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Differences in D2 dopamine receptor binding in the neostriatum between cats hemidecorticated neonatally or in adulthood
Developmental Brain Research 107 Ž1998. 113–122
Research report
Differences in D 2 dopamine receptor binding in the neostriatum between cats hemidecorticated neonatally or in adulthood Louise D. Loopuijt a
a,1,)
, David A. Hovda b,d,e, Ali Ebrahim c , Jaime R. Villablanca Harry T. Chugani c,2
a,e
,
Departments of Psychiatry and BiobehaÕioral Sciences and of Neurobiology, UCLA School of Medicine, Los Angeles, USA b DiÕision of Neurosurgery, UCLA School of Medicine, Los Angeles, USA c Department of Pediatric Neurology and DiÕision of Nuclear Medicine, UCLA School of Medicine, Los Angeles, USA d Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, USA e Brain Research Institute, Los Angeles, USA Accepted 16 December 1997
Abstract In order to study differences in response to neocortical injury sustained at different ages at the neurotransmitter level, we examined the density in D 2 dopamine receptors in the neostriatum of cats hemidecorticated neonatally Ž N s 4. or in adulthood Ž N s 4., as well as in intact brains Ž N s 6.. Receptor densities were measured using quantitative autoradiography and w 3 Hx-spiperone binding in 12 regions of the neostriatum and nucleus accumbens septi. We found that the anterior lateral caudate nucleus on both sides of the brain contained a higher D 2 receptor density in neonatal-lesioned as compared to adult-lesioned brains. Ipsilateral to the lesion, the increase was 101% Ž P - 0.05. and contralaterally it amounted to 77% Ž P - 0.05.. Moreover, this region of the ipsilateral caudate nucleus of neonatal-lesioned cats tended to be more densely labeled than that of intact brain by 58% Ž P - 0.1.. D 2 receptor densities in adult-lesioned cats did not differ from that of intact controls. Comparison of these data with those of a former morphological study using the same animals suggested that this bilateral elevation of D 2 receptor density in neonatally lesioned brains represents a higher mean density of binding sites per neuron. The elevation in the neonatal-lesioned cats might be a response of the striatum to neuroplastic changes in the striatal neuropil, including the corticostriatal afferents, since such changes are different in neonatal- as compared to adult-lesioned cats. q 1998 Elsevier Science B.V. Keywords: D 2 dopamine receptor; Caudate-putamen; Age-at-lesion; Hemidecortication; Cat
1. Introduction Brain damage sustained at an early age results in more functional recovery than when the brain is damaged during adulthood w33,76x. Morphological studies of such animals with cortical ablation show that areas with secondary damage exhibit less degeneration of neurons and less gliosis than adult-lesioned brains w25,66,75x. For example,
) Corresponding author. Department of Neuropharmacology, Zoological Institute, University of Tubingen, Germany. Fax: q49-7071-29-87815; ¨ E-mail: [email protected] 1 Present address: Department of Neuropharmacology, Mediz. Naturw. Forsch. Zentr., University of Tubingen, 72074 Tubingen, Germany. ¨ ¨ 2 Present address: Pediatric NeurologyrPET Center, Children’s Hospital of Michigan, Wayne State University, Detroit, MI 48201, USA.
0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 0 0 4 - 2
in the cat caudate nucleus neuron loss and increase in glial cell density are more prominent in adult-lesioned than in neonatal-lesioned animals w37x. Moreover, anatomical reorganization and axonal sprouting is more extensive in neonatal- than adult-lesioned brains. For example, early lesioned cats were shown to have contralateral corticorubral w78x, corticothalamic w77x and corticospinal w22x projections, which were not found in intact animals or occurred to a lesser extent in adult-lesioned brains. Similarly, in rats and monkeys, the corticostriatal crossed projection, which is minor in intact animals w39,79x, was much more dense in brains in which the cortex was unilaterally ablated just after Žrat w32x. or before Žmonkey w21x. birth. Moreover, in a PET scan study, human infants were shown to have reduced glucose uptake Žmetabolic activity. in the caudate nucleus 6 months after hemidecortication, while 2 1r2 years after surgery, the glucose uptake had increased
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to normal levels w11,12x, suggesting reinnervation and normalization of glucose metabolic activity in the caudate at this time. Also in experimentally decorticated cats, metabolic activity was shown to recover, if the lesion was inflicted one week after birth w26x. In terms of neurotransmitters, not only the glutamatergic corticostriatal system is affected by hemidecortication, but also the dopaminergic system. Thus, in adult-hemidecorticated rats, the amount of striatal dopamine is decreased w53,68x, indicating a decrease of release of dopamine by nigrostriatal terminals. These data prompted us to investigate dopamine receptor densities in the caudate nucleus at long survival times in neonatal- and adult-hemidecorticated brains to verify, whether differences in anatomical reorganization might also be reflected in changes of striatal dopamine receptor densities. We measured D 2 dopamine receptor binding in neonatal- and adult-hemidecorticated brains of cats, using w 3 Hxspiperone, a ligand which binds almost exclusively to the D 2 receptor family. Since the distribution of dopamine receptors in the striatum is heterogeneous w4,28,36x, and since there is evidence that the response of the caudateputamen to an unilateral lesion of the substantia nigra pars compacta can vary for the various regions within the caudate-putamen w62x, we divided the caudate-putamen and the nucleus accumbens into 12 different regions and measured the density of w 3 Hx-spiperone binding in the presence of ketanserine in order to assess the density of binding sites for the D 2 dopamine receptor family in these nuclei.
and since the w 14 Cx2-deoxyglucose was injected in vivo, the frozen sections, which were to be used for w 3 Hxspiperone binding, contained w 14 Cx2-deoxyglucose label. To remove the w 14 Cx2-deoxyglucose-6-phosphate, the sections were preincubated for 20 min in phosphate buffered saline ŽPBS, pH 7.4. at room temperature, then incubated in 1 nM w 3 Hx-spiperone ŽNEN, specific radioactivity 32.1 Cirmmol. in buffer Ž0.17 M Tris–HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 . in the presence of 40 nM ketanserine ŽSigma. for 30 min at room temperature, rinsed in distilled water Ž2 = 5 s., then in ice-cold buffer Ž2 = 5 min. and blown dry with cold air. For non-specific binding, sections were incubated in 1 nM w 3 Hx-spiperone in buffer in the presence of 40 nM ketanserine and 1 m m Žq.butaclamol ŽSigma. for 30 min at room temperature. Sections were dried overnight at room temperature.
2. Materials and methods Four kittens between the age of 7 and 14 days had the neocortex of the left side removed following procedures described in Loopuijt et al. w37x, and survived into young adulthood. Four adult cats were similarly hemidecorticated and survived for at least 6 months after surgery Žmean survival time 358 days, range 195–586 days.. Six adult cats served as controls. All animals were used in experiments to measure cerebral glucose utilization with the w 14 Cx2-deoxyglucose method w69x. At the time of sacrifice Žmean age of intact controls 1925 days, range 278–5219; of adult-hemidecorticated, mean age 1052 days, range 900–1195; of neonatal-hemidecorticated, mean age 374 days, range 294–451., cats received an overdose of Nembutal Ž100 mgrkg., then the brain was quickly removed from the skull and immediately frozen in dry ice. All brains were cut on a cryostat microtome into coronal sections at 20 m m. The sections were mounted on coverslips and stored at y208C until further use. 2.1. Spiperone binding assay Part of the sections of the above-mentioned brains were used in experiments to measure local glucose utilization,
Fig. 1. Schematic representation of sampling sites for optical density measurements in the caudate nucleus, nucleus accumbens and putamen. Abbreviations and symbols: L s lateral; M s medial; ca s caudate nucleus; ps putamen; na s nucleus accumbens septi; I ssample area. V146 is the number of the cat and 88b, 84b and 77b indicate the numbers of the histological sections.
