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Early social isolation decreases the expression of calbindin D-28k in rat cerebellar Purkinje cells
Neuroscience Letters 272 (1999) 171±174 www.elsevier.com/locate/neulet
Early social isolation decreases the expression of calbindin D-28k in rat cerebellar Purkinje cells Rodrigo Pascual*, Enrique VerduÂ, Antoni Valero, Xavier Navarro Neuroplasticity Group, Department of Cell Biology, Physiology and Immunology, Universitat AutoÁnoma de Barcelona, Barcelona, Spain Received 6 July 1999; received in revised form 12 July 1999; accepted 13 July 1999
Abstract The main objective of this study was to analyze the effects of early social isolation on the expression of calbindin D-28k (CAD-28k) in cerebellar Purkinje cells. Sprague±Dawley rats were reared either in isolation (IC) or socially housed conditions (SC) from postnatal days 18±32. Half of the rats of each group were sacri®ced at 32 days of age and the cerebella processed for immunocytochemical labeling against CAD-28k. The remaining IC animals were housed under standard conditions for re-socialization between postnatal days 32 and 62. The results indicate that Purkinje cells of rats reared under early isolation show a marked loss of CAD-28k immunoreactivity, and that this de®ciency is recovered by later social interaction, although the thickness of the molecular layer remains reduced. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calbindin; Cerebellum; Development; Isolation; Purkinje cells; Rat
Social deprivation imposed during the early postnatal period in laboratory animals is known to cause profound and long-lasting CNS abnormalities. These alterations involve dopaminergic [14], serotonergic [13] and noradrenergic [17] systems. In addition, structural sequelae include a signi®cant decrease in mesolimbic innervation of medial prefrontal cortex [22] and hippocampal neural degeneration [21]. These impairments are associated with behavioral disorders, such as locomotor hyperactivity [6,14], anxiety [9] and de®cits in memory tasks [22]. Some decades ago, Prescott [19] hypothesized that abnormal behaviors observed in animals reared under early social deprivation are related to developmental alterations in the cerebellar cytoarchitecture and function. This hypothesis received support from neurochemical studies indicating that rodents reared in isolation showed alterations in cerebellar noradrenaline and serotonin metabolism [6], associated with a signi®cant decrease in benzodiazepine binding sites [16]. In a recent study [18] we demonstrated that Golgi-impregnated Purkinje cells of rats subjected to early social isolation showed a delayed dendritic arborization; however, it is not known if such structural impairment is correlated with cellular functional abnormalities. Taking * Corresponding author. Tel.: 134-93-581-1966; fax: 134-935812-986. E-mail address: [email protected] (R. Pascual)
into account that the intracellular protein calbindin D-28k (CAD-28k) regulates Ca 21-dependent aspects of neuronal function [5] and that Purkinje cells highly express CAD28k [20], the main objective of the present study was to evaluate the effect of early social isolation on the expression of CAD-28k by using an immunocytochemical approach. Twenty-eight Sprague±Dawley female rats born from multiparous mothers were weaned at 18 days and divided in two groups counterbalanced by weight: isolated rats (IC, n 14), housed singly in small opaque plastic cages (10 £ 10 £ 10 cm), and socially-reared rats (SC, n 14) maintained in groups of three to four animals per cage (50 £ 28 £ 20 cm). Both groups had food and water available ad libitum and were maintained in the same room with regulated environmental conditions (temperature: 21 ^ 28C, humidity: 65±70%, 12:12 h light/dark cycle). Half of the IC and SC rats were sacri®ced at 32 postnatal days for histological evaluation, while the remaining IC rats were rehoused socially (three to four animals per cage) until 62 postnatal days. At either 32 or 62 postnatal days the rats were weighed, deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused transcardially with cold 0.9% saline for 1 min followed by 4% paraformaldehyde in 0.1 M phosphate buffer solution (PBS; pH 7.4) for 6 min. The cerebella were removed from the skull and post®xed in 4% paraformaldehyde for 24 h and then cryoprotected in 0.1 M PBS containing 20% sucrose and stored at 48C. Parasa-
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 60 8- 4
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R. Pascual et al. / Neuroscience Letters 272 (1999) 171±174
gital vermal sections, cut at 40 mm thickness with a freezing sliding microtome, were collected and washed free-¯oating in 0.1 M PBS with 0.3% Triton X-100 (Fluka, Buchs, Switzerland) and 1% fetal calf serum (FCS) (ICN, Costa Mesa, USA) for 1 h and then incubated for 48 h with primary polyclonal antibody generated against calbindin D-28k (rabbit; 1:2000; Chemicon). After washes with the same diluted solution, sections were incubated in secondary antisera goat anti-rabbit cyanine 3.18 labeled IgG (1:2000; Jackson Immunoresearch, West Grove, USA) overnight at 48C. Following additional washes, sections were mounted on gelatin-coated slides, dehydrated in ethanol, cleared in methylsalicylate, and coverslipped with DPX (Fluka). CAD-28k immunoreactivity was analyzed under an Olympus BX-40 microscope equipped for epi¯uorescence. Selected images (10£ magni®cation) were taken with the aid of a digital camera (Olympus DP20) attached to the microscope and analyzed using NIH Image software (freely distributed from NIH). One hundred and sixty-six parasagital vermal sections (IC: 87 sections; SC: 79 sections) were evaluated, measuring both the density of Purkinje cells (cells/mm 2) immunoreactivity to CAD-28k and the thickness of the molecular layer (where Purkinje cell dendrite arborization is distributed). Measurements were performed in the subvermal lobule VId (according to Larsell's terminology) in all the samples, because morphology is clearly distinguishable and varies little from section to section in
the vermis. Furthermore, hematoxylin-eosin alternate sections were obtained in order to evaluate possible cytoarchitectonic abnormalities. All measurements were performed on coded slides in a blind manner. Data are expressed as mean ^ SD and were compared by unpaired t-test (Stat View 4.5) and differences considered signi®cant when P , 0:05. Low power magni®cation (10£) images of cerebellar sections immunostained against CAD-28k show that 32 days old IC rats exhibit a heterogeneous pattern of Purkinje cell ¯uorescence with wide patches lacking immunoreactivity (Fig. 1A). In contrast, a highly ordered pattern of CAD28k expression was observed in age-matched SC rats (Fig. 1B). On the other hand, IC rats submitted to 30 days of socially normal experiences undergo an almost complete restoration of the CAD-28k de®cit (Fig. 1C). The density of CAD-28k immunoreactive Purkinje cells was markedly reduced in isolated versus socially reared rats by 32 days of age (P , 0:001), a difference not found after 30 days of social housing (Fig. 2A). In addition, the thickness of the molecular layer was signi®cantly reduced in IC rats versus SC rats at both 32 (IC: 165:2 ^ 17:3 mm; SC: 189:1 ^ 14:1 mm; P , 0:001) and 62 (IC: 211:9 ^ 21:9; SC: 222:9 ^ 24:3 mm; P , 0:05) postnatal days (Fig. 2B). The lack of CAD-28k-immunoreactive Purkinje cells was not due to cell death, as evidenced in cerebellar alternate sections stained with hematoxylin-eosin (Fig. 3). Body weight gain
Fig. 1. Immuno¯uorescence staining demonstrating changes in calbindin-D28k levels on representative vermal cerebellar sections of isolated (IC; (A,C)) and socially-reared (SC; (B,D)) rats, at 32 (P32) and 62 (P62) postnatal days. ML: molecular layer; PL: Purkinje cell layