral 2 m m and 3.5 mm. The occipital injections were m a d e at - 5 m m from the bregma and lateral 2.5, 3.5, and 4.5 mm.

Fig. 2. Photograph of a brain of a rat with a frontal lesion in the left hemisphere at PN1 and in the right hemisphere at PN10. The left hemisphere is visibly smaller than the right one.

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2.3.3. Tyrosine-hydroxylase staining. Sections for immunohistochemistry were incubated for 48 h at 4°C in a 1 : 100 dilution of rabbit anti-TH antibodies (Eugene Technical) containing 0.3% Triton X-100 and 1% normal goat serum. After washing in phosphate-buffered saline (with 1% normal goat serum and 0.3% Triton X-100) for 10 rain, sections were incubated for 1 h in a 1 : 30 dilution of goat antirabbit antibodies conjugated to fluorescein isothiocyanate (FITC), at room temperature. Following a final wash in 0.1 M phosphate buffer, sections were mounted and coverslipped with 1:1 glycerol and water (pH 7.8), and viewed under excitation light of 470 n m wavelength to illuminate the FITC-labeled TH. Control sections had the primary antibody omitted. This prevented staining.

2.4. Experiment 4 The animals in this experiment were given True Blue injections into the cortex as infants. Since it proved difficult to make selective

cortical injections into the cortex on PN1, we made our injections on PN4 and PN10. The animals were then allowed to live to adulthood before sacrifice (Fig. 1, groups 4,5)

2.4.1. Subjects Forty-four Long-Evans rats derived from Charles River stock received injections of True Blue on either postnatal day 4 (n = 20) or postnatal day 10 (n = 24). The animals were maintained as in Expts. 1-3.

2.4.2. Surgical and anatomical procedures The animals were anesthetized by cooling and then fixed in place on the stereotaxic base by using masking tape. After the skin was incised and the skull was exposed a small puncture was made in the skull by using a 20 gauge needle. The blunt-tipped microsyringe was lowered through the puncture hole under visual guidance with the aid of an operating microscope. The syringe was lowered so that it just punctured the pial surface and each animal was given a 0.1 /xl

MOTOR

Day 1

Day 10

\

~______Day 1

PARIETAL__

Day 10

Fig. 3. Examples of layer I I / I I I pyramidal cells taken from motor and parietal cortex of a rat that had a frontal lesion in one hemisphere on day 1 and in the other hemisphere on day 10. The cells in the day 10 hemisphere have more dendritic arbor than those in the day 1 hemisphere.

B. Kolb et aL / Brain Research 645 (1994) 85-97

TWO-STAGE LESIONS

injection of a 5% True Blue solution. These animals were allowed to survive until adulthood when they were sacrificed and processed in the same m a n n e r as the adult operates.

40

3. Results

The main findings were that (1) hemispheres with PN10 frontal lesions had greater dendritic arbor than normal hemispheres whereas hemispheres with PN1 frontal lesions had a reduction in dendritic branching relative to normal hemispheres; (2) rats with PN10 frontal lesions had no obvious abnormalities in cortical connections; (3) rats with PN1 frontal lesions had abnormal adult thalamo-cortical, amygdalo-cortical and nigro-cortical connections; and (4) many of these abnormal connections were present in the brains of 4day-old normal rats. These will each be discussed in detail separately. 3.1. Gross morphological changes observed in adult rats The frontal decortications removed most of the prefrontal cortex as well as the frontal pole, including part of the motor cortex (Fig. 2). The differential effect of the day 1 and day 10 lesions on cerebral size is easily seen in Fig. 2 in which the left hemisphere received a lesion on day 1 whereas the right hemisphere received a lesion on day 10. The day 1 lesion produced a visibly smaller hemisphere in all cases than did the day 10 lesion. 3.2. Dendritic branching The overall effect was that hemispheres with PN10 frontal lesions had greater dendritic branching than controls whereas hemispheres with PN1 frontal lesions had a small decline in branching relative to Controls. The contrasting effects of PN1 and PN10 lesions is illustrated in Fig. 3, which compares the dendritic arborization layer I I / I I I pyramidal cells in the motor and parietal cortex of the two hemispheres of a rat with a PN 1 lesion in one hemisphere and a PN10 lesion in the other. It is evident that in the same animal the lesions had a differential effect on dendritic branching in the two hemispheres. A similar pattern of results was visible in all three serial lesion cases and is numerically summarized in Fig. 4. In order to statistically analyze the dendritic results it was decided to perform an overall analysis of variance in which hemisphere (day 1 vs. day 10), dendritic measure (apical vs. basilar), and cortical location were treated as factors. Such an analysis would thus have sufficient power to find interactions that might dissociate the effects of the day 1 and day 10 lesions on the dendritic morphology in specific cortical locations. The

89

30

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APICAL

BASILAR

PLACE Fig. 4. Summary of dendritic branching in rats with two-stage neonatal frontal lesions (unilateral lesions at 1 and 10 days). The 10 day hemisphere had significantly more dendritic branching than did the 1 day hemisphere. (The error bars indicate S.E.M.s.)

analysis showed significant main effects of hemisphere and dendritic measure (F(1,24)= 17.5, P = 0 . 0 0 0 3 ; F(1,24) = 12.71, P = 0 . 0 0 1 6 ) , whereas neither place (Par 1, Ocl, T e l ) nor any of the interactions reached significance ( P s > 0.40). Thus, it appears that 10 day and 1 day frontal lesions have a grossly different effect upon the subsequent development of dendritic arbor in the same brain. In order to further examine the generality of the dendritic effects across different cortical regions, we examined the dendritic changes in five neocortical areas in a larger series of animals that had bilateral frontal lesions at either 1 or 10 days of age. The results confirmed the serial lesion results except that the increase after day 10 lesions appeared to be larger in the motor and parietal regions, which are nearer to the lesion, than in the more distant visual or temporal regions (Fig. 5). Analyses of variance were performed on each cortical area to compare the day 1 frontals and their littermate controls, who were all processed using a celloidin-embedding technique, and separate analyses were performed on the day 10 frontals and their littermate controls, who were processed using a frozen-tissue technique. Analyses of the day 10 group revealed that basilar branches in areas FL, Par 1, Ocl, and Te and the apical branches in areas FL and Par 1 were significantly increased in the lesion vs. the control animals (Ps < 0.005 for the significant comparisons or > 0.05 for the nonsignificant ones). In contrast, comparison of the day 1 frontal and control groups showed a significant decrease in branching in the Par 1 and

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Ocl basilar branches (Ps < 0.05) and Ocl and Oc2 apical branches (Ps < 0.05) in the brains with lesions. No other comparisons reached significance (Ps > 0.05). 3.3. Parietal connections in adult rats

Since we had little way to be certain that any injection would include the same precise cytoarchitectonic region in normal or operated brains, we opted to make multiple injections to increase the likelihood that the injections included some comparable tissue (Figs. 6B, 7A). Eight day 1 bilateral frontal, four day 10 bilateral frontal, and five control rats had comparable unilateral parietal cortex injections that did not include subcortical structures nor show evidence of leakage into the APICAL DENDRITIC BRANCHING 200180 O

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Fig. 6. A - C : Photomicrographs illustrating the True Blue injections in two PN4 brains (A,C), and an adult control brain (B). D: Higher power magnification of the staining along the ventricle shown in A. This staining was visible in all brains with PN4 injections into parietal cortex. It is separated from the injection site by white matter and is restricted to the dorsomedial region of the ventricular wall.

FR 10

C413J~

Fig. 5. Summary of the total dendritic branching observed in five cortical regions of normal control (CON), day 1 frontal (FR 1), and day 10 frontal (FR 10) operates. The general result was that the F R 10 animals had an increase in dendritic branching across all five areas, but especially in the forelimb (FL) and parietal (PAR 1) regions. In contrast, the FR 1 animals showed a slight decline in dendritic branching. The data are expressed as percent of control value. Nomenclature after Zilles [40].

ventricular system. All injections retrogradely labeled the ipsilateral ventrolateral thalamus, reticular thalamus, basal forebrain, insular cortex, and various ipsilateral and contralateral neocortical regions (Fig. 8). In addition, adult injection of True Blue into adult PN1 frontal operates produced several consistent differences with injections into adult PN10 frontals or normal animals. First, there was more extensive thalamic labeling in the PN1 operates (Fig. 9). Thus, in addition to the normal labeling in the ventrolateral and the reticular nuclei, there was label in MD (3 of 8 cases), the lateral dorsal nucleus (4 of 8 cases), lateral geniculate (dorsal) nucleus (4 of 8 cases), medial geniculate nucleus (5 of 8 cases), and lateral posterior nucleus (2 of 8 cases). The pattern of label in the different thalamic regions was idiosyncratic as every day 1 lesion case showed at least one abnormal connection and some showed all of them. There was no obvious relationship between the injections and the abnormal thalamic labeling. Second, there were labeled cells in the substantia nigra and ventral tegmentum of all day 1 cases (Fig. 7C). Third, seven of eight cases had labeled cells in the amygdala. Finally, there appeared to be group differences in the extent of label observed in the posterior cortex. Thus, the day 1 animals had far more extensive

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cortico-cortical labeling in the parietal, visual and temporal cortex than either the control or day 10 animals. In order to further examine the abnormal midbrain, presumably dopaminergic, projections to the parietal cortex, [3H]leucine was injected into the ventral t e g m e n t a l / s u b s t a n t i a nigra region of an adult day 1 frontal rat (Fig. 9). Labeled fibers could be seen leaving the striatum and penetrating the parietal cortex near the edge of the lesion, which included the region of injection in the True Blue cases. In addition, inspection of tyrosine hydroxylase immunofluorescence in the same [3H]leucine animals showed dopaminergic fibers leaving the heavily stained striatum and entering the parietal cortical white matter (Fig. 10). In sum, bifrontal decortication at day 1 resulted in a variety of abnormal connections in adulthood that were not observed in rats with similar lesions on day 10 or in adulthood. These connections included a variety of abnormal thalamocortical, amygdalocortical, and midbrain dopaminergic projections to the cortex. 3.4. Visual cortex injections into adult rats

Adult injections into the visual cortex of unoperated controls led to labeling in the lateral posterior, lateral

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geniculate, and posterior thalamic nuclei, as well as the basal forebrain, insular cortex, and various ipsilateral and contralateral neocortical areas (Fig. 11). In contrast to the adult parietal injections, however, the pattern of labeling after adult visual cortex injections failed to show obvious abnormalities in any of the cases with neonatal lesions at either day 1 or day 10. Thus, it appears that the normal cortical connectivity is disrupted in the remaining anterior, but not posterior, cortex after neonatal frontal lesions. 3.5. True Blue injections into neonatal rats

In order to determine whether the abnormal projections observed in adult rats with day 1 frontal lesions represented (1) the growth of new abnormal connections after the frontal injury, or (2) the failure of 'normal' exuberant connections to retract after the lesion, we made True Blue injections into neonates on day 4 or 10 days of life. Most of the injections into the 4 day or 10 day cortex were restricted to the neocortex, although it proved difficult to match the injections exactly in the adult and neonatal groups (Figs. 6, 7). However, there were 5 parietal and 3 occipital injections in the PN4 group and 5 parietal and 5 occipital

L

Fig. 7. A,B: Photomicrographs illustrating the True Blue injection sites in an adult rat with a frontal lesion at PN1 (A) and an adult rat with a PN4 injection. C: Photomicrograph illustrating the retrograde label in the substantia nigra of the brain with the injection shown in A. D: Photomicrograph illustrating the retrograde label in the substantia nigra of the brain with the injection shown in B.

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injections in the PN10 group that were comparable to the adult injections. The overall result was that the parietal injections at PN4 led to much more extensive labeling of both subcortical and cortical structures than did the 10-day injections, which had a pattern of label that was rather similar to that observed in the adults. Fig. 12 summarizes a neonatal parietal injection and shows that like the adult injection into a day 1 frontal rat, it resulted in 'abnormal' label in both the dorsolateral (LD), posterolateral (LP) and lateral genciulate (LGN) of the thalamus and the substantia nigra/ventral tegmentum (SN/VTA) (Fig. 7D). Again, like the injections into adult rats with day 1 frontal lesions, the pattern of abnormal labeling was idiosyncratic as 4/5 cases

ADULT C O N T R O L

... i~6 pv MHb

DAY 1 F R O N T A L

Fig. 9. Reconstruction of the pattern of retrograde labeling in the diencephalon after True Blue injections into the sensorimotor cortex of a rat with a frontal lesion at day i. Like the case illustrated in Fig. 7, there was abnormal label in MD, LD, LP, DLG, Po, MG, SNR, and VTA. The presence of retrogradely labeled cells in MD may account, in part, for the failure of MD to degenerate following neonatal frontal lesions. The thalamus was shrunken and malformed so the identification of some thalamic nuclei was questionable (indicated by '?'). Injection sites are illustrated in the bottom right panel. Abbreviations as in Fig. 3 and: PT, anterior pretectal nucleus; CG, central grey; CM, central medial nucleus; DLG, lateral geniculate, dorsal component; FR, fasiculus retroflexus; IC, internal capsule; LHb, lateral habenula; MHb, medial habenula; ML, medial lemniscus; MT, mammillothalamic tract; PC, posterior commissure; PF, parafasicular nucleus; SC, superior colliculus; SN, substantia nigra; VLG, lateral geniculate, ventral component; VTA, ventral tegmental area; ZI, zona incerta; 3V, third ventricle.

Fig. 8. Reconstructions of the pattern of retrograde labeling after True Blue injections into the sensorimotor cortex of an adult control rat and an adult rat with a Day 1 Frontal lesion. All injections retrogradely labeled the ventrolateral thalamus, basal forebrain, insular cortex, and various ipsilateral and contralateral neocortical regions. The Day 1 Frontal injections also produced labeling in LD, LP, LGN, MGN, amygdala, ventral tegmental area and substantia nigra. There are also more ipsilateral cortico-cortical connections in the Day 1 Frontal animal relative to the control. Abbreviations as in Fig. 3.

showed L D / L P label, 2/5 LGN label, 1/5 medial geniculate nucleus (MGN) label, and 3/5 SN/VTA label. One additional peculiar result of the PN4 parietal injections was label along the dorsomedial edge of the ventricular wall, as illustrated in Fig. 6. Thus, in every case there was a short band of label that was separated from the cortical injection site by the white matter. This is unlikely to reflect direct leakage of the dye into the ventricle because leakage would have led to label along the entire ventricular wall, but this did not occur. This label was never seen in any animals with injec-

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tions at PN10 or adulthood. It is interesting to note that the location of the dye corresponds to the region of the ventricular wall that has been shown to have proliferating subependymal cells in adulthood [32]. True Blue injections into the visual cortex of unoperated PN4, PN10 and adult rats produced a rather similar pattern of subcortical labeling, the lone anomaly being the presence of labeled cells in the medial geniculate nucleus in all 3 cases with day 4 injections. On the other hand, the day 4 neonatal injections into the visual cortex produced an abnormal pattern of corticocortical labeling both ipsilaterally and contralaterally across the corpus callosum (Fig. 12). Hence, all 3 cases had a similar pattern of label that included (1) the contralateral prefrontal cortex, (2) primary visual cortex on the contralateral side, and (3) more extensive ipsilateral parietal cortex.

ADULT CONTRO'L

DAY 1 F R O N T A L

Fig. 11. Reconstructions of the pattern of retrograde labeling after True Blue injections into the visual cortex of an adult control rat and an adult rat with a day 1 frontal lesion. The general pattern of labeling in thalamic areas LP, LGN, and Po as well as the insular cortex and basal forebrain is similar in both cases.

Fig. 10. A: Darkfield photomicrograph showing the distribution of anterogradely transported [3H]leucine in a coronal forebrain section ipsilateral to a large injection (24 h earlier) centered in the substantia nigra - ventral tegmental area. The frontal cortex was ablated in this animal on day 1. Labeled fibers can be seen leaving the striatum (arrow) and running through the white matter of the parietal cortex in long bundles. B: Higher power photomicrographof area similar to that arrowed in A. The tyrosine hydroxylase immunofluorescence shown here demonstrates that in animals receiving neonatal frontal decortication some dopaminergic fibei-s leave the densely immunofluorescent striatum and enter the cortical white matter.

In sum, injections into the parietal cortex of normal 4-day-old, but not normal 10-day-old, rats led to a pattern of retrograde labeling that was strikingly similar to that observed in adult-injected animals that received frontal lesions on day 1. In contrast, injections into the parietal cortex of normal 10-day-old animals revealed a pattern of labeling that was similar to normal adult-injected rats or adult-injected rats with day 10 lesions. These results suggest that the day 1 frontal lesions interfere with the normal process of retraction of exuberant cortical afferents. This normal retraction would appear to be complete by day 10 since the 'abnormal' cortical afferents were not visible in normal animals injected at 10 days of age or in adult-injected animals with day 10 lesions. Curiously, the day 1 lesions do not appear to interfere with the normal loss of callosal connections. Thus, day 4 injections into normal animals revealed a much more extensive callosal connection than observed in day 1 lesioned-rats injected in

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adulthood or in normal rats injected on day 10 or in adulthood. 3.6. Cortico-striatal connections in adult rats

There were two significant observations in adult rats who sustained unilateral frontal lesions on day 1 and retrograde tracer injections in the striatum as adults. First, relative to normal adult rats there was an abnormally large contralateral prefrontal projection to the striatum in the unilateral neonatal operates but no contralateral projection at all in the adult frontal operates (Fig. 13). Furthermore, as reported elsewhere for rats with neonatal hemidecortication, this increased

Fig. 13. Reconstruction of the pattern of retrograde labeling after a True Blue injection into the adult striatum ipsilateral to a frontal lesion at one day of age. There is label, which is largely confined to the superficial layers, in the contralateral prefrontal cortex. There is also label in the shrunken ipsilateral thalamus, including MD. This remaining thalamo-cortical projection from MD may account, in part, for the failure of MD to degenerate following neonatal frontal removal.

projection had its origin in the more superficial layers ( I I / I I I ) as well as the normal layer V [26]. Note, however, that the abnormal contralateral projection to the striatum was limited to the prefrontal cortex as layer V neurons in the intact posterior cortex in the lesion hemisphere still projected to the ipsilateral striatum (Fig. 13). Second, the striatal injections labeled the ipsilateral dorsomedial nucleus of the thalamus (MD) in all cases. Thus, although the normal cortical afferents of MD were completely removed, this nucleus still had viable projections to the striatum. Label was not observed in other thalamic nuclei. In sum, the striatum of adult rats who had sustained a neonatal unilateral frontal lesion showed an abnormal projection from the intact, contralateral, prefrontal cortex and a normal projection from the ipsilateral MD. Fig. 12. Reconstruction of the pattern of retrograde labeling after True Blue injections into the parietal (left) or visual (right) cortex of a day 4 rat, which was sacrificed as an adult. Note the similarity between the adult injection into a day 1 frontal (Fig. 7) and the day 4 parietal injection illustrated here. The one clear difference is the greater ipsilateral cortical-cortical labeling shown in the adult day 1 frontal in Fig. 7. In contrast, injection of True Blue into the visual cortex on day 4 produced more ipsilateral and contralateral label than observed in either the normal adult control or adult day 1 frontal illustrated in Fig. 10. In addition, the day 4 injection labeled cells in the MGN.

4. Discussion

Damage to the frontal cortex of rats has different behavioral effects depending upon the developmental age of the brain at the time of injury. Thus, rats with frontal cortical damage in the first few days of life have behavioral deficits that are more severe than those observed in adult rats with similar lesions. In contrast,

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rats with frontal cortical injury at 7-10 days show sparing of functions lost or impaired after similar lesions in adulthood. In fact, on many behavioral tests that are sensitive to the effects of adult frontal lesions, the 7-10 day frontal rats are nearly indistinguishable from controls [21,31]. Kolb and Whishaw [31] first reported that day 7 lesions were associated with a smaller adult brain, reduced diencephalic volume, and thinner remaining neocortex relative to adult operates. Subsequent studies confirmed this finding and demonstrated that the extent of reduction in each of these measures relative to control brains was directly related to the age at which the brain was injured: the earlier the injury, the thinner the cortex [21]. This relationship held up until about 25 days of age by which time the remaining cortex was approximately the same thickness as adult operates (for a review, see Kolb and Gibb [22]). The changes in cortical thickness had a gross correlation to behavior in the younger animals as the 10 day animals had a better behavioral outcome and thicker cortex than did animals with earlier lesions, but cortical thickness failed to correlate with behavior in animals with lesions at 25 days of age since they had cortex of normal thickness but failed to show sparing of function. We were also puzzled as to how the brains of 10 day animals could support spared behaviors with a smaller brain and thinner cortex than older animals who showed larger behavioral loss. The current study therefore sought to find other neuroanatomical changes that would better predict the behavioral outcomes. We focused our attention on changes in connectivity and dendritic arborization, which we shall consider separately. 4.1. Dendritic arborization Rats with day 10 lesions showed a dramatic and widespread increase in dendritic arborization, especially in the sensorimotor cortex, whereas rats with day 1 lesions showed a slight decrease in dendritic arbor. Similar frontal lesions in adulthood have previously been shown to produce a small increase in parietal, but not in visual, cortex [24,25]. The changes in dendritic arbor correlate well with the observed behavioral changes and it is reasonable to postulate that the dendritic changes reflect some mechanism underlying the presence of sparing from neonatal neocortical injury. There is little precedence for this claim as lesions are usually assumed to produced a decrease in dendritic complexity [17]. Steward and his colleagues have shown in the hippocampus that this decrease in dendritic complexity is followed by a compensatory increase, which is assumed to underlie recovery [38,39]. Furthermore, increased dendritic arborization is associated with other forms of cortical plasticity, such as in the cortical response to environmental enrichment or

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the acquisition of specific maze or motor tasks [15]. We have also found that sparing after neonatal hemidecortication in rats is also correlated with an increase in dendritic arborization [26]. One challenge in the interpretation of dendritic changes is that it is necessary to account for the contrasting effects of increased dendritic branching after day 10 and decreased dendritic branching after day 1 lesions. This is unlikely to result from some artifact of the trauma of having brain damage at the two ages since the two-stage animals showed different effects in the same hemispheres, yet the animals experienced the trauma at both ages. One explanation for the differential effects at the two ages is that it is directly related to the very different developmental states of the brain on the first and tenth day of life. The dendritic trees have barely begun to develop at the former age whereas by day 10 the principal branches have already been formed [18,36]. It is also curious that we have found that the dendritic response is slow to develop after day 10 lesions: it is not present on day 22 but is present by day 60 [25]. Another explanation for the day 1 / d a y 10 difference may be related to the development of astrocytes. Prior to about day 5 there are few cortical astrocytes as the principal time of astrocyte growth is roughly postnatal day 7-15 [33]. Thus, it may be that the brain's response to damage at day 10, but not at day 1, includes trophic factors released by astrocytes. The problem then becomes one of why the astrocytes should have a greater effect upon dendritic growth after lesions at 10 days than after lesions later in life. Perhaps the trophic effects of the astrocytes are more beneficial for dendritic growth during the period of maximal dendritic growth (day 10) than later in development. A second challenge for the dendritic studies is to determine what the changes in dendritic arborization reflect functionally. There are undoubtedly changes in cortical connectivity in the day 10 brains, especially in the intrinsic cortical connections that synapse upon the available dendritic space. These changes remain to be shown, however. In addition, there are likely to be other physiological a n d / o r biochemical measures that will be found to covary with the changes in dendritic branching. Nonetheless, we believe that increases in cortical neuropil may help support functional sparing. 4.2. Cortical connectiL, ity Examination of the pattern of cortical connectivity to both subcortical and cortical structures revealed that although neonatal frontal lesions led to significant alterations in cortical connectivity, these changes were most extensive in animals with frontal injuries on the day of birth. Thus, rats with day 1 lesions had abnormal thalamo-cortical, amygdalo-cortical, cortico-cortical, and dopaminergic connections whereas similar ab-

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normalities were not found in the day 10 operates. Furthermore, like rats with neonatal hemidecortications [26], rats with day 1 frontal lesions also showed an enhanced contralateral prefrontal-striatal connection that originated from the more superficial layers of the cortex. Several points are germane here. First, it seems unlikely that the brains of the day 1 frontal operates grew all of these connections in response to the injuries. Indeed, early neonatal tracer injections revealed that exactly these exuberant projections are present in normal unoperated neonates [6] Thus, it seems likely that the thalamocortical and dopaminergic connections normally present in the developing brain will later retract. The very early frontal lesions may interfere with this normal process of retraction, leaving what appear to be anomalous connections. The loss of normal retraction could be due to the reduced competition for synaptic space after the frontal decortication, or from some other reason. It is worth emphasizing here that for the most part these exuberant connections did not represent the presence of large novel projections such as reported following neonatal tectal lesions in hamsters [11,37] or corticospinal system lesions in rats [4]. For the most part the abnormal connections were found in regions that receive projections in infancy and the connections were characterized by the presence of a relatively small number labeled cells. Both these observations would seem to be consistent with the hypothesis that the 'abnormal' connections in adulthood resulted from the maintenance of exuberant projections present during development. Second, the demonstration of the most extensive anomalous thalamo-cortical and dopaminergic connections in the day 1 rats, who show greater behavioral loss than do adult animals with similar lesions, implies that these abnormal connections may interfere with normal behavioral function. Thus, rather than being an advantage to the animal, the presence of these abnormal connections could actually be a disadvantage. For example, the presence of abnormal thalamo-cortical connections into the somatosensory cortex could interfere with normal somatosensory function. Since the rat relies heavily upon vibrissal information this could be disruptive, although this remains to be proven. Third, the failure to find significant qualitative changes in subcortical-cortical connectivity of the rats with lesions at day 10 suggests that the spared behavioral capacities of these animals is not likely to result from qualitative changes in forebrain connectivity. On the other hand, our data do not speak to local changes in cortical connectivity, nor do they address the possibility of quantitative changes in normal connectivity, either of which could still contribute to the behavioral sparing.

Our findings of significant abnormalities in parietal connectivity following very early prefrontal ablation has little precedence in the literature. In one of the few previous studies Goldman [13] reported an abnormal crossed striatal projection from prefrontal cortex after a unilateral neonatal prefrontal lesion in monkeys. Our studies confirm this observation in rats. We know of no previous evidence of altered cortical dopaminergic projections, although de Brabander [2,3] found an increase in the concentration of dopamine in the sensorimotor cortex adjacent to neonatal prefrontal lesions in rats. In view of our tracing studies it is reasonable to suppose that the increased dopamine reflects the abnormal nigral a n d / o r tegmental projections in the early operates. Finally, our tracing data shed light on the finding that MD does not degenerate after neonatal lesions in either rats [21,30,34] or monkeys [14]. Hence, we found labeled cells in MD after injections of True Blue into either the striatum or the parietal cortex adjacent to the lesion. Thus, it seems likely that MD survives neonatal prefrontal decortication because of remaining sustaining projections to the striatum and the parietal cortex. The former connections are seen in normal adult and neonatal brains as well, whereas the latter ones may be retained exuberant neonatal projections.

4.3. Neonatal injections The data from the neonatal injections are generally consistent with previous studies indicating that the general topography of thalamo-cortical connectivity is attained early in development and that the neonatal projection is somewhat more divergent than that of the adult [7]. Similarly, the clear reduction in callosal transport with age is consistent with a substantial literature suggesting that there is a major retraction of callosal fibers, possibly in the second week of the rat's life [16,35]. An important technical limitation of studies of this type concerns the duration of time that the tracer is available for uptake. The clear reduction in the extent of transport in the day 10 vs. day 4 cases suggests that the dye injected on day 4 was not available on day 10, which is consistent with previous studies [10]. Thus, the changes in connectivity we have observed during development are not an artifact of prolonged tracer availability.

Acknowledgements This research was supported by a Natural Science and Engineering Research Council of Canada award to B.K., a Medical Research Council of Canada award to D.v.d.K., and National Centre for Excellence grants to B.K. and D.v.d.K.

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