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Bram Research, 568 (1991) 24-34 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50
BRES 17270
Neuroanatomical correlates of sparing of function after neonatal medial prefrontal cortex lesions in rats J.M. de Brabander, C.G. van Eden and J.P.C. de Bruin Netherlands lnstuute for Brain Research, Amsterdam (Netherlands)
(Accepted 6 August 1991) Key words. Frontal cortex; Recovery; Brmn damage; Mediodorsal nucleus of the thalamus; Doparmne; Rat
In rats, the possibility of neuroanatomical changes in response to partial medial prefrontal cortex lesions at postnatal day 6, concomitant with behavioural sparing, was investigated. The projections from the mediodorsal nucleus of the thalamus (MD) and the mesocortlcal dopaminergic (DA) projection were examined. No indications were found for a changed pattern of projection from MD in response to either a neonatal or an adult medial prefrontal cortex (mPFC) lesion. However, the DA innervatlon was changed after neonatal mPFC lesions. In the remainder of the mPFC, the DA fibre network proved to be denser, fibres were thicker, had more varicosities, and often the background staining was higher. None of these phenomena were seen m operated adult rats or in controls. It ~s postulated that the changes in DA innervation might contribute to the sparing of function observed in the spatial delayed alternation task. INTRODUCTION The concept that the immature central nervous system possesses a greater ability to compensate for loss of function after brain damage compared with the adult brain is generally known and often referred to as the 'Kennard principle'. Several investigators have reported that after neonatal damage of the medial prefrontal cortex (mPFC) animals showed sparing of function in the performance of food hoarding (rat4°), spatial delayed alternation beh a v i o u r (rat6'27"29'31-33'4°), and delayed responding or alternation (monkey2'12'14's3). In contrast, damaging the mPFC in adult animals resulted in a permanent impairment of these behavioural tasks 3'6'13'15'26'29'3°'4°. An intriguing question is which neural systems are involved in recovery of function. Many hypotheses have been proposed to explain behavioural recovery after neonatal lesions on the basis of neural reorganisation. Several of the proposed mechanisms imply reactive synaptogenesis, rerouting of axons, collateral sprouting and the consolidation of otherwise transient fibres 8'12A8'33'35' 36,38,39,42,43. All these mechanisms would lead to a changed projection pattern in combination with functional recovery at the behavioural level. Our earlier studies have demonstrated clear sparing of spatial delayed alternation behaviour after neonatal mPFC lesions 6. The present study focuses on possible changes in two systems which project to the mPFC and which might underlie behavioural sparing: the projection
from the mediodorsal nucleus of the thalamus (MD) and the dopaminergic (DA) projection from the ventral tegmental area (VTA). Both systems have a dense and specific innervation pattern within the prefrontal cortical areas 17'22'34'37'55'56 and both systems are crucial to the functioning of the prefrontal cortex. In the rat, the PFC is defined as the cortical projection area of the MD. Damaging this thalamic nucleus in adulthood leads to impairment of PFC-related tasks 3'24' 50 Additionally, the MD is a possible candidate mediating the neural reorganisation after PFC damage, since the retrograde degeneration in this nucleus frequently observed after mPFC lesions in adult rats could not be detected after mPFC lesions in neonatal rats t2'29'4°. This might indicate that the immature MD fibres that were severed by the lesions are capable of making new contacts in an undamaged part of the brain, and thus contribute to the survival of the MD cells and possibly to the observed sparing of function. Another candidate for mediating neural reorganisation after damage is the dense D A innervation of the PFC, which forms part of the mesocorticolimbic pathway. The cells of origin are situated in the VTA and project also to limbic structures, such as the nucleus accumbens. Several studies have supported a close functional relationship of this D A pathway with the PFC: lesions of this pathway in adult monkeys and rats resulted in a diminished spatial delayed alternation behaviour while in adult rats food hoarding performance was also
Correspondence. J.M. de Brabander, Netherlands Institute for Brmn Research, Melbergdreef 33, 1105 AZ Amsterdam, The Netherlands.
25 d e c r e a s e d 4'5'21'23'34'46'47'49'52,
i.e.
behavioural
impair-
m e n t s similar to t h o s e resulting f r o m m P F C lesions in adult animals. T h e s e o b s e r v a t i o n s and t h e fact that treatm e n t w i t h D A agonists a f t e r d a m a g e to the n e o c o r t e x in adult rats facilitates b e h a v i o u r a l r e c o v e r y 11'16'19'25'51 m a k e it w o r t h w h i l e to i n v e s t i g a t e t h e possible i n v o l v e m e n t o f this D A p a t h w a y in f u n c t i o n a l sparing a f t e r n e o natal m P F C lesions.
MATERIALS AND METHODS
Animals All rats studied (n = 66) were males of the albino Wistar strain (CPB-WU). Two groups were distinguished: the first was obtained from Harlan-TNO (Zeist, The Netherlands) and arrived at our institute at the age of 7 weeks (average body weight 190 g). The rats of the second group were born at our institute from timed pregnant females also obtained from Harlan TNO. At birth these rats were cross-fostered and weaned between PN20 and PN22 (the day of birth is postnatal day 0, PNO). The rats were housed in macroIon cages (75 x 55 x 23 era) in groups of 4 and kept at a 12 h light-dark cycle (lights on 15.30-03.30 h); the average temperature was between 22 °C and 24 *C and humidity between 50% and 60%. Just prior to and during the period of behavioural testing, food availability was reduced to achieve a body weight of approximately 85% of that in an ad libitum feeding situation. After completion of behavioural testing, food was again available ad libitum (standard rodent pellets, Hope Farms, Woerden, The Netherlands).
Surgery Neonatal lesions. Neonatal lesions were inflicted at the age of 6 days. The animals were randomly divided into an experimental group (n = 13), a sham-operated group (n = 12) and a control group (n = 8). Under ether anaesthesia, the skull of the experimental and sham-operated groups was opened and the anterior sinus sagittalis superior ligatured. In the experimental rats, the mPFC was subsequently bilaterally ablated by aspiration (for a detailed description of the surgleai procedures, see De Brabander et al.6). From every litter rats were allocated to all 3 groups and after weanmg every cage contained animals of every group. In the behav~oural and anatomical results, the sham-operated and control did not differ from each other, thus, we have regarded them as one group, to be referred to as the controls. Young adult lesions. In this group surgery was performed at the age of 8 weeks. The rats were randomly divided into two groups: an experimental group (n = 19) and a sham-operated group (the controls; n = 14). The surgical procedures were essentially the same as in the neonatal groups, except that the adult rats were anaesthetised with Hypoorm (0.1 ml/100 g body weight i.m.; Janssen Pharmaceutic.a, Beerse, Belgium). As with the rats treated neonatally, each cage contained animals of both conditions.
Behavtoural testing At the age of 15 weeks, all rats were subjected to the spatial delayed alternation test. A T-maze was used consisting of a startbox plus main alley (65 cm) and two side arms of 40 cm (the apparatus is described in detail in Stare et al.47). In this test rats must alternate between the left and right side arms to obtain a reward (a Noyes food pellet, 45 nag). Testing was preceded by a period of adaptation and autoshaping. During the fast 10 testing days 16 trials per day were performed at intertrial intervals of 0 s (ITI 0). Testing was continued for another 10 days of 16 trials per day with 15 s intertnal intervals (ITI 15) (procedures described in detail in De Brabander et al.6). Spatial delayed alternation tests were conducted during the dark penod (between 08.30 and 15.00 h). Animals were considered to have reached criterion with more than 80%
correct responses for at least 3 consecutive days. The non-parametric Chi-Square (g2) test4S was used to analyse the number of rats which had reached criterion on the last testing days at ITI 0 and ITI 15.