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2.2. Autoradiography The mounted sections were covered with w 3 Hx sensitive film ŽLKB, Sweden. and were developed after 6–8 weeks of exposure in the dark at 48C. All sheets of film also covered a plastic standard with 14 samples of different concentrations of w 3 Hx label ŽARC.. 2.3. Analysis of films Optical density measurements were performed with a high-voltage lightbox ŽAristo, Roslyn, NY. and a high-resolution video camera ŽCohu, Japan. connected to a computer with image analysis software ŽJandel Scientific, CA, USA.. Because of a mediolateral gradient of dopamine D 2 receptor density in the striatum in the cat w4x and rat w5,36x, the caudate nucleus was divided into 3 regions Žmedial, intermediate and lateral. along the dorsolateral–ventromedial axis and sampled according to the scheme illustrated in Fig. 1. In addition, the caudate nucleus was divided along the anterioposterior axis in three parts, anterior, middle and posterior, which each contained a similar amount of sections. The putamen was divided into 2 regions along its anteroposterior axis and, in addition, one
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sample was taken in the nucleus accumbens septi. Thus, the 12 different sample areas were taken in 10 different sections per animal, the sections being equally spaced along the anteroposterior axis. The mean values of these 10 measurements were used for further analysis Žsee Section 2.4.. Optical densities were converted to fmolrmg wet tissue with the aid of the standards, data from the literature w19,20x and a computer program ŽLIGAND w41x.. Values for non-specific binding were obtained by subtracting the values of total bound spiperone from that of sections that were incubated with w 3 Hx-spiperone with 40 nM ketanserine and 1 m m Žq.butaclamol. 2.4. Statistical analysis The internal distribution of label in the caudate nucleus was described by means of regression analysis w15x. Comparisons of the caudate nucleus between the three groups of experimental animals were done with a repeated measures analysis of variance ŽANOVA., which was conducted with the individual region serving as the repeated variable. Comparisons were made to determine the significance of the main effect Žhemidecortication. with simple main effect comparisons to intact values using appropriate contrasts w44x. The values for putamen and nucleus accum-
Fig. 2. Autoradiographs of w 3 Hx-spiperone binding. ŽA. Coronal section of intact brain through the caudate nucleus, putamen and nucleus accumbens septi. ŽB. Non-specific binding, i.e., binding of 1 nM w 3 Hx-spiperone and 40 nM ketanserine in the presence of 1 m m Žq.butaclamol. ŽC. Total binding Žw 3 Hx-spiperone plus 40 nm ketanserine. in adult-hemidecorticated brain. ŽD. Total binding of w 3 Hx-spiperone plus ketanserine in neonatal hemidecorticated brain. C and D also show the similar extent of the hemidecortication in the two age-at-lesion groups. Abbreviations; ca s caudate nucleus; p s putamen; na s nucleus accumbens septi. Bar s 9 mm.
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bens septi were analyzed with the non-parametric Mann– Whitney U-test.
3. Results
1r3–1r2 of the medial sylvian gyrus. Dorsally, there were cortical remnants only in the midline, rostral to the genu of the corpus callosum, including variable portions of the gyri rectus and cingulum. The caudate nucleus was intact in all brains, as well as the entire contralateral hemisphere.
3.1. Description of lesions 3.2. D2 dopamine receptor binding The lesions of these brains have been described in detail by Loopuijt et al. w37x. The nature and extent of the resection were similar, in all aspects, for the two age-at-lesion groups. In all animals, almost the entire left neocortex was removed ŽFig. 2C,D; see also Ref. w37x, Fig. 1, cats a160 and a334.. However, since the caudate nucleus was spared, portions of the neocortex lateral and ventral to the head of this nucleus were not removed, including the lower
w 3 Hx-spiperone binding at 1 nM resulted in a high amount of labeling in the caudate nucleus, putamen and nucleus accumbens septi, in all intact, as well as hemidecorticated cats ŽFig. 2A,C,D.. This labeling was very high as compared to the surrounding tissue, e.g., the cortex ŽFig. 2A,C,D.. Non-specific binding was always very low ŽFig. 2B..
Fig. 3. Specific binding of spiperone Žfmolrmg wet weight. in different regions of the caudate nucleus. Error bars represent standard errors of the mean ŽS.E.M... ` s Intact; I s adult hemidecorticated ŽAHDC.; ^ s neonatal-hemidecorticated ŽNHDC.. Note that there is a dorsolateral–ventromedial gradient in w 3 Hx-spiperone binding, which stretches from the anterior through the posterior end of the caudate nucleus. Note also, that in NHDC cats, the specific binding in the lateral region of the anterior caudate on both sides of the brain is significantly higher than in AHDC animals Ž P - 0.05., while there is a trend for the left lateral region of neonatal-hemidecorticated brains to be higher than that of the intact brains Ž P - 0.1.. Intact N s 6; AHDC: N s 4; NHDC: N s 4. Abbreviations: AHDCs adult hemidecorticated brains; NHDCs neonatal hemidecorticated brains. ) P - 0.05 NHDC compared to AHDC; qP - 0.1 NHDC compared to Intact.
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3.3. Intact cats Quantification of w 3 Hx-spiperone binding showed that the caudate nucleus in intact cats contained more labeling at its dorsolateral than ventromedial side ŽFig. 3., thereby displaying a gradient from dorsolateral to ventromedial. This gradient was present in the anterior, middle and posterior caudate nucleus and showed a significant linear relationship, taking into account that the distance between the medial and intermediate sample was identical to the distance between the intermediate and lateral sample ŽFig. 1.. The slope of the regression line of the left anterior caudate was 9.60 Ž r 2 s 0.96; FŽ1,4. s 99.14; P - 0.0005., for the left middle caudate 14.81 Ž r 2 s 0.99; FŽ1,4. s 230.37; P - 0.0005. and the left posterior caudate it was 8.78 Ž r 2 s 0.66; FŽ1,4. s 7.84; P - 0.05.. On the right side, the regression coefficients were, for anterior, middle and posterior caudate nucleus, respectively: 15.02 Ž r 2 s 0.99; FŽ1,4. s 481.91; P - 0.0005.; 17.07 Ž r 2 s 0.99; FŽ1,4. s 183.91; P - 0.0005. and 8.8 Ž r 2 s 0.97, FŽ1,4. s 153.11; P - 0.0005.. Within each of the lateral, intermediate and medial regions, in a rostrocaudal direction, there was no difference in the amount of spiperone binding between the anterior, middle and posterior caudate. 3.4. Adult and neonatal hemidecorticated animals The linear, dorsolateral–ventromedial gradient of spiperone binding described above was preserved in brains that had undergone adult or neonatal hemidecortication ŽFig. 3A,B.. In the anterior left caudate nucleus Žon the decorticated side., the regression slopes of adult-hemidecorticated ŽAHDC. and neonatal-hemidecorticated ŽNHDC. cats were, respectively: 6.73 Ž r 2 s 0.81; FŽ1,2. s 74.23; P - 0.05. and 30.58 Ž r 2 s 0.98; FŽ1,2. s 97.91; P - 0.05.. In the anterior left caudate, the three groups of experimental animals appeared to have significantly different relationships between their lateral, intermediate and medial values Ž P - 0.05, repeated measurements ANOVA.. This discrepancy was caused predominantly by the high mean values of the lateral region in NHDC brains, which showed a higher value than in AHDC by 101% Ž P - 0.05; post-hoc analysis. and tended to be higher than that of intact brains, by 58.2% Ž P - 0.1.. The middle and posterior caudate at the left side did not show significant changes in the distribution of label in NHDC as compared to AHDC or intact in any of the medio-lateral region ŽFig. 3A.. On the right side, the label in the anterior lateral region of the caudate nucleus of NHDC cats was significantly more dense than in AHDC cats by 77% Ž P - 0.05., but the apparent difference of 26% between NHDC and intact cats was not statistically significant ŽFig. 3B.. Also on the right side, the middle and posterior caudate nucleus of NHDC cats did not show differences in any of the three Žlateral, intermediate or medial. regions.
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Table 1 Specific binding of spiperone in the nucleus accumbens septi ŽN. accumb.. and putamen Žfmolrmg wet weight.. Intact N s6, adult-hemidecorticate ŽAHDC. N s 4, neonatal hemidecorticate ŽNHDC. N s 4. Left
Intact AHDC NHDC
N. accumb. mean SEM
Putamen anterior mean SEM
Putamen posterior mean SEM
21.69 22.48 22.85
34.85 39.98 47.74
41.64 54.89 34.68
3.04 6.28 0.83
5.79 10.70 7.97
9.93 17.31 4.97
Right
Intact AHDC NHDC
N. accumb. mean SEM
Putamen anterior mean SEM
Putamen posterior mean SEM
22.98 21.98 29.52
39.95 40.09 62.58
44.49 44.07 46.50
1.93 4.59 2.90
5.91 8.90 10.76
9.67 11.98 8.28
In case of the AHDC brains, the values in the lateral caudate nucleus of the anterior, middle and posterior regions were not different from that of the intact brains ŽFig. 3A,B.. Similarly, no such differences were observed in the intermediate and medial regions of the caudate nucleus. The concentration of D 2 dopamine receptor binding sites in the putamen of intact brains were similar to that of the lateral part of the anterior and middle caudate nucleus ŽTable 1.. However, there were no differences between NHDC, AHDC and intact brains in the anterior or posterior half of the putamen ŽTable 1, Mann–Whitney U-test.. Finally, the amount of binding in the nucleus accumbens septi of hemidecorticated cats was not changed on either side relative to intact controls.
4. Discussion The main finding of the present report is the local difference in D 2 receptor density between neonatal and adult cat brains after hemidecortication: after a minimum of 6 months survival time, the D 2 receptor density of the ipsilateral anterior lateral caudate nucleus is higher in NHDC than in AHDC cats, by 101% at the ipsilateral and 77% at the contralateral side. 4.1. Technical considerations These conclusions are based on autoradiographic measurements of in vitro incubations of w 3 Hx-spiperone at a concentration of 1 nM in the presence of ketanserine. The data of our study represent specific binding at 1 nM w 3 Hx-spiperone, which is obtained by subtracting total spiperone binding from non-specific binding Žincubations with spiperone, ketanserine and Žq.butaclamol Žsee Section 2.. These last measurements indicated that non-specific binding accounted for 15% of the total binding Ždata not shown., which is similar to that in other in vitro w 3 Hxspiperone incubations systems w5x. Thus it appears, that
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there was very little or no residual w 14 Cx2-deoxy-glucose6-phosphate left in our sections Žsee Section 2.. Our values, measured at 1 nM w 3 Hx-spiperone, do not represent an estimation of total available binding sites Ž Bmax ., but by comparison with data from the literature, these values can be estimated to be 15% lower than that of the Bmax . In addition, we did not correct for tritium absorption in the tissue. However, we kept our incubation and exposure conditions the same throughout all experiments, often incubating sections of the three animal groups at the same time, so that we are confident that comparisons within and between animals in this study are reliable. Comparing our quantitative data with those of other investigators, we found in the intermediate anterior caudate nucleus of intact brains, a binding of 35 fmolrmg wet tissue Žapproximately 350 fmolrmg protein. at 1 nM spiperone, which likely represents 85% of the Bmax . Richfield et al. w59x, also in the cat, found 500 fmolrmg protein Žprot. at 0.25 nM spiperone, which probably represents 35% of the Bmax . In rats, Bmax values for spiperone binding autoradiography vary between groups of investigators: 843 fmolrmg prot w5x, 860 fmolrmg prot w59x, and 458 " 51 fmolrmg prot w29x. Thus, our data are within the range of those reported by other investigators. All the brains that were used in this study were thoroughly examined using thionin-stained sections. Brains in which the caudate nucleus appeared to be damaged were not included. For more documentation on the extent of the lesion, see Loopuijt et al. w37x. 4.2. Response of D2 receptor density to hemidecortication Several groups of investigators have reported on the response of striatal D 2 receptors to unilateral cortical ablation in adult rats. Some performed complete hemidecortications, comparable to those of the present study, and reported a decrease in Bmax w43,54,60,64,71,73x. Others performed restricted lesions and found no difference between the striatum ipsilateral to the lesion and the striatum in control rats w29,74x. In contrast, in neonatally hemidecorticated brains, we observed a bilateral regional increase in receptor density. At the time that the hemidecortications were performed ŽP7–P14., all the afferents to the striatum have established their connections, although the neurons that have already established their contacts are still migrating w18x. In rats there is a sevenfold increase of D 2 receptor density in the striatum between postnatal days P7 and P49 w6–8,17,49,51x. Assuming that the maturity of the cat brain at P7 is equivalent that of approximately P18 in the rat brain w61x, the cat striatum is probably in a phase of rapid increase of D 2 receptor density at the time of surgery. w 3 Hx-spiperone binds to all subunits of the dopamine D 2 receptor family, comprising the D 2 , D 3 and D4 receptor proteins. These subtypes not only differ in molecular structure, but also in localization. In the striatum, D 2 receptors
can be found throughout w67x, while D 3 receptors are predominantly found in the ventral striatum, nucleus accumbens septi and islands of Calleja w70x. D4 receptor binding has been reported to be present w42,65x or absent w58x in the striatum. D4 mRNA could be revealed in the striatum by some authors w38x, but not by others w42x, and D4 receptor protein could be found in cell bodies of corticostriatal neurons in the cortex and also in the striatum, although in the latter, it was found in low amounts w2x. Finally, removal of the prefrontal cortex in adult rats resulted in a decrease in D4 receptor binding in the nucleus accumbens, but not a significant decrease in the striatum w72x. Taken together, these results might suggest the presence of D4 receptors on corticostriatal nerve endings. If so, one would expect a cortical ablation to result in a small decrease in w 3 Hx-spiperone binding in the striatum. In our AHDC animals, a small decrease in w 3 Hx-spiperone binding could have been present, although the values were not significantly different from the controls. Such a decrease would have been minor; therefore, the significant difference between NHDC and AHDC in the anterolateral striatum cannot be attributed to a decrease in w 3 Hx-spiperone binding in AHDC animals, but mainly to an increase in w 3 Hx-spiperone binding in NHDC cats. 4.3. Age-at-lesion effects: neuron density and neuron loss We reported morphological differences in the caudate nucleus between cats that have sustained cortex ablation as an adult or neonate w37x. In AHDC, there was a loss of 21.8% Ž P - 0.05. of the neurons, while in NHDC there was only a tendency for neuronal loss Ž17.9%, P - 0.1.. Neuronal cell loss in ADHC was accompanied by shrinkage of volume without change in neuronal cell density, while the caudate of NHDC cats did not show a change in volume, but did show a decrease in neuronal cell packing density w37x. Since we measured and expressed spiperone binding as D 2 receptor binding sites per mg wet weight, our values represent concentrations of binding sites. Such values can be compared to values for cell packing density, because they represent the concentration of cells. Since we did not find any changes in the density of neurons or spiperone D 2 receptor binding sites in AHDC, the average number of spiperone binding sites per neuron in these animals was not changed. We found a reduction in total number of neurons however, and this resulted in a shrinkage of the total caudate volume w37x. Therefore, in AHDC animals, there was a loss of neurons, while the surviving neurons maintained the same average D 2 receptor density as before the insult. In contrast, in NHDC animals, the cell packing density was decreased by 25.8% w37x. Therefore, an increase in D 2 receptor binding site concentration in the anterior lateral caudate and the lack of change in binding site concentration in the other regions of the caudate nucleus mean, that
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the average number of binding sites per neuron in the caudate of these animals was increased. Thus, it is reasonable to propose that if the neocortical resection is performed when the cats are young, i.e., at a time when the density of D 2 receptors is still increasing w6–8,49,51x, such densities can still adapt to changes in the structure of the brain. We would like to suggest that this process might be related to the presence of higher levels of the neurotrophin TGFalpha. In fact, in neonatal hemidecorticated rats, the levels of TGFalpha are much higher than in adult-lesioned rat brains w34x. One parameter, which was not measured in the present nor the other mentioned study w37x, was the morphology of the dopaminergic system. This could be important, because dopamine depletion has been shown to be accompanied by up-regulation of D 2 receptors w13,14x. 4.4. Regional differences in the caudate nucleus The increase in D 2 receptor binding sites in the NHDC cats of the present study was only observed in the anterior lateral region of the caudate and not in the other regions of this nucleus. This might be related to the fact that the caudate nucleus receives there its most dense cortricostriatal projection Žwhich comes from the sensorimotor cortex w39,79x.. Thus, hemidecortication probably causes the most dramatic effects in the anterior lateral caudate as compared to other caudate nucleus regions. Several groups of investigators have reported differences between the medial and lateral caudate. In 6OHDA-lesioned rats, Keefe and Gerfen w30x described differences in the response of lateral and medial striatum to blockade of NMDA receptors. Savasta et al. w62x found that haloperidol treatment resulted in up-regulation of D 2 receptors in only the lateral part of the striatum. And in the intact brain, D 2 dopamine receptors w4,5,16,36x, muscarinic receptors w46x and AChE staining w57x are not homogeneously distributed, but have a higher concentration in the lateral as compared to the medial striatum. Such regional differences might have functional significance: many investigators now believe that the circuit of the basal ganglia is composed of parallel loops with different neuroanatomical connections. The separate striatal regions belong to either ‘sensorimotor’, ‘associative’ or ‘limbic’ loops w1,23,52x. Thus, the processing of information might have different characteristics in the respective loops of the basal ganglia. 4.5. Bilateral Õs. unilateral response The corticostriatal projection is bilateral: from all cortical regions, neurons project to a restricted area of the ipsilateral striatum and to a homological counterpart of this nucleus at the opposite side w39,79x. Although the contralateral corticostriatal projection is somewhat less dense than the ipsilateral one, it projects, in comparison to other
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caudal regions, at its most dense to the anterior lateral caudate. Therefore, it is not surprising that the elevation of D 2 receptors occurred in the anterodorsal part of the caudate nucleus on both sides of the brain. 4.6. Response from the contralateral cortex When the neocortex is removed, the striatum is deprived of the bulk of its neocortical afferents. However, part of the corticostriatal afferents, i.e., those which originate in the contralateral cortex, still remain. In the rat, after neonatal unilateral removal of the neocortex, the contralateral cortex will extend its projection to the caudate nucleus at the side of the ablation, whereas this effect is almost negligible in adult-lesioned rats w32x. In the cat, similar neonatal hemidecortication results in contralateral cortical projections to the thalamus, red nucleus and dorsal column nuclei w22,77,78x. Thus, replacement of corticostriatal connections by projections originating in the contralateral cortex is also likely in cats. This reorganization might compensate, at least partly, for the loss of glutamatergic neurotransmission in the caudate nucleus, ipsilateral to the hemidecortication in NHDC cats. 4.7. Interaction of glutamatergic and dopaminergic systems in the striatum Up-regulation of striatal dopamine receptors have been described after destruction of all dopaminergic fibers w13,14x or blockade of dopamine receptors w9x. However, on the one hand, in the lesioned brains of the present study, the striatum was not deprived of dopaminergic fibers, but of glutamatergic afferents. On the other hand, there is considerable evidence that there is, in caudate and putamen, direct interaction between dopaminergic and glutamatergic neurotransmission. Thus, at the behavioral level, blockade of glutamatergic neurotransmission via striatal NMDA receptors results in attenuation of cocaine-induced behavior w31x and neuroleptic-induced catalepsy w63x. In contrast, behaviors induced by unilateral stimulation of striatal NMDA receptors are blocked by haloperidol Ža D 2 receptor antagonist w50x.. Physiological studies have revealed that striatal dopamine can modulate postsynaptic signals generated by impulses from the glutamatergic corticostriatal system w10,24,27x, and that there is a reciprocal regulation of the release of glutamate and dopamine from their respective fibers w35,40,3x. Moreover, the density of striatal NMDA receptors is decreased after destruction of dopaminergic fibers w55x and, conversely, D 2 dopamine receptor density is slightly reduced after NMDA receptor blockade w56x. Thus, if functional recovery from cortical damage occurs Žas documented for neonatal-hemispherectomized cats w76x., adaptation of the dopaminergic system in the basal ganglia is required after the ipsilateral corticostriatal system has been destroyed. Regrowth of corticostriatal fibers from the contralateral cortex Žsee Section 4.6.
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could have contributed substantially to recovery of striatal function, but might not have been sufficient in the dorsolateral anterior caudate nucleus. Therefore, we would like to suggest another aspect of reorganization of the basal ganglia, which might be involved.
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4.8. Possible mechanism of D2 receptor up-regulation in NHDC cats Since the brain, including that of the cat, possesses a glutamatergic unilateral cortico-nigral projection w45,47,48x, ablation of the left neocortex, as carried out in this study, will also interrupt the corticonigral projection. Thus, this ablation deprives dopaminergic neurons in the substantia nigra pars compacta of glutamatergic excitation. This could result in lack of release of dopamine from nerve endings in the striatum. Moreover, the lack of facilitory control of dopamine release that is normally mediated through AMPA and NMDA receptors located on dopaminergic nerve terminals also could be responsible for the up-regulation of D 2 receptor binding. In adult hemidecorticated brains, the level of dopamine in the striatum has been shown to decrease w53,68x. If such a decrease would also occur in neonatal-lesioned animals, this also might have contributed to the increase in D 2 receptors.
Acknowledgements We thank Sima Sorour and Keith Tatsukawa for expert histological work. Grants USPHS HD-05958, HD-04612, 2P01-NS15654-13.
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Research report
Differences in D 2 dopamine receptor binding in the neostriatum between cats hemidecorticated neonatally or in adulthood Louise D. Loopuijt a
a,1,)
, David A. Hovda b,d,e, Ali Ebrahim c , Jaime R. Villablanca Harry T. Chugani c,2
a,e
,
Departments of Psychiatry and BiobehaÕioral Sciences and of Neurobiology, UCLA School of Medicine, Los Angeles, USA b DiÕision of Neurosurgery, UCLA School of Medicine, Los Angeles, USA c Department of Pediatric Neurology and DiÕision of Nuclear Medicine, UCLA School of Medicine, Los Angeles, USA d Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, USA e Brain Research Institute, Los Angeles, USA Accepted 16 December 1997
Abstract In order to study differences in response to neocortical injury sustained at different ages at the neurotransmitter level, we examined the density in D 2 dopamine receptors in the neostriatum of cats hemidecorticated neonatally Ž N s 4. or in adulthood Ž N s 4., as well as in intact brains Ž N s 6.. Receptor densities were measured using quantitative autoradiography and w 3 Hx-spiperone binding in 12 regions of the neostriatum and nucleus accumbens septi. We found that the anterior lateral caudate nucleus on both sides of the brain contained a higher D 2 receptor density in neonatal-lesioned as compared to adult-lesioned brains. Ipsilateral to the lesion, the increase was 101% Ž P - 0.05. and contralaterally it amounted to 77% Ž P - 0.05.. Moreover, this region of the ipsilateral caudate nucleus of neonatal-lesioned cats tended to be more densely labeled than that of intact brain by 58% Ž P - 0.1.. D 2 receptor densities in adult-lesioned cats did not differ from that of intact controls. Comparison of these data with those of a former morphological study using the same animals suggested that this bilateral elevation of D 2 receptor density in neonatally lesioned brains represents a higher mean density of binding sites per neuron. The elevation in the neonatal-lesioned cats might be a response of the striatum to neuroplastic changes in the striatal neuropil, including the corticostriatal afferents, since such changes are different in neonatal- as compared to adult-lesioned cats. q 1998 Elsevier Science B.V. Keywords: D 2 dopamine receptor; Caudate-putamen; Age-at-lesion; Hemidecortication; Cat
1. Introduction Brain damage sustained at an early age results in more functional recovery than when the brain is damaged during adulthood w33,76x. Morphological studies of such animals with cortical ablation show that areas with secondary damage exhibit less degeneration of neurons and less gliosis than adult-lesioned brains w25,66,75x. For example,
) Corresponding author. Department of Neuropharmacology, Zoological Institute, University of Tubingen, Germany. Fax: q49-7071-29-87815; ¨ E-mail: [email protected] 1 Present address: Department of Neuropharmacology, Mediz. Naturw. Forsch. Zentr., University of Tubingen, 72074 Tubingen, Germany. ¨ ¨ 2 Present address: Pediatric NeurologyrPET Center, Children’s Hospital of Michigan, Wayne State University, Detroit, MI 48201, USA.
0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 0 0 4 - 2
in the cat caudate nucleus neuron loss and increase in glial cell density are more prominent in adult-lesioned than in neonatal-lesioned animals w37x. Moreover, anatomical reorganization and axonal sprouting is more extensive in neonatal- than adult-lesioned brains. For example, early lesioned cats were shown to have contralateral corticorubral w78x, corticothalamic w77x and corticospinal w22x projections, which were not found in intact animals or occurred to a lesser extent in adult-lesioned brains. Similarly, in rats and monkeys, the corticostriatal crossed projection, which is minor in intact animals w39,79x, was much more dense in brains in which the cortex was unilaterally ablated just after Žrat w32x. or before Žmonkey w21x. birth. Moreover, in a PET scan study, human infants were shown to have reduced glucose uptake Žmetabolic activity. in the caudate nucleus 6 months after hemidecortication, while 2 1r2 years after surgery, the glucose uptake had increased
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to normal levels w11,12x, suggesting reinnervation and normalization of glucose metabolic activity in the caudate at this time. Also in experimentally decorticated cats, metabolic activity was shown to recover, if the lesion was inflicted one week after birth w26x. In terms of neurotransmitters, not only the glutamatergic corticostriatal system is affected by hemidecortication, but also the dopaminergic system. Thus, in adult-hemidecorticated rats, the amount of striatal dopamine is decreased w53,68x, indicating a decrease of release of dopamine by nigrostriatal terminals. These data prompted us to investigate dopamine receptor densities in the caudate nucleus at long survival times in neonatal- and adult-hemidecorticated brains to verify, whether differences in anatomical reorganization might also be reflected in changes of striatal dopamine receptor densities. We measured D 2 dopamine receptor binding in neonatal- and adult-hemidecorticated brains of cats, using w 3 Hxspiperone, a ligand which binds almost exclusively to the D 2 receptor family. Since the distribution of dopamine receptors in the striatum is heterogeneous w4,28,36x, and since there is evidence that the response of the caudateputamen to an unilateral lesion of the substantia nigra pars compacta can vary for the various regions within the caudate-putamen w62x, we divided the caudate-putamen and the nucleus accumbens into 12 different regions and measured the density of w 3 Hx-spiperone binding in the presence of ketanserine in order to assess the density of binding sites for the D 2 dopamine receptor family in these nuclei.
and since the w 14 Cx2-deoxyglucose was injected in vivo, the frozen sections, which were to be used for w 3 Hxspiperone binding, contained w 14 Cx2-deoxyglucose label. To remove the w 14 Cx2-deoxyglucose-6-phosphate, the sections were preincubated for 20 min in phosphate buffered saline ŽPBS, pH 7.4. at room temperature, then incubated in 1 nM w 3 Hx-spiperone ŽNEN, specific radioactivity 32.1 Cirmmol. in buffer Ž0.17 M Tris–HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 . in the presence of 40 nM ketanserine ŽSigma. for 30 min at room temperature, rinsed in distilled water Ž2 = 5 s., then in ice-cold buffer Ž2 = 5 min. and blown dry with cold air. For non-specific binding, sections were incubated in 1 nM w 3 Hx-spiperone in buffer in the presence of 40 nM ketanserine and 1 m m Žq.butaclamol ŽSigma. for 30 min at room temperature. Sections were dried overnight at room temperature.
2. Materials and methods Four kittens between the age of 7 and 14 days had the neocortex of the left side removed following procedures described in Loopuijt et al. w37x, and survived into young adulthood. Four adult cats were similarly hemidecorticated and survived for at least 6 months after surgery Žmean survival time 358 days, range 195–586 days.. Six adult cats served as controls. All animals were used in experiments to measure cerebral glucose utilization with the w 14 Cx2-deoxyglucose method w69x. At the time of sacrifice Žmean age of intact controls 1925 days, range 278–5219; of adult-hemidecorticated, mean age 1052 days, range 900–1195; of neonatal-hemidecorticated, mean age 374 days, range 294–451., cats received an overdose of Nembutal Ž100 mgrkg., then the brain was quickly removed from the skull and immediately frozen in dry ice. All brains were cut on a cryostat microtome into coronal sections at 20 m m. The sections were mounted on coverslips and stored at y208C until further use. 2.1. Spiperone binding assay Part of the sections of the above-mentioned brains were used in experiments to measure local glucose utilization,
Fig. 1. Schematic representation of sampling sites for optical density measurements in the caudate nucleus, nucleus accumbens and putamen. Abbreviations and symbols: L s lateral; M s medial; ca s caudate nucleus; ps putamen; na s nucleus accumbens septi; I ssample area. V146 is the number of the cat and 88b, 84b and 77b indicate the numbers of the histological sections.
L.D. Loopuijt et al.r DeÕelopmental Brain Research 107 (1998) 113–122
2.2. Autoradiography The mounted sections were covered with w 3 Hx sensitive film ŽLKB, Sweden. and were developed after 6–8 weeks of exposure in the dark at 48C. All sheets of film also covered a plastic standard with 14 samples of different concentrations of w 3 Hx label ŽARC.. 2.3. Analysis of films Optical density measurements were performed with a high-voltage lightbox ŽAristo, Roslyn, NY. and a high-resolution video camera ŽCohu, Japan. connected to a computer with image analysis software ŽJandel Scientific, CA, USA.. Because of a mediolateral gradient of dopamine D 2 receptor density in the striatum in the cat w4x and rat w5,36x, the caudate nucleus was divided into 3 regions Žmedial, intermediate and lateral. along the dorsolateral–ventromedial axis and sampled according to the scheme illustrated in Fig. 1. In addition, the caudate nucleus was divided along the anterioposterior axis in three parts, anterior, middle and posterior, which each contained a similar amount of sections. The putamen was divided into 2 regions along its anteroposterior axis and, in addition, one
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sample was taken in the nucleus accumbens septi. Thus, the 12 different sample areas were taken in 10 different sections per animal, the sections being equally spaced along the anteroposterior axis. The mean values of these 10 measurements were used for further analysis Žsee Section 2.4.. Optical densities were converted to fmolrmg wet tissue with the aid of the standards, data from the literature w19,20x and a computer program ŽLIGAND w41x.. Values for non-specific binding were obtained by subtracting the values of total bound spiperone from that of sections that were incubated with w 3 Hx-spiperone with 40 nM ketanserine and 1 m m Žq.butaclamol. 2.4. Statistical analysis The internal distribution of label in the caudate nucleus was described by means of regression analysis w15x. Comparisons of the caudate nucleus between the three groups of experimental animals were done with a repeated measures analysis of variance ŽANOVA., which was conducted with the individual region serving as the repeated variable. Comparisons were made to determine the significance of the main effect Žhemidecortication. with simple main effect comparisons to intact values using appropriate contrasts w44x. The values for putamen and nucleus accum-
Fig. 2. Autoradiographs of w 3 Hx-spiperone binding. ŽA. Coronal section of intact brain through the caudate nucleus, putamen and nucleus accumbens septi. ŽB. Non-specific binding, i.e., binding of 1 nM w 3 Hx-spiperone and 40 nM ketanserine in the presence of 1 m m Žq.butaclamol. ŽC. Total binding Žw 3 Hx-spiperone plus 40 nm ketanserine. in adult-hemidecorticated brain. ŽD. Total binding of w 3 Hx-spiperone plus ketanserine in neonatal hemidecorticated brain. C and D also show the similar extent of the hemidecortication in the two age-at-lesion groups. Abbreviations; ca s caudate nucleus; p s putamen; na s nucleus accumbens septi. Bar s 9 mm.
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bens septi were analyzed with the non-parametric Mann– Whitney U-test.
3. Results
1r3–1r2 of the medial sylvian gyrus. Dorsally, there were cortical remnants only in the midline, rostral to the genu of the corpus callosum, including variable portions of the gyri rectus and cingulum. The caudate nucleus was intact in all brains, as well as the entire contralateral hemisphere.
3.1. Description of lesions 3.2. D2 dopamine receptor binding The lesions of these brains have been described in detail by Loopuijt et al. w37x. The nature and extent of the resection were similar, in all aspects, for the two age-at-lesion groups. In all animals, almost the entire left neocortex was removed ŽFig. 2C,D; see also Ref. w37x, Fig. 1, cats a160 and a334.. However, since the caudate nucleus was spared, portions of the neocortex lateral and ventral to the head of this nucleus were not removed, including the lower
w 3 Hx-spiperone binding at 1 nM resulted in a high amount of labeling in the caudate nucleus, putamen and nucleus accumbens septi, in all intact, as well as hemidecorticated cats ŽFig. 2A,C,D.. This labeling was very high as compared to the surrounding tissue, e.g., the cortex ŽFig. 2A,C,D.. Non-specific binding was always very low ŽFig. 2B..
Fig. 3. Specific binding of spiperone Žfmolrmg wet weight. in different regions of the caudate nucleus. Error bars represent standard errors of the mean ŽS.E.M... ` s Intact; I s adult hemidecorticated ŽAHDC.; ^ s neonatal-hemidecorticated ŽNHDC.. Note that there is a dorsolateral–ventromedial gradient in w 3 Hx-spiperone binding, which stretches from the anterior through the posterior end of the caudate nucleus. Note also, that in NHDC cats, the specific binding in the lateral region of the anterior caudate on both sides of the brain is significantly higher than in AHDC animals Ž P - 0.05., while there is a trend for the left lateral region of neonatal-hemidecorticated brains to be higher than that of the intact brains Ž P - 0.1.. Intact N s 6; AHDC: N s 4; NHDC: N s 4. Abbreviations: AHDCs adult hemidecorticated brains; NHDCs neonatal hemidecorticated brains. ) P - 0.05 NHDC compared to AHDC; qP - 0.1 NHDC compared to Intact.
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3.3. Intact cats Quantification of w 3 Hx-spiperone binding showed that the caudate nucleus in intact cats contained more labeling at its dorsolateral than ventromedial side ŽFig. 3., thereby displaying a gradient from dorsolateral to ventromedial. This gradient was present in the anterior, middle and posterior caudate nucleus and showed a significant linear relationship, taking into account that the distance between the medial and intermediate sample was identical to the distance between the intermediate and lateral sample ŽFig. 1.. The slope of the regression line of the left anterior caudate was 9.60 Ž r 2 s 0.96; FŽ1,4. s 99.14; P - 0.0005., for the left middle caudate 14.81 Ž r 2 s 0.99; FŽ1,4. s 230.37; P - 0.0005. and the left posterior caudate it was 8.78 Ž r 2 s 0.66; FŽ1,4. s 7.84; P - 0.05.. On the right side, the regression coefficients were, for anterior, middle and posterior caudate nucleus, respectively: 15.02 Ž r 2 s 0.99; FŽ1,4. s 481.91; P - 0.0005.; 17.07 Ž r 2 s 0.99; FŽ1,4. s 183.91; P - 0.0005. and 8.8 Ž r 2 s 0.97, FŽ1,4. s 153.11; P - 0.0005.. Within each of the lateral, intermediate and medial regions, in a rostrocaudal direction, there was no difference in the amount of spiperone binding between the anterior, middle and posterior caudate. 3.4. Adult and neonatal hemidecorticated animals The linear, dorsolateral–ventromedial gradient of spiperone binding described above was preserved in brains that had undergone adult or neonatal hemidecortication ŽFig. 3A,B.. In the anterior left caudate nucleus Žon the decorticated side., the regression slopes of adult-hemidecorticated ŽAHDC. and neonatal-hemidecorticated ŽNHDC. cats were, respectively: 6.73 Ž r 2 s 0.81; FŽ1,2. s 74.23; P - 0.05. and 30.58 Ž r 2 s 0.98; FŽ1,2. s 97.91; P - 0.05.. In the anterior left caudate, the three groups of experimental animals appeared to have significantly different relationships between their lateral, intermediate and medial values Ž P - 0.05, repeated measurements ANOVA.. This discrepancy was caused predominantly by the high mean values of the lateral region in NHDC brains, which showed a higher value than in AHDC by 101% Ž P - 0.05; post-hoc analysis. and tended to be higher than that of intact brains, by 58.2% Ž P - 0.1.. The middle and posterior caudate at the left side did not show significant changes in the distribution of label in NHDC as compared to AHDC or intact in any of the medio-lateral region ŽFig. 3A.. On the right side, the label in the anterior lateral region of the caudate nucleus of NHDC cats was significantly more dense than in AHDC cats by 77% Ž P - 0.05., but the apparent difference of 26% between NHDC and intact cats was not statistically significant ŽFig. 3B.. Also on the right side, the middle and posterior caudate nucleus of NHDC cats did not show differences in any of the three Žlateral, intermediate or medial. regions.
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Table 1 Specific binding of spiperone in the nucleus accumbens septi ŽN. accumb.. and putamen Žfmolrmg wet weight.. Intact N s6, adult-hemidecorticate ŽAHDC. N s 4, neonatal hemidecorticate ŽNHDC. N s 4. Left
Intact AHDC NHDC
N. accumb. mean SEM
Putamen anterior mean SEM
Putamen posterior mean SEM
21.69 22.48 22.85
34.85 39.98 47.74
41.64 54.89 34.68
3.04 6.28 0.83
5.79 10.70 7.97
9.93 17.31 4.97
Right
Intact AHDC NHDC
N. accumb. mean SEM
Putamen anterior mean SEM
Putamen posterior mean SEM
22.98 21.98 29.52
39.95 40.09 62.58
44.49 44.07 46.50
1.93 4.59 2.90
5.91 8.90 10.76
9.67 11.98 8.28
In case of the AHDC brains, the values in the lateral caudate nucleus of the anterior, middle and posterior regions were not different from that of the intact brains ŽFig. 3A,B.. Similarly, no such differences were observed in the intermediate and medial regions of the caudate nucleus. The concentration of D 2 dopamine receptor binding sites in the putamen of intact brains were similar to that of the lateral part of the anterior and middle caudate nucleus ŽTable 1.. However, there were no differences between NHDC, AHDC and intact brains in the anterior or posterior half of the putamen ŽTable 1, Mann–Whitney U-test.. Finally, the amount of binding in the nucleus accumbens septi of hemidecorticated cats was not changed on either side relative to intact controls.
4. Discussion The main finding of the present report is the local difference in D 2 receptor density between neonatal and adult cat brains after hemidecortication: after a minimum of 6 months survival time, the D 2 receptor density of the ipsilateral anterior lateral caudate nucleus is higher in NHDC than in AHDC cats, by 101% at the ipsilateral and 77% at the contralateral side. 4.1. Technical considerations These conclusions are based on autoradiographic measurements of in vitro incubations of w 3 Hx-spiperone at a concentration of 1 nM in the presence of ketanserine. The data of our study represent specific binding at 1 nM w 3 Hx-spiperone, which is obtained by subtracting total spiperone binding from non-specific binding Žincubations with spiperone, ketanserine and Žq.butaclamol Žsee Section 2.. These last measurements indicated that non-specific binding accounted for 15% of the total binding Ždata not shown., which is similar to that in other in vitro w 3 Hxspiperone incubations systems w5x. Thus it appears, that
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there was very little or no residual w 14 Cx2-deoxy-glucose6-phosphate left in our sections Žsee Section 2.. Our values, measured at 1 nM w 3 Hx-spiperone, do not represent an estimation of total available binding sites Ž Bmax ., but by comparison with data from the literature, these values can be estimated to be 15% lower than that of the Bmax . In addition, we did not correct for tritium absorption in the tissue. However, we kept our incubation and exposure conditions the same throughout all experiments, often incubating sections of the three animal groups at the same time, so that we are confident that comparisons within and between animals in this study are reliable. Comparing our quantitative data with those of other investigators, we found in the intermediate anterior caudate nucleus of intact brains, a binding of 35 fmolrmg wet tissue Žapproximately 350 fmolrmg protein. at 1 nM spiperone, which likely represents 85% of the Bmax . Richfield et al. w59x, also in the cat, found 500 fmolrmg protein Žprot. at 0.25 nM spiperone, which probably represents 35% of the Bmax . In rats, Bmax values for spiperone binding autoradiography vary between groups of investigators: 843 fmolrmg prot w5x, 860 fmolrmg prot w59x, and 458 " 51 fmolrmg prot w29x. Thus, our data are within the range of those reported by other investigators. All the brains that were used in this study were thoroughly examined using thionin-stained sections. Brains in which the caudate nucleus appeared to be damaged were not included. For more documentation on the extent of the lesion, see Loopuijt et al. w37x. 4.2. Response of D2 receptor density to hemidecortication Several groups of investigators have reported on the response of striatal D 2 receptors to unilateral cortical ablation in adult rats. Some performed complete hemidecortications, comparable to those of the present study, and reported a decrease in Bmax w43,54,60,64,71,73x. Others performed restricted lesions and found no difference between the striatum ipsilateral to the lesion and the striatum in control rats w29,74x. In contrast, in neonatally hemidecorticated brains, we observed a bilateral regional increase in receptor density. At the time that the hemidecortications were performed ŽP7–P14., all the afferents to the striatum have established their connections, although the neurons that have already established their contacts are still migrating w18x. In rats there is a sevenfold increase of D 2 receptor density in the striatum between postnatal days P7 and P49 w6–8,17,49,51x. Assuming that the maturity of the cat brain at P7 is equivalent that of approximately P18 in the rat brain w61x, the cat striatum is probably in a phase of rapid increase of D 2 receptor density at the time of surgery. w 3 Hx-spiperone binds to all subunits of the dopamine D 2 receptor family, comprising the D 2 , D 3 and D4 receptor proteins. These subtypes not only differ in molecular structure, but also in localization. In the striatum, D 2 receptors
can be found throughout w67x, while D 3 receptors are predominantly found in the ventral striatum, nucleus accumbens septi and islands of Calleja w70x. D4 receptor binding has been reported to be present w42,65x or absent w58x in the striatum. D4 mRNA could be revealed in the striatum by some authors w38x, but not by others w42x, and D4 receptor protein could be found in cell bodies of corticostriatal neurons in the cortex and also in the striatum, although in the latter, it was found in low amounts w2x. Finally, removal of the prefrontal cortex in adult rats resulted in a decrease in D4 receptor binding in the nucleus accumbens, but not a significant decrease in the striatum w72x. Taken together, these results might suggest the presence of D4 receptors on corticostriatal nerve endings. If so, one would expect a cortical ablation to result in a small decrease in w 3 Hx-spiperone binding in the striatum. In our AHDC animals, a small decrease in w 3 Hx-spiperone binding could have been present, although the values were not significantly different from the controls. Such a decrease would have been minor; therefore, the significant difference between NHDC and AHDC in the anterolateral striatum cannot be attributed to a decrease in w 3 Hx-spiperone binding in AHDC animals, but mainly to an increase in w 3 Hx-spiperone binding in NHDC cats. 4.3. Age-at-lesion effects: neuron density and neuron loss We reported morphological differences in the caudate nucleus between cats that have sustained cortex ablation as an adult or neonate w37x. In AHDC, there was a loss of 21.8% Ž P - 0.05. of the neurons, while in NHDC there was only a tendency for neuronal loss Ž17.9%, P - 0.1.. Neuronal cell loss in ADHC was accompanied by shrinkage of volume without change in neuronal cell density, while the caudate of NHDC cats did not show a change in volume, but did show a decrease in neuronal cell packing density w37x. Since we measured and expressed spiperone binding as D 2 receptor binding sites per mg wet weight, our values represent concentrations of binding sites. Such values can be compared to values for cell packing density, because they represent the concentration of cells. Since we did not find any changes in the density of neurons or spiperone D 2 receptor binding sites in AHDC, the average number of spiperone binding sites per neuron in these animals was not changed. We found a reduction in total number of neurons however, and this resulted in a shrinkage of the total caudate volume w37x. Therefore, in AHDC animals, there was a loss of neurons, while the surviving neurons maintained the same average D 2 receptor density as before the insult. In contrast, in NHDC animals, the cell packing density was decreased by 25.8% w37x. Therefore, an increase in D 2 receptor binding site concentration in the anterior lateral caudate and the lack of change in binding site concentration in the other regions of the caudate nucleus mean, that
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the average number of binding sites per neuron in the caudate of these animals was increased. Thus, it is reasonable to propose that if the neocortical resection is performed when the cats are young, i.e., at a time when the density of D 2 receptors is still increasing w6–8,49,51x, such densities can still adapt to changes in the structure of the brain. We would like to suggest that this process might be related to the presence of higher levels of the neurotrophin TGFalpha. In fact, in neonatal hemidecorticated rats, the levels of TGFalpha are much higher than in adult-lesioned rat brains w34x. One parameter, which was not measured in the present nor the other mentioned study w37x, was the morphology of the dopaminergic system. This could be important, because dopamine depletion has been shown to be accompanied by up-regulation of D 2 receptors w13,14x. 4.4. Regional differences in the caudate nucleus The increase in D 2 receptor binding sites in the NHDC cats of the present study was only observed in the anterior lateral region of the caudate and not in the other regions of this nucleus. This might be related to the fact that the caudate nucleus receives there its most dense cortricostriatal projection Žwhich comes from the sensorimotor cortex w39,79x.. Thus, hemidecortication probably causes the most dramatic effects in the anterior lateral caudate as compared to other caudate nucleus regions. Several groups of investigators have reported differences between the medial and lateral caudate. In 6OHDA-lesioned rats, Keefe and Gerfen w30x described differences in the response of lateral and medial striatum to blockade of NMDA receptors. Savasta et al. w62x found that haloperidol treatment resulted in up-regulation of D 2 receptors in only the lateral part of the striatum. And in the intact brain, D 2 dopamine receptors w4,5,16,36x, muscarinic receptors w46x and AChE staining w57x are not homogeneously distributed, but have a higher concentration in the lateral as compared to the medial striatum. Such regional differences might have functional significance: many investigators now believe that the circuit of the basal ganglia is composed of parallel loops with different neuroanatomical connections. The separate striatal regions belong to either ‘sensorimotor’, ‘associative’ or ‘limbic’ loops w1,23,52x. Thus, the processing of information might have different characteristics in the respective loops of the basal ganglia. 4.5. Bilateral Õs. unilateral response The corticostriatal projection is bilateral: from all cortical regions, neurons project to a restricted area of the ipsilateral striatum and to a homological counterpart of this nucleus at the opposite side w39,79x. Although the contralateral corticostriatal projection is somewhat less dense than the ipsilateral one, it projects, in comparison to other
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caudal regions, at its most dense to the anterior lateral caudate. Therefore, it is not surprising that the elevation of D 2 receptors occurred in the anterodorsal part of the caudate nucleus on both sides of the brain. 4.6. Response from the contralateral cortex When the neocortex is removed, the striatum is deprived of the bulk of its neocortical afferents. However, part of the corticostriatal afferents, i.e., those which originate in the contralateral cortex, still remain. In the rat, after neonatal unilateral removal of the neocortex, the contralateral cortex will extend its projection to the caudate nucleus at the side of the ablation, whereas this effect is almost negligible in adult-lesioned rats w32x. In the cat, similar neonatal hemidecortication results in contralateral cortical projections to the thalamus, red nucleus and dorsal column nuclei w22,77,78x. Thus, replacement of corticostriatal connections by projections originating in the contralateral cortex is also likely in cats. This reorganization might compensate, at least partly, for the loss of glutamatergic neurotransmission in the caudate nucleus, ipsilateral to the hemidecortication in NHDC cats. 4.7. Interaction of glutamatergic and dopaminergic systems in the striatum Up-regulation of striatal dopamine receptors have been described after destruction of all dopaminergic fibers w13,14x or blockade of dopamine receptors w9x. However, on the one hand, in the lesioned brains of the present study, the striatum was not deprived of dopaminergic fibers, but of glutamatergic afferents. On the other hand, there is considerable evidence that there is, in caudate and putamen, direct interaction between dopaminergic and glutamatergic neurotransmission. Thus, at the behavioral level, blockade of glutamatergic neurotransmission via striatal NMDA receptors results in attenuation of cocaine-induced behavior w31x and neuroleptic-induced catalepsy w63x. In contrast, behaviors induced by unilateral stimulation of striatal NMDA receptors are blocked by haloperidol Ža D 2 receptor antagonist w50x.. Physiological studies have revealed that striatal dopamine can modulate postsynaptic signals generated by impulses from the glutamatergic corticostriatal system w10,24,27x, and that there is a reciprocal regulation of the release of glutamate and dopamine from their respective fibers w35,40,3x. Moreover, the density of striatal NMDA receptors is decreased after destruction of dopaminergic fibers w55x and, conversely, D 2 dopamine receptor density is slightly reduced after NMDA receptor blockade w56x. Thus, if functional recovery from cortical damage occurs Žas documented for neonatal-hemispherectomized cats w76x., adaptation of the dopaminergic system in the basal ganglia is required after the ipsilateral corticostriatal system has been destroyed. Regrowth of corticostriatal fibers from the contralateral cortex Žsee Section 4.6.
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could have contributed substantially to recovery of striatal function, but might not have been sufficient in the dorsolateral anterior caudate nucleus. Therefore, we would like to suggest another aspect of reorganization of the basal ganglia, which might be involved.
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4.8. Possible mechanism of D2 receptor up-regulation in NHDC cats Since the brain, including that of the cat, possesses a glutamatergic unilateral cortico-nigral projection w45,47,48x, ablation of the left neocortex, as carried out in this study, will also interrupt the corticonigral projection. Thus, this ablation deprives dopaminergic neurons in the substantia nigra pars compacta of glutamatergic excitation. This could result in lack of release of dopamine from nerve endings in the striatum. Moreover, the lack of facilitory control of dopamine release that is normally mediated through AMPA and NMDA receptors located on dopaminergic nerve terminals also could be responsible for the up-regulation of D 2 receptor binding. In adult hemidecorticated brains, the level of dopamine in the striatum has been shown to decrease w53,68x. If such a decrease would also occur in neonatal-lesioned animals, this also might have contributed to the increase in D 2 receptors.
Acknowledgements We thank Sima Sorour and Keith Tatsukawa for expert histological work. Grants USPHS HD-05958, HD-04612, 2P01-NS15654-13.
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