Anatomy Tracer injections and immunocytochemical staining. Following T-maze testing, the rats were operated again for the tracer experiment. The anterograde tracer, Phaseohis Vulgaris Leucoagglutinin 1° [PHA-L, 2.5% dissolved in 0.01 M phosphate buffer (pH 7.6); Vector laboratories, U.S.A.] was injected iontophoretically into the MD of the fight hemisphere, using the following coordinates, with Bregma as reference point: AP -2.5 ram, L +1.7 nun, V -5.4 ram, under an angle of + 12". The tip diameter of the glass pipette was 50/tm and an alternating 6 mA DC current was applied for 30 min (5 s on, 5 s off). After a survival period of 7 days, the animals were intracardially perfnsed under deep pentobarbital anaesthesia (1 ml/kg body weight, i.p.) with saline followed by fixative. The fixation consisted of 5% glutaraldehyde in 0.05 M acetate buffer (pH 4.0). After perfusion the brains were removed from the skull and postfixed for 30 rain. Then the brains were immersed in 0.05 M Tris-buffered saline (TBS) containing 1% NazS20 s (pH 7.2). Using a vibroslice, coronal serial sections (50 /~m) were gathered for PHA-L, D A and Nissl staining. The PHA-L and Nissl-sections were collected in 0.05 M TBS and the D A sections in TBS containing 1% NazS20 5. Alternating sections were used for immunocytoehemieal staining for PHA-L and DA. All antibodies were raised in our institute and the specificity of the staining for PHA-L and D A has been demonstrated previously9'2°. The PHA-L and DA sections were incubated using the peroxidase-antiperoxidase (PAP) method *s, with 0.3% and 0.5% ammonium nickel sulphate, respectively, to enhance the reaction with diaminobenzidine (DAB). For the Nissl staining, the sections were first mounted on glass slides and dried, then rinsed in a solution of ethanol and chloroform (1:1) to remove the hpids, thus brightening the staining with 0.5% thionine. The DA innervation of control animals and animals with neonatal and adult lesions was investigated at 3 levels, corresponding with +3.0, +2.5 and +2.0 mm anterior of bregma of the Zilles atlas 59. Together, these levels cover the extent of the lesions of most of the animals and they are easily distinguishable in the sections. RESULTS
Morphology of the lesion site M a c r o s c o p i c a l l y t h e r e was a r e m a r k a b l e d i f f e r e n c e in t h e a p p e a r a n c e o f t h e lesion b e t w e e n n e o n a t a l a n d adult e x p e r i m e n t a l rats. M o s t o f t h e lesions inflicted n e o n a tally s h o w e d n o o b v i o u s i n d i c a t i o n o f a lesion at t h e o u t e r cortical surface o f t h e b r a i n , e x c e p t for a scar, b u t all lesions in adult rats w e r e visible as a cavity ( m o r p h o l ogy d e s c r i b e d in detail in D e B r a b a n d e r et al.6). In pilot studies ( u n p u b l i s h e d o b s e r v a t i o n s ) , w e f o u n d that after a survival t i m e o f up to o n e w e e k , n e o n a t a l and adult lesions a p p e a r e d to b e essentially similar. H o w e v e r , after a survival t i m e o f 5 - 8 m o n t h s ( p r e s e n t study), in 11 o u t o f 13 rats with n e o n a t a l lesions n o gross modification o f the b r a i n was visible any l o n g e r , w h e r e a s o n l y two s h o w e d a cavity in t h e c e r e b r u m in r e s p o n s e to t h e n e o n a t a l lesion.
Neonatal lesion. A l t h o u g h m a c r o s c o p i c a l l y m o s t n e o natal lesions w e r e n o t visible as a cavity, m i c r o s c o p i c a l l y the l o c a t i o n o f the lesion was m a r k e d by t h e p r e s e n c e o f
26
Fig. 1. Photomicrographs of coronal Nissl-stained sections of the brains of rats with a representative adult (a) me&al prefrontal cortex (mPFC) lesion and neonatal (PN6) mPFC lesion (b). Note the cawty in the cerebrum m case of the adult lesion. Location and extent of the lesion in the neonatally operated animal are only visible by the scar (indicated by arrows) and the cytoarchitectonical abnormalities.
27 A DULT
NEONATAL
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Fig. 2. Reconstructions of the smallest (hatched) and the largest (dotted) adult (left panel) and neonatal (right panel) mPFC lesions41 of the animals used for behavioural analysis. Both groups had comparable lesions: the mPFC subareas frontal area 2 (Fr2) and dorsal anterior eingulate area (ACd) were either completely removed or severely damaged.
glial scar tissue. Parallel to the scar, cortical cells formed a thin homogeneous band with only cortical layer I being clearly distinguishable. Fig. l b shows a photomicrograph of a representative section of the frontal cortex of a rat with a neonatal lesion. Ventral and dorsal of the scar cytoarchitectonic criteria 34'54'57'59 could be applied
NEONA TAL IT115
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Fig. 3. Cumulative percentage of animals, which had reached the criterion of more than 80% correct responses in the spatial delayed alternation (SDA) tests during at least 3 consecutive days with intertrial intervals (ITI) of 0 and 15 s.
to identify remaining cortical areas. For instance, the presence of a dysgranular cortical layer IV, dorsolaterally of the scar tissue, indicated that this cortical area was the frontal area 1 (Frl) and, therefore, implied that the frontal area 2 (Fr2) had been removed. Ventral of the scar tissue, the cortical area was identified by the pronounced differences between the dorsal anterior cingulate area (ACd) and the prelimbic area (PL), such as, the difference in thickness of the layers II/III, the degree of extension of layers V and VI and the morphology of layer VI. Thus, lesion site and location were reconstructed and it appeared that in all rats with neonatal lesions the subareas Fr2 and A C d were almost completely removed; in two animals P L and infralimbic area (IL) were also damaged. Young adult lesion. In the adult experimental group (n = 19), lesions were clearly visible as cavities lined by glial tissue (Fig. la). The frontal areas that were not re-
28 a Control
/ /--I
b. Neonatal les=on
Fig. 4. The injection site in mediodorsal nucleus of the thalamus (MD) and distribution of the transported label in the PFC m a control animal (a) and an animal with a neonatal lesion (b). The Phaseolns Vulgaris Leucoaggluunin (PHA-L) spot m the control animal is located m the laterocentrai segment of MD (extension from Bregma -2.5 to -4.0). Labelled fibres are present in Fr2, ACd and prellmbic area (PL) and the lateral PFC, as expected on the basis of the topograplucal relation of MD projections to the PFC. The PHA-L spot in the animal with a neonatal lesion includes all 3 segments of MD (extension from Bregma -2.5 to -3.3): a larger injection site overlapping the one in the control animal. Labelled fibres are located in the rostral part of ACd and in the caudal half of Fr2, which are not ablated, PL and in both subareas of the lateral PFC. The distribution within these PFC areas is as expected on the basis of the topographical relation of MD-PFC projections.
moved showed no changes in cytoarchitecture and could be identified as described above. The lesions involved the two dorsal m P F C subareas, Fr2 and A C d , and in 13 rats also PL, IL, or parts of the forceps minor and caudate putamen.
Observation of sparing of function All rats were tested in the T-maze to assess their ability to learn to alternate between the arms, first with a 0 s intertrial interval (ITI 0; 10 sessions), then with a delay of 15 s between trials (ITI 15; 10 sessions). In order to compare the performance of rats with neonatal and adult lesions, only those animals in which the damage was restricted to the two dorsal m P F C subareas Fr2 and A C d were used for behavioural analysis (n = 11 and n = 6 for the rats with neonatal and adult lesions, respectively; see Fig. 2 for reconstructions of the smallest and the largest lesions). On the last ITI 0 testing day, the criterion (more than 80% correct responses on 3 consecutive testing days) was met by 100% (n = 20) of the neonatal control rats and 79% (11 out of 14) of the adult controls (Fig. 3). After
lengthening the ITI to 15 s the number of control animals, which responded during the first 3 days with more than 80% correct responses was strongly reduced to about 35%. But on testing day 6 again more than 50% of the controls of both the neonatally and adult-operated rats reached criterion, and on the last testing day 95% (19 out of 20) of the neonatal and 93% (13 out of 14) of the adult controls had reached criterion. The 11 rats with neonatal lesions performed as well as their controls. This was obvious from the number of rats per testing day that had reached criterion: both at ITI 0 and at ITI 15 the percentage was similar to the control animals (Fig. 3). In contrast, the 6 rats with adult lesions performed poorly during the ITI 0 and ITI 15 testing days. At ITI 0 only two (33%) animals managed to reach criterion on the last testing day. However, this difference just failed to be significant. At ITI 15 only one rat with an adult lesion (17%) reached criterion and this differed significantly (Z2, P ~ 0.001) from the adult controis. The other 5 animals with adult lesions, which did not reach the criterion, performed with 60-70% correct responses during the last 5 ITI 15 sessions.
29
Neuroanatomical correlates of sparing off'unction In order to investigate possible neuroanatomical changes in MD and DA projection pattern in response to the lesions, a combination of an anterograde tracer
technique and immunocytochemical staining was used for all rats, following spatial delayed alternation testing. 1. Projection from the mediodorsal nucleus
Fig. 5. Photomicrograph of the dopaminergic (DA) innervation of PL at a standard level corresponding with Bregma + 2.5 mm of the Zilles atlas sg. The boxed areas a, b and c indicate the location of the photomicrographs a, b and c in Fig. 6. P, pial surface; W, white matter. Bar = 0.2 mm.
CytoarchitectonicaUy, no signs of gross retrograde alterations were observed in the MD in response to either neonatal or adult mPFC lesions. The subnuclei of MD could be discerned using the cytoarchitectonic criteria of Groenewegen ~7 and Krettek and Price 34. The projection of the MD to the PFC was studied by iontophoretical injections of the anterograde tracer Phaseolus Vulgaris Leucoagglutinin (PHA-L) aimed at the MD of the fight hemisphere. After a survival period of 7 days the distribution of labelled fibres in PFC and adjacent frontal areas was examined.
CONTROL
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Fig. 6. Photomicrographs of the D A innervation of the PL regions (portrayed in Fig. 5) of a control rat, a rat with an adult mPFC lesion and a rat with a neonatal mPFC lesion: (a) superficial layers I, II and III; (b) layer V; (c) basal layer V and layer VI. Bar ffi 0.05 ram.
30
Fig. 7. Photormcrograph of DA fibres at the ventral border of the neonatal lesion site. Note the lugh density of DA fibres, which run parallel to the scar, the thickness of the fibres, the number of varicosities and the difference in background staining between PL and Frl. Bar = 0.05 mm.
Eight control rats with successful injections, i.e. with the P H A - L spot restricted to MD, were analysed. These injections resulted in labelled fibres in layers I, III, V, and VI of the PFC. Fibres in layers V and VI had occasional branches and varicosities, while in layer III and in upper layer V the fibres terminated in an extensive fibre plexus, consisting of numerous fine axons, which branched profusely and contained many varicosities. The injections with P H A - L usually did not fill the entire MD, but the injections of all animals taken together covered the entire MD. Most of the injections were confined to the lateral and central segments of either the rostral half of the MD (from Bregma -1.8 to -3.3; n = 3) or the caudal half (Bregma -2.5 to -4.0; n = 3). In two rats the injection was confined to the medial and central segments of MD (Bregma -1.8 to -3.3). When the spots included the lateral and central segments, labelled fibres were primarily found in Fr2 and ACd of the mPFC and in both dorsal and ventral agranular insular areas (Aid and AIv, respectively) of the lateral PFC. When the injection site involved the caudal part of the central MD, labelled fibres were also observed in PL (Fig. 4). P H A - L injections in the medial and central segments gave rise to labelled fibres in PL and IL (mPFC) and in the agranular insular PFC, However, in two rats with P H A - L spots restricted to the dorsal part of mediocentral MD no labelled fibres were observed in Aid. These P H A - L injections into MD of control rats confirmed the topography of MD projection within the PFC previously described by Groenewegen 17, Krettek and Price 34 and Van Eden 55. Besides these projections to the PFC subareas some injections also labeled a few fibres in the striatum, both dorsally and ventrally. No clear topographical pattern
of MD projection within the striatum could be detected. In 6 rats with neonatal mPFC lesions, P H A - L injections were restricted to MD. The injection sites involved the lateral, central, and medial segments (between Bregma -1.8 to -3.3, n = 3 and Bregma -2.5 to -4.0, n = 3). In none of these cases labelled fibres were found in areas where they were not expected on the basis of the topography of MD projections (see description of MD-PFC projection in controls). Within the undamaged PFC areas no changes were observed in the topographical relationship between PFC subareas and MD subnuclei, nor were changes observed in the density of labelled MD fibres within the PFC. Although, in this group also some injections labeled fibres in the striatum, no clear differences were found in comparison with the control group. In Fig. 4 the results of overlapping MD injections are juxtaposed in a control rat (a) and in a rat with a neonatal lesion (b). In the rat with a neonatal lesion, the large PHA-L injection included the middle part of the centrolateral MD segments, encroaching upon the medial segment. Labelled fibres were found in the lateral PFC and in those parts of the mPFC that were not damaged. None of these termination plexuses were particularly dense in comparison with the projections in controis, nor were fibres observed outside the PFC. In 9 rats with adult lesions, P H A - L injections were confined to the MD. The injections involved lateral, medial and central segments of MD (Bregma -1.8 to -4.0). In none of these rats, projections outside the original MD projection areas (see above) were observed, nor were there changes in the MD projection pattern within the striatum and the remaining parts of the PFC.
2. The dopaminergic innervation of the prefrontal cortex After mPFC lesions inflicted either neonatally or in adulthood, sections at the level of the VTA revealed no detectable loss of D A cells in that area. In 20 control rats coronal sections of the frontal cortex were immunocytochemically stained for DA, thus visualising the pattern of distribution and morphology of D A fibres in the PFC. D A fibres were seen in all subareas of medial and lateral PFC. Within the mPFC, the density of fibres was considerably higher in the basal cortical layers (VI and V) than in the more superficial layers III, II and I. In addition, regional differences in fibre density were seen within the mPFC, corresponding with the cytoarchitectonic subareas PL, ACd and Fr2. The density of D A fibres was highest in PL, in both basal and superficial cortical layers. ACd contained fewer fibres in the superficial layers compared with PL, whereas the density in the basal layers was similar. Of the 3 subareas, the lowest fibre density was found in Fr2, in all of its cortical layers. In this subarea, fibres were primarily
31 limited to layer VI and the lower part of layer V. Furthermore, the D A fibres had a specific course within different conical layers: the fibres in layer VI ran parallel to the pial surface and in layer V, III and II perpendicular to the pial surface. In layer I, where a few fibres were found, they ran parallel to the medial cerebral surface in a ventrodorsal direction. In the 9 rats with lesions inflicted in adulthood the distribution and morphology of D A fibres within the remaining parts of the PFC was similar to that of the DA fibres in control animals. Figs. 5 and 6 show photomicrographs of the prefrontal area PL. When the density of the DA fibres of controls and animals with adult lesions was compared in the superficial layers, in layer V and in the basal layer VI (Fig. 6), no differences were noticeable, except for the tissue in the direct vicinity of the lesion site. Here fewer D A fibres were observed in animals with adult lesions compared with the controls. However, when a lesion was inflicted at PN6, there was a remarkable change in D A innervation in the remaining mPFC, PL and IL (Fig. 6). In all of the 8 animals with neonatal lesions, in which the D A innervation was investigated, an increase in the density of D A fibres was observed in all conical layers of the remaining PL and IL. The dopaminergic response was highest at a distance of 2.5 mm anterior of bregma, with a lower response at more anterior (+3.0) and/or posterior (+2.0) levels. The increased density was accompanied by an increased branching of fibres. The fibres contained more varicosities than observed in control animals. In many animals the diameter of both intervaricose segments and varicosities was increased. The course of the DA fibres in the PFC had not changed, except for the fibres adjacent to the scar. At the ventral border of the lesion site an unusually high density of D A fibres was observed running perpendicular to the pial surface, i.e. parallel to the scar. These fibres were also very thick and had numerous varicosities (Fig. 7). In many animals the high background staining of the mPFC was also remarkable, whereas the staining of the background in the frontal areas in the same section dorsolateral to the lesion was comparable to that in control animals. In one rat an extension of the supragenual D A system was observed. The fine fibres of this D A system extended rostral of the genu of the corpus callosum. These fibres terminated and ramified in layers II and III, and appeared thinner than in the basal layers. Further examination of other parts of the brain revealed that the D A input might also be increased in the lateral part of the PFC and in other brain areas, such as the nucleus accumbens, MD, septum, and other cortical areas. However, the increased innervation was by far the most conspicuous in the mPFC.
DISCUSSION
Sparing of function In the present study it was established that rats with bilateral mPFC lesion inflicted at postnatal day 6 showed sparing of spatial delayed alternation behaviour, whereas rats with a similar adult mPFC lesion were impaired in the performance of this learning task. This behavioural sparing after neonatal lesions was accompanied by an increased D A immunoreactivity in the remaining mPFC. Other investigators observed sparing of function in learning tasks after neonatal PFC lesions in rats and monkeys. In rats, it was shown that lesions of the mPFC inflicted between PN6 and PN24 resulted in clear sparing in learning tasks, whereas lesions after PN24 had an impairing effect on the performance of these tasks 6'29'31' 32,40. In monkeys the age of 12 months seems to be criticalTM. With respect to other PFC-related tasks, e.g. species-typical tasks, less is known about the critical period in which sparing of function occurs. Results described by Nonneman and Corwin40 showed sparing of food hoarding performance with mPFC lesions inflicted at PN9, but we could not confirm this with similar lesions at PN66.
Neuroanatomical correlates of sparing of function Many neuroanatomical changes have been described in response to neonatal cortical lesions s'27'31'32. Following neonatal medial frontal cortical lesions, Kolb and coworkers27'31'32 reported a decrease in thickness and number of neurons in several parts of the cortex, along with an increased dendritic arborisation. In addition, retrograde tracers injected in the visual and somatosensory cortex revealed projections from some thalamic nuclei, amygdala and catecholaminergic nuclei (substantia nigra and VTA), which are normally not seen in adulthood. However, these abnormal connections were not accompanied by functional sparing 2s (B. Kolb, personal communication). The present study was focused on two systems known to be crucial for PFC functioning: the projection from MD to the PFC and the mesocortical DA system, originating in the VTA 3'5'21'23'24'46'47'49'50'52. We used anterograde tracing and immunocytochemical methods to reveal possible changes in these afferent systems resulting from neonatal mPFC lesions. They also gave us an opportunity to search for subtle differences in the projection pattern within the remaining PFC areas.
1. Innervation from the mediodorsal nucleus of the thalamus The projection from MD to the PFC subareas was examined using the anterograde tracer Phaseolus Vulgaris
32 Leucoagglutinin (PHA-L). In control rats the topographical projection from MD to the PFC subareas extensively described by Groenewegen 17, Krettek and P r i c e 34 and Van Eden s5 was confirmed. In rats with neither neonatal nor adult lesions, P H A - L injections in MD showed a projection different from that expected on the basis of the MD projection pattern in control animals. In none of the rats of the lesion groups MD fibres were observed in other cortical or subcortical areas normally devoid of this input. Also within the PFC areas, the MD projections showed no changes in density or in their laminar distribution. We also paid attention to the projection to the striaturn, because the striatum is a component of a functional system, together with MD and PFC 33'5s and therefore was suggested as a candidate for taking over the PFC function 13'14'27'53. However, although the injections analysed were confined to MD, we can not exclude that some of the P H A - L was taken up by cells of the intralaminar nuclei, since cells of intralaminar areas, especially the central lateral nucleus of the thalamus, intermingle with MD cells. Therefore, according to Berendse and Groenewegen 1, it is possible that these few projections originate in intralaminar nuclei and not in MD. Whatever the precise origin of these thalamostriatal projections may be, no changes in these projections between animals with lesions of either age and controls were observed. Among several possible mechanisms that could account for sparing, Goldman and Galkin 12 and Kolb and Nonneman 29 considered the possibility that MD could be the neuroanatomical substrate of sparing of function following early PFC lesions. This hypothesis was based on the observation that there was no sign of degenerated cells in the thalamus after neonatal lesions, whereas after an adult lesion retrograde degeneration in MD was observed. These authors postulated that these cells, which had not degenerated, might have established new connections with other areas in response to the lesion and thus had compensated for loss of PFC function. Although the number of rats with mPFC lesions and tracer injections in MD was not sufficient for a systematic analysis of MD projections, tracer injections within each of the groups did cover the entire MD. Since none of these injections gave rise to a projection pattern which was not described previously 17'34's5 it is unlikely, that sprouting or rerouting of MD projections had occurred, and therefore making it also unlikely that a change in this system would be responsible for the observed sparing of function.
2. The dopaminergic innervation of the prefrontal cortex The main finding of our study is that the D A innervation in response to a neonatal mPFC lesion is in-
creased compared with the innervation in control rats. In the entire part of the non-ablated mPFC areas (mainly PL and IL), more and thicker fibres with more varicosities were observed often along with an increased background staining. A more dense innervation of the remaining prefrontal area (PL and IL) could be the result of a thinning of the cortical thickness (see e.g. Kolb and Whishawa2). Such a difference in cortical thickness of PL and IL does not explain the thickening of the fibres, the more and larger varicosities and the higher background. These morphological changes indicate changes in the D A system, in response to neonatal but not to adult lesions. These immunocytochemical changes can not be elusive with regard to the state of activity of the system. Thickening of fibres and varicosities can be an indication of a higher activity as well as of an accumulation of presynaptic DA. Biochemical methods are needed for further determining the state of activity of the D A system. The importance of the D A system in PFC functioning appears from studies in monkeys 4 and rats T M in which the D A innervation of the PFC was depleted by 6-hydroxydopamine. The DA-depleted animals showed an impaired performance of PFC-related behaviour. Also electrophysiological studies indicate that D A plays an important role by facilitating ~ask-specific neuronal activity in the PFC 44. Additionally, there is evidence that the D A system could play a role in behavioural sparing: amphetamine, which leads to an increased extracellular D A concentration, and apomorphine, which acts as a postsynaptic D A agonist, can both improve recovery after neocortex lesions in adulthood x1'16"19'25'51. Furthermore, neonatal lesions of VTA cells result in a lasting impairment of PFC function zx, indicating that when this mesolimbocortical DA-system is absent no sparing occurs. Therefore, the presently reported response of the D A system in the PFC after neonatal lesions, could very well be causally related to concomitant behavioural sparing. This mechanism can act most efficiently in an area functionally related to the ablated areas (Fr2 and ACd): the infralimbic and prelimbic areas of the mPFC. However, further studies are necessary to prove that the D A activity is increased in response to neonatal mPFC lesions and that it is of functional significance for behavioural sparing.
Acknowledgements.We thank Elhs Jansen, Nora Geerts and Annet Louwerse for their assistance in behavioural testing, Eileen van Vulpen and Ria Matthijssen for their histological assistance We also want to thank Wendy van Noppen and Dinie Kok-Noorman for hngmstlc and other corrections, Henk Stoffels for drawing the figures and Gerben van der Meulen for preparing the photographs. The comments of Harry Uyhngs and Dick Swaab are gratefully acknowledged.
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Brain Research, 142 (1978) 1-24. 38 Loesche, J. and Stewart, O., Behavioral correlates of denervation and reinnervation of the hippocampal formation of the rat: recovery of alternaaon performance following unilateral entorhinal cortex lesions, Brain Res. Bull., 2 (1977) 31-39. 39 Lynch, G., Deadwyler, S. and Cotman, C., Postlesion axonal growth produces permanent functional connections, Saence, 180 (1973) 1364-1366. 40 Nonneman, A.J. and Corwm, J.V., Differential effects of prefrontal cortex ablation in neonatal, juvenile and young adult rats, J. Comp. Physwl. Psychol., 95 (1981) 588-602. 41 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982 42 Ralsman, G., Neuronal plasticity in the septal nuclei of the adult rat, Brain Research, 14 (1969) 25-48. 43 Ramirez, J.J. and Stein, D.G., Sparing and recovery of spatial alternation performance after entorhinal cortex lesions in rats, Behav. Brain Res., 13 (1984) 53-61. 44 Sawaguchl, T., Matsumura, M. and Kubota, K., Catecholanunergic effects on neuronal activity related to a delayed response task in monkey prefrontal cortex, J. Neurophysiol., 63 (1990) 1385-1400. 45 Siegel, S. and Castellan, N.J., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1988, 399 pp. 46 Simon, H., Scatton, B. and Le Moal, M., Dopaminergic A10 neurons are involved in cognitive functions, Nature, 286 (1980) 150-151. 47 Stare, C.J., De Bruin, J.EC., Van Haelst, A.M., Van der Gugten, J. and Kalsbeek, A., Influence of the mesocortical dopaminergic system on activity, food hoarding, social-agonistic behavior, and spatial delayed alternation in male rats, Behav. Neurosci., 103 (1989) 24-35. 48 Sternberger, L.A., lmmunocytochemistry, Prentice-Hall, Englewood Cliffs, 1979, 246 pp. 49 Stinus, L., Gaffori, O., Simon, H. and Le Moal, M., Disappearance of hoarding and disorganization of eating behavior after ventral mesencephalic tegmentum lesion in rats, J. Comp. Physiol. Psychol., 92 (1978) 289-296.
50 Stokes, K.A. and Best, P.J., Response biases do not underly the radial maze deficit in rats with mediodorsal thalamus lesions, Behav. Biol., 53 (1990) 334-345. 51 Sutton, R.L., Hovda, D.A. and Feeney, D.N., Amphetanune accelerates recovery of locomotor function following bilateral frontal cortex ablation in cats, Behav. Neurosci., 103 (1989) 837-841. 52 Taghzouti, K., Louflot, A., Herman, J.P., Le Moal, M. and S1mon, H., Alternation behavior, spatial discrimination, and reversal disturbances following 6-hydroxydopamine lesions in the nucleus accumbens of the rat, Behav Biol., 44 (1985) 354-363. 53 Tucker, T.J. and Kling, A., Differential effects of early and late lesions of frontal granular cortex in the monkey, Brain Research, 5 (1967) 377-389. 54 Uylings, H.B.M. and Van Eden, C.G., Qualitative and quantitative comparison of the prefrontal cortices m rat and in primates, including humans. In H.B.M. Uylings, C.G. Van Eden, J.EC. De Bruin, M.A. Corner and M.G.P. Feenstra (Eds.), The Prefrontal Cortex: Its Structure, Function and Pathology, Progress In Brain Research, Vol. 85, Elsevier, Amsterdam, 1990, pp. 31-62. 55 Van Eden, C.G., Development of thalamortical connectaons between the mediodorsal nucleus and the prefrontal cortex in the rat, J. Comp. Neurol., 244 (1986) 349-359. 56 Van Eden, C.G., Hoorneman, E.M.D., Buys, R.M., Matthljssen, M.A.H., Geffard, M. and Uylings, H.B.M., Immunocytochemical localization of doparmne m the prefrontal cortex of the rat at the light and electron microscopical level, Neuroscience, 22 (1987) 849-862. 57 Van Eden, C.G. and Uylings, H.B.M., Cytoarchitectomcal development of the prefrontal cortex in the rat, J Comp. Neurol., 241 (1985) 253-267. 58 Vlcedomim, J.P., Corwm, J.V. and Nonneman, A.J., Behavioral effects of lesions to the caudate nucleus or mediodorsal thalamus m neonatal, juvenile, and adult rats, Physzol. Psychol., 10 (1982) 246-250. 59 ZiUes, K., The Cortex of the Rat, A Stereotaxlc Atlas, SpringerVerlag, Berlin, 1985, 121 pp.
Bram Research, 568 (1991) 24-34 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50
BRES 17270
Neuroanatomical correlates of sparing of function after neonatal medial prefrontal cortex lesions in rats J.M. de Brabander, C.G. van Eden and J.P.C. de Bruin Netherlands lnstuute for Brain Research, Amsterdam (Netherlands)
(Accepted 6 August 1991) Key words. Frontal cortex; Recovery; Brmn damage; Mediodorsal nucleus of the thalamus; Doparmne; Rat
In rats, the possibility of neuroanatomical changes in response to partial medial prefrontal cortex lesions at postnatal day 6, concomitant with behavioural sparing, was investigated. The projections from the mediodorsal nucleus of the thalamus (MD) and the mesocortlcal dopaminergic (DA) projection were examined. No indications were found for a changed pattern of projection from MD in response to either a neonatal or an adult medial prefrontal cortex (mPFC) lesion. However, the DA innervatlon was changed after neonatal mPFC lesions. In the remainder of the mPFC, the DA fibre network proved to be denser, fibres were thicker, had more varicosities, and often the background staining was higher. None of these phenomena were seen m operated adult rats or in controls. It ~s postulated that the changes in DA innervation might contribute to the sparing of function observed in the spatial delayed alternation task. INTRODUCTION The concept that the immature central nervous system possesses a greater ability to compensate for loss of function after brain damage compared with the adult brain is generally known and often referred to as the 'Kennard principle'. Several investigators have reported that after neonatal damage of the medial prefrontal cortex (mPFC) animals showed sparing of function in the performance of food hoarding (rat4°), spatial delayed alternation beh a v i o u r (rat6'27"29'31-33'4°), and delayed responding or alternation (monkey2'12'14's3). In contrast, damaging the mPFC in adult animals resulted in a permanent impairment of these behavioural tasks 3'6'13'15'26'29'3°'4°. An intriguing question is which neural systems are involved in recovery of function. Many hypotheses have been proposed to explain behavioural recovery after neonatal lesions on the basis of neural reorganisation. Several of the proposed mechanisms imply reactive synaptogenesis, rerouting of axons, collateral sprouting and the consolidation of otherwise transient fibres 8'12A8'33'35' 36,38,39,42,43. All these mechanisms would lead to a changed projection pattern in combination with functional recovery at the behavioural level. Our earlier studies have demonstrated clear sparing of spatial delayed alternation behaviour after neonatal mPFC lesions 6. The present study focuses on possible changes in two systems which project to the mPFC and which might underlie behavioural sparing: the projection
from the mediodorsal nucleus of the thalamus (MD) and the dopaminergic (DA) projection from the ventral tegmental area (VTA). Both systems have a dense and specific innervation pattern within the prefrontal cortical areas 17'22'34'37'55'56 and both systems are crucial to the functioning of the prefrontal cortex. In the rat, the PFC is defined as the cortical projection area of the MD. Damaging this thalamic nucleus in adulthood leads to impairment of PFC-related tasks 3'24' 50 Additionally, the MD is a possible candidate mediating the neural reorganisation after PFC damage, since the retrograde degeneration in this nucleus frequently observed after mPFC lesions in adult rats could not be detected after mPFC lesions in neonatal rats t2'29'4°. This might indicate that the immature MD fibres that were severed by the lesions are capable of making new contacts in an undamaged part of the brain, and thus contribute to the survival of the MD cells and possibly to the observed sparing of function. Another candidate for mediating neural reorganisation after damage is the dense D A innervation of the PFC, which forms part of the mesocorticolimbic pathway. The cells of origin are situated in the VTA and project also to limbic structures, such as the nucleus accumbens. Several studies have supported a close functional relationship of this D A pathway with the PFC: lesions of this pathway in adult monkeys and rats resulted in a diminished spatial delayed alternation behaviour while in adult rats food hoarding performance was also
Correspondence. J.M. de Brabander, Netherlands Institute for Brmn Research, Melbergdreef 33, 1105 AZ Amsterdam, The Netherlands.
25 d e c r e a s e d 4'5'21'23'34'46'47'49'52,
i.e.
behavioural
impair-
m e n t s similar to t h o s e resulting f r o m m P F C lesions in adult animals. T h e s e o b s e r v a t i o n s and t h e fact that treatm e n t w i t h D A agonists a f t e r d a m a g e to the n e o c o r t e x in adult rats facilitates b e h a v i o u r a l r e c o v e r y 11'16'19'25'51 m a k e it w o r t h w h i l e to i n v e s t i g a t e t h e possible i n v o l v e m e n t o f this D A p a t h w a y in f u n c t i o n a l sparing a f t e r n e o natal m P F C lesions.
MATERIALS AND METHODS
Animals All rats studied (n = 66) were males of the albino Wistar strain (CPB-WU). Two groups were distinguished: the first was obtained from Harlan-TNO (Zeist, The Netherlands) and arrived at our institute at the age of 7 weeks (average body weight 190 g). The rats of the second group were born at our institute from timed pregnant females also obtained from Harlan TNO. At birth these rats were cross-fostered and weaned between PN20 and PN22 (the day of birth is postnatal day 0, PNO). The rats were housed in macroIon cages (75 x 55 x 23 era) in groups of 4 and kept at a 12 h light-dark cycle (lights on 15.30-03.30 h); the average temperature was between 22 °C and 24 *C and humidity between 50% and 60%. Just prior to and during the period of behavioural testing, food availability was reduced to achieve a body weight of approximately 85% of that in an ad libitum feeding situation. After completion of behavioural testing, food was again available ad libitum (standard rodent pellets, Hope Farms, Woerden, The Netherlands).
Surgery Neonatal lesions. Neonatal lesions were inflicted at the age of 6 days. The animals were randomly divided into an experimental group (n = 13), a sham-operated group (n = 12) and a control group (n = 8). Under ether anaesthesia, the skull of the experimental and sham-operated groups was opened and the anterior sinus sagittalis superior ligatured. In the experimental rats, the mPFC was subsequently bilaterally ablated by aspiration (for a detailed description of the surgleai procedures, see De Brabander et al.6). From every litter rats were allocated to all 3 groups and after weanmg every cage contained animals of every group. In the behav~oural and anatomical results, the sham-operated and control did not differ from each other, thus, we have regarded them as one group, to be referred to as the controls. Young adult lesions. In this group surgery was performed at the age of 8 weeks. The rats were randomly divided into two groups: an experimental group (n = 19) and a sham-operated group (the controls; n = 14). The surgical procedures were essentially the same as in the neonatal groups, except that the adult rats were anaesthetised with Hypoorm (0.1 ml/100 g body weight i.m.; Janssen Pharmaceutic.a, Beerse, Belgium). As with the rats treated neonatally, each cage contained animals of both conditions.
Behavtoural testing At the age of 15 weeks, all rats were subjected to the spatial delayed alternation test. A T-maze was used consisting of a startbox plus main alley (65 cm) and two side arms of 40 cm (the apparatus is described in detail in Stare et al.47). In this test rats must alternate between the left and right side arms to obtain a reward (a Noyes food pellet, 45 nag). Testing was preceded by a period of adaptation and autoshaping. During the fast 10 testing days 16 trials per day were performed at intertrial intervals of 0 s (ITI 0). Testing was continued for another 10 days of 16 trials per day with 15 s intertnal intervals (ITI 15) (procedures described in detail in De Brabander et al.6). Spatial delayed alternation tests were conducted during the dark penod (between 08.30 and 15.00 h). Animals were considered to have reached criterion with more than 80%
correct responses for at least 3 consecutive days. The non-parametric Chi-Square (g2) test4S was used to analyse the number of rats which had reached criterion on the last testing days at ITI 0 and ITI 15.
Anatomy Tracer injections and immunocytochemical staining. Following T-maze testing, the rats were operated again for the tracer experiment. The anterograde tracer, Phaseohis Vulgaris Leucoagglutinin 1° [PHA-L, 2.5% dissolved in 0.01 M phosphate buffer (pH 7.6); Vector laboratories, U.S.A.] was injected iontophoretically into the MD of the fight hemisphere, using the following coordinates, with Bregma as reference point: AP -2.5 ram, L +1.7 nun, V -5.4 ram, under an angle of + 12". The tip diameter of the glass pipette was 50/tm and an alternating 6 mA DC current was applied for 30 min (5 s on, 5 s off). After a survival period of 7 days, the animals were intracardially perfnsed under deep pentobarbital anaesthesia (1 ml/kg body weight, i.p.) with saline followed by fixative. The fixation consisted of 5% glutaraldehyde in 0.05 M acetate buffer (pH 4.0). After perfusion the brains were removed from the skull and postfixed for 30 rain. Then the brains were immersed in 0.05 M Tris-buffered saline (TBS) containing 1% NazS20 s (pH 7.2). Using a vibroslice, coronal serial sections (50 /~m) were gathered for PHA-L, D A and Nissl staining. The PHA-L and Nissl-sections were collected in 0.05 M TBS and the D A sections in TBS containing 1% NazS20 5. Alternating sections were used for immunocytoehemieal staining for PHA-L and DA. All antibodies were raised in our institute and the specificity of the staining for PHA-L and D A has been demonstrated previously9'2°. The PHA-L and DA sections were incubated using the peroxidase-antiperoxidase (PAP) method *s, with 0.3% and 0.5% ammonium nickel sulphate, respectively, to enhance the reaction with diaminobenzidine (DAB). For the Nissl staining, the sections were first mounted on glass slides and dried, then rinsed in a solution of ethanol and chloroform (1:1) to remove the hpids, thus brightening the staining with 0.5% thionine. The DA innervation of control animals and animals with neonatal and adult lesions was investigated at 3 levels, corresponding with +3.0, +2.5 and +2.0 mm anterior of bregma of the Zilles atlas 59. Together, these levels cover the extent of the lesions of most of the animals and they are easily distinguishable in the sections. RESULTS
Morphology of the lesion site M a c r o s c o p i c a l l y t h e r e was a r e m a r k a b l e d i f f e r e n c e in t h e a p p e a r a n c e o f t h e lesion b e t w e e n n e o n a t a l a n d adult e x p e r i m e n t a l rats. M o s t o f t h e lesions inflicted n e o n a tally s h o w e d n o o b v i o u s i n d i c a t i o n o f a lesion at t h e o u t e r cortical surface o f t h e b r a i n , e x c e p t for a scar, b u t all lesions in adult rats w e r e visible as a cavity ( m o r p h o l ogy d e s c r i b e d in detail in D e B r a b a n d e r et al.6). In pilot studies ( u n p u b l i s h e d o b s e r v a t i o n s ) , w e f o u n d that after a survival t i m e o f up to o n e w e e k , n e o n a t a l and adult lesions a p p e a r e d to b e essentially similar. H o w e v e r , after a survival t i m e o f 5 - 8 m o n t h s ( p r e s e n t study), in 11 o u t o f 13 rats with n e o n a t a l lesions n o gross modification o f the b r a i n was visible any l o n g e r , w h e r e a s o n l y two s h o w e d a cavity in t h e c e r e b r u m in r e s p o n s e to t h e n e o n a t a l lesion.
Neonatal lesion. A l t h o u g h m a c r o s c o p i c a l l y m o s t n e o natal lesions w e r e n o t visible as a cavity, m i c r o s c o p i c a l l y the l o c a t i o n o f the lesion was m a r k e d by t h e p r e s e n c e o f
26
Fig. 1. Photomicrographs of coronal Nissl-stained sections of the brains of rats with a representative adult (a) me&al prefrontal cortex (mPFC) lesion and neonatal (PN6) mPFC lesion (b). Note the cawty in the cerebrum m case of the adult lesion. Location and extent of the lesion in the neonatally operated animal are only visible by the scar (indicated by arrows) and the cytoarchitectonical abnormalities.
27 A DULT
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Fig. 2. Reconstructions of the smallest (hatched) and the largest (dotted) adult (left panel) and neonatal (right panel) mPFC lesions41 of the animals used for behavioural analysis. Both groups had comparable lesions: the mPFC subareas frontal area 2 (Fr2) and dorsal anterior eingulate area (ACd) were either completely removed or severely damaged.
glial scar tissue. Parallel to the scar, cortical cells formed a thin homogeneous band with only cortical layer I being clearly distinguishable. Fig. l b shows a photomicrograph of a representative section of the frontal cortex of a rat with a neonatal lesion. Ventral and dorsal of the scar cytoarchitectonic criteria 34'54'57'59 could be applied
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Fig. 3. Cumulative percentage of animals, which had reached the criterion of more than 80% correct responses in the spatial delayed alternation (SDA) tests during at least 3 consecutive days with intertrial intervals (ITI) of 0 and 15 s.
to identify remaining cortical areas. For instance, the presence of a dysgranular cortical layer IV, dorsolaterally of the scar tissue, indicated that this cortical area was the frontal area 1 (Frl) and, therefore, implied that the frontal area 2 (Fr2) had been removed. Ventral of the scar tissue, the cortical area was identified by the pronounced differences between the dorsal anterior cingulate area (ACd) and the prelimbic area (PL), such as, the difference in thickness of the layers II/III, the degree of extension of layers V and VI and the morphology of layer VI. Thus, lesion site and location were reconstructed and it appeared that in all rats with neonatal lesions the subareas Fr2 and A C d were almost completely removed; in two animals P L and infralimbic area (IL) were also damaged. Young adult lesion. In the adult experimental group (n = 19), lesions were clearly visible as cavities lined by glial tissue (Fig. la). The frontal areas that were not re-
28 a Control
/ /--I
b. Neonatal les=on
Fig. 4. The injection site in mediodorsal nucleus of the thalamus (MD) and distribution of the transported label in the PFC m a control animal (a) and an animal with a neonatal lesion (b). The Phaseolns Vulgaris Leucoaggluunin (PHA-L) spot m the control animal is located m the laterocentrai segment of MD (extension from Bregma -2.5 to -4.0). Labelled fibres are present in Fr2, ACd and prellmbic area (PL) and the lateral PFC, as expected on the basis of the topograplucal relation of MD projections to the PFC. The PHA-L spot in the animal with a neonatal lesion includes all 3 segments of MD (extension from Bregma -2.5 to -3.3): a larger injection site overlapping the one in the control animal. Labelled fibres are located in the rostral part of ACd and in the caudal half of Fr2, which are not ablated, PL and in both subareas of the lateral PFC. The distribution within these PFC areas is as expected on the basis of the topographical relation of MD-PFC projections.
moved showed no changes in cytoarchitecture and could be identified as described above. The lesions involved the two dorsal m P F C subareas, Fr2 and A C d , and in 13 rats also PL, IL, or parts of the forceps minor and caudate putamen.
Observation of sparing of function All rats were tested in the T-maze to assess their ability to learn to alternate between the arms, first with a 0 s intertrial interval (ITI 0; 10 sessions), then with a delay of 15 s between trials (ITI 15; 10 sessions). In order to compare the performance of rats with neonatal and adult lesions, only those animals in which the damage was restricted to the two dorsal m P F C subareas Fr2 and A C d were used for behavioural analysis (n = 11 and n = 6 for the rats with neonatal and adult lesions, respectively; see Fig. 2 for reconstructions of the smallest and the largest lesions). On the last ITI 0 testing day, the criterion (more than 80% correct responses on 3 consecutive testing days) was met by 100% (n = 20) of the neonatal control rats and 79% (11 out of 14) of the adult controls (Fig. 3). After
lengthening the ITI to 15 s the number of control animals, which responded during the first 3 days with more than 80% correct responses was strongly reduced to about 35%. But on testing day 6 again more than 50% of the controls of both the neonatally and adult-operated rats reached criterion, and on the last testing day 95% (19 out of 20) of the neonatal and 93% (13 out of 14) of the adult controls had reached criterion. The 11 rats with neonatal lesions performed as well as their controls. This was obvious from the number of rats per testing day that had reached criterion: both at ITI 0 and at ITI 15 the percentage was similar to the control animals (Fig. 3). In contrast, the 6 rats with adult lesions performed poorly during the ITI 0 and ITI 15 testing days. At ITI 0 only two (33%) animals managed to reach criterion on the last testing day. However, this difference just failed to be significant. At ITI 15 only one rat with an adult lesion (17%) reached criterion and this differed significantly (Z2, P ~ 0.001) from the adult controis. The other 5 animals with adult lesions, which did not reach the criterion, performed with 60-70% correct responses during the last 5 ITI 15 sessions.
29
Neuroanatomical correlates of sparing off'unction In order to investigate possible neuroanatomical changes in MD and DA projection pattern in response to the lesions, a combination of an anterograde tracer
technique and immunocytochemical staining was used for all rats, following spatial delayed alternation testing. 1. Projection from the mediodorsal nucleus
Fig. 5. Photomicrograph of the dopaminergic (DA) innervation of PL at a standard level corresponding with Bregma + 2.5 mm of the Zilles atlas sg. The boxed areas a, b and c indicate the location of the photomicrographs a, b and c in Fig. 6. P, pial surface; W, white matter. Bar = 0.2 mm.
CytoarchitectonicaUy, no signs of gross retrograde alterations were observed in the MD in response to either neonatal or adult mPFC lesions. The subnuclei of MD could be discerned using the cytoarchitectonic criteria of Groenewegen ~7 and Krettek and Price 34. The projection of the MD to the PFC was studied by iontophoretical injections of the anterograde tracer Phaseolus Vulgaris Leucoagglutinin (PHA-L) aimed at the MD of the fight hemisphere. After a survival period of 7 days the distribution of labelled fibres in PFC and adjacent frontal areas was examined.
CONTROL
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Fig. 6. Photomicrographs of the D A innervation of the PL regions (portrayed in Fig. 5) of a control rat, a rat with an adult mPFC lesion and a rat with a neonatal mPFC lesion: (a) superficial layers I, II and III; (b) layer V; (c) basal layer V and layer VI. Bar ffi 0.05 ram.
30
Fig. 7. Photormcrograph of DA fibres at the ventral border of the neonatal lesion site. Note the lugh density of DA fibres, which run parallel to the scar, the thickness of the fibres, the number of varicosities and the difference in background staining between PL and Frl. Bar = 0.05 mm.
Eight control rats with successful injections, i.e. with the P H A - L spot restricted to MD, were analysed. These injections resulted in labelled fibres in layers I, III, V, and VI of the PFC. Fibres in layers V and VI had occasional branches and varicosities, while in layer III and in upper layer V the fibres terminated in an extensive fibre plexus, consisting of numerous fine axons, which branched profusely and contained many varicosities. The injections with P H A - L usually did not fill the entire MD, but the injections of all animals taken together covered the entire MD. Most of the injections were confined to the lateral and central segments of either the rostral half of the MD (from Bregma -1.8 to -3.3; n = 3) or the caudal half (Bregma -2.5 to -4.0; n = 3). In two rats the injection was confined to the medial and central segments of MD (Bregma -1.8 to -3.3). When the spots included the lateral and central segments, labelled fibres were primarily found in Fr2 and ACd of the mPFC and in both dorsal and ventral agranular insular areas (Aid and AIv, respectively) of the lateral PFC. When the injection site involved the caudal part of the central MD, labelled fibres were also observed in PL (Fig. 4). P H A - L injections in the medial and central segments gave rise to labelled fibres in PL and IL (mPFC) and in the agranular insular PFC, However, in two rats with P H A - L spots restricted to the dorsal part of mediocentral MD no labelled fibres were observed in Aid. These P H A - L injections into MD of control rats confirmed the topography of MD projection within the PFC previously described by Groenewegen 17, Krettek and Price 34 and Van Eden 55. Besides these projections to the PFC subareas some injections also labeled a few fibres in the striatum, both dorsally and ventrally. No clear topographical pattern
of MD projection within the striatum could be detected. In 6 rats with neonatal mPFC lesions, P H A - L injections were restricted to MD. The injection sites involved the lateral, central, and medial segments (between Bregma -1.8 to -3.3, n = 3 and Bregma -2.5 to -4.0, n = 3). In none of these cases labelled fibres were found in areas where they were not expected on the basis of the topography of MD projections (see description of MD-PFC projection in controls). Within the undamaged PFC areas no changes were observed in the topographical relationship between PFC subareas and MD subnuclei, nor were changes observed in the density of labelled MD fibres within the PFC. Although, in this group also some injections labeled fibres in the striatum, no clear differences were found in comparison with the control group. In Fig. 4 the results of overlapping MD injections are juxtaposed in a control rat (a) and in a rat with a neonatal lesion (b). In the rat with a neonatal lesion, the large PHA-L injection included the middle part of the centrolateral MD segments, encroaching upon the medial segment. Labelled fibres were found in the lateral PFC and in those parts of the mPFC that were not damaged. None of these termination plexuses were particularly dense in comparison with the projections in controis, nor were fibres observed outside the PFC. In 9 rats with adult lesions, P H A - L injections were confined to the MD. The injections involved lateral, medial and central segments of MD (Bregma -1.8 to -4.0). In none of these rats, projections outside the original MD projection areas (see above) were observed, nor were there changes in the MD projection pattern within the striatum and the remaining parts of the PFC.
2. The dopaminergic innervation of the prefrontal cortex After mPFC lesions inflicted either neonatally or in adulthood, sections at the level of the VTA revealed no detectable loss of D A cells in that area. In 20 control rats coronal sections of the frontal cortex were immunocytochemically stained for DA, thus visualising the pattern of distribution and morphology of D A fibres in the PFC. D A fibres were seen in all subareas of medial and lateral PFC. Within the mPFC, the density of fibres was considerably higher in the basal cortical layers (VI and V) than in the more superficial layers III, II and I. In addition, regional differences in fibre density were seen within the mPFC, corresponding with the cytoarchitectonic subareas PL, ACd and Fr2. The density of D A fibres was highest in PL, in both basal and superficial cortical layers. ACd contained fewer fibres in the superficial layers compared with PL, whereas the density in the basal layers was similar. Of the 3 subareas, the lowest fibre density was found in Fr2, in all of its cortical layers. In this subarea, fibres were primarily
31 limited to layer VI and the lower part of layer V. Furthermore, the D A fibres had a specific course within different conical layers: the fibres in layer VI ran parallel to the pial surface and in layer V, III and II perpendicular to the pial surface. In layer I, where a few fibres were found, they ran parallel to the medial cerebral surface in a ventrodorsal direction. In the 9 rats with lesions inflicted in adulthood the distribution and morphology of D A fibres within the remaining parts of the PFC was similar to that of the DA fibres in control animals. Figs. 5 and 6 show photomicrographs of the prefrontal area PL. When the density of the DA fibres of controls and animals with adult lesions was compared in the superficial layers, in layer V and in the basal layer VI (Fig. 6), no differences were noticeable, except for the tissue in the direct vicinity of the lesion site. Here fewer D A fibres were observed in animals with adult lesions compared with the controls. However, when a lesion was inflicted at PN6, there was a remarkable change in D A innervation in the remaining mPFC, PL and IL (Fig. 6). In all of the 8 animals with neonatal lesions, in which the D A innervation was investigated, an increase in the density of D A fibres was observed in all conical layers of the remaining PL and IL. The dopaminergic response was highest at a distance of 2.5 mm anterior of bregma, with a lower response at more anterior (+3.0) and/or posterior (+2.0) levels. The increased density was accompanied by an increased branching of fibres. The fibres contained more varicosities than observed in control animals. In many animals the diameter of both intervaricose segments and varicosities was increased. The course of the DA fibres in the PFC had not changed, except for the fibres adjacent to the scar. At the ventral border of the lesion site an unusually high density of D A fibres was observed running perpendicular to the pial surface, i.e. parallel to the scar. These fibres were also very thick and had numerous varicosities (Fig. 7). In many animals the high background staining of the mPFC was also remarkable, whereas the staining of the background in the frontal areas in the same section dorsolateral to the lesion was comparable to that in control animals. In one rat an extension of the supragenual D A system was observed. The fine fibres of this D A system extended rostral of the genu of the corpus callosum. These fibres terminated and ramified in layers II and III, and appeared thinner than in the basal layers. Further examination of other parts of the brain revealed that the D A input might also be increased in the lateral part of the PFC and in other brain areas, such as the nucleus accumbens, MD, septum, and other cortical areas. However, the increased innervation was by far the most conspicuous in the mPFC.
DISCUSSION
Sparing of function In the present study it was established that rats with bilateral mPFC lesion inflicted at postnatal day 6 showed sparing of spatial delayed alternation behaviour, whereas rats with a similar adult mPFC lesion were impaired in the performance of this learning task. This behavioural sparing after neonatal lesions was accompanied by an increased D A immunoreactivity in the remaining mPFC. Other investigators observed sparing of function in learning tasks after neonatal PFC lesions in rats and monkeys. In rats, it was shown that lesions of the mPFC inflicted between PN6 and PN24 resulted in clear sparing in learning tasks, whereas lesions after PN24 had an impairing effect on the performance of these tasks 6'29'31' 32,40. In monkeys the age of 12 months seems to be criticalTM. With respect to other PFC-related tasks, e.g. species-typical tasks, less is known about the critical period in which sparing of function occurs. Results described by Nonneman and Corwin40 showed sparing of food hoarding performance with mPFC lesions inflicted at PN9, but we could not confirm this with similar lesions at PN66.
Neuroanatomical correlates of sparing of function Many neuroanatomical changes have been described in response to neonatal cortical lesions s'27'31'32. Following neonatal medial frontal cortical lesions, Kolb and coworkers27'31'32 reported a decrease in thickness and number of neurons in several parts of the cortex, along with an increased dendritic arborisation. In addition, retrograde tracers injected in the visual and somatosensory cortex revealed projections from some thalamic nuclei, amygdala and catecholaminergic nuclei (substantia nigra and VTA), which are normally not seen in adulthood. However, these abnormal connections were not accompanied by functional sparing 2s (B. Kolb, personal communication). The present study was focused on two systems known to be crucial for PFC functioning: the projection from MD to the PFC and the mesocortical DA system, originating in the VTA 3'5'21'23'24'46'47'49'50'52. We used anterograde tracing and immunocytochemical methods to reveal possible changes in these afferent systems resulting from neonatal mPFC lesions. They also gave us an opportunity to search for subtle differences in the projection pattern within the remaining PFC areas.
1. Innervation from the mediodorsal nucleus of the thalamus The projection from MD to the PFC subareas was examined using the anterograde tracer Phaseolus Vulgaris
32 Leucoagglutinin (PHA-L). In control rats the topographical projection from MD to the PFC subareas extensively described by Groenewegen 17, Krettek and P r i c e 34 and Van Eden s5 was confirmed. In rats with neither neonatal nor adult lesions, P H A - L injections in MD showed a projection different from that expected on the basis of the MD projection pattern in control animals. In none of the rats of the lesion groups MD fibres were observed in other cortical or subcortical areas normally devoid of this input. Also within the PFC areas, the MD projections showed no changes in density or in their laminar distribution. We also paid attention to the projection to the striaturn, because the striatum is a component of a functional system, together with MD and PFC 33'5s and therefore was suggested as a candidate for taking over the PFC function 13'14'27'53. However, although the injections analysed were confined to MD, we can not exclude that some of the P H A - L was taken up by cells of the intralaminar nuclei, since cells of intralaminar areas, especially the central lateral nucleus of the thalamus, intermingle with MD cells. Therefore, according to Berendse and Groenewegen 1, it is possible that these few projections originate in intralaminar nuclei and not in MD. Whatever the precise origin of these thalamostriatal projections may be, no changes in these projections between animals with lesions of either age and controls were observed. Among several possible mechanisms that could account for sparing, Goldman and Galkin 12 and Kolb and Nonneman 29 considered the possibility that MD could be the neuroanatomical substrate of sparing of function following early PFC lesions. This hypothesis was based on the observation that there was no sign of degenerated cells in the thalamus after neonatal lesions, whereas after an adult lesion retrograde degeneration in MD was observed. These authors postulated that these cells, which had not degenerated, might have established new connections with other areas in response to the lesion and thus had compensated for loss of PFC function. Although the number of rats with mPFC lesions and tracer injections in MD was not sufficient for a systematic analysis of MD projections, tracer injections within each of the groups did cover the entire MD. Since none of these injections gave rise to a projection pattern which was not described previously 17'34's5 it is unlikely, that sprouting or rerouting of MD projections had occurred, and therefore making it also unlikely that a change in this system would be responsible for the observed sparing of function.
2. The dopaminergic innervation of the prefrontal cortex The main finding of our study is that the D A innervation in response to a neonatal mPFC lesion is in-
creased compared with the innervation in control rats. In the entire part of the non-ablated mPFC areas (mainly PL and IL), more and thicker fibres with more varicosities were observed often along with an increased background staining. A more dense innervation of the remaining prefrontal area (PL and IL) could be the result of a thinning of the cortical thickness (see e.g. Kolb and Whishawa2). Such a difference in cortical thickness of PL and IL does not explain the thickening of the fibres, the more and larger varicosities and the higher background. These morphological changes indicate changes in the D A system, in response to neonatal but not to adult lesions. These immunocytochemical changes can not be elusive with regard to the state of activity of the system. Thickening of fibres and varicosities can be an indication of a higher activity as well as of an accumulation of presynaptic DA. Biochemical methods are needed for further determining the state of activity of the D A system. The importance of the D A system in PFC functioning appears from studies in monkeys 4 and rats T M in which the D A innervation of the PFC was depleted by 6-hydroxydopamine. The DA-depleted animals showed an impaired performance of PFC-related behaviour. Also electrophysiological studies indicate that D A plays an important role by facilitating ~ask-specific neuronal activity in the PFC 44. Additionally, there is evidence that the D A system could play a role in behavioural sparing: amphetamine, which leads to an increased extracellular D A concentration, and apomorphine, which acts as a postsynaptic D A agonist, can both improve recovery after neocortex lesions in adulthood x1'16"19'25'51. Furthermore, neonatal lesions of VTA cells result in a lasting impairment of PFC function zx, indicating that when this mesolimbocortical DA-system is absent no sparing occurs. Therefore, the presently reported response of the D A system in the PFC after neonatal lesions, could very well be causally related to concomitant behavioural sparing. This mechanism can act most efficiently in an area functionally related to the ablated areas (Fr2 and ACd): the infralimbic and prelimbic areas of the mPFC. However, further studies are necessary to prove that the D A activity is increased in response to neonatal mPFC lesions and that it is of functional significance for behavioural sparing.
Acknowledgements.We thank Elhs Jansen, Nora Geerts and Annet Louwerse for their assistance in behavioural testing, Eileen van Vulpen and Ria Matthijssen for their histological assistance We also want to thank Wendy van Noppen and Dinie Kok-Noorman for hngmstlc and other corrections, Henk Stoffels for drawing the figures and Gerben van der Meulen for preparing the photographs. The comments of Harry Uyhngs and Dick Swaab are gratefully acknowledged.
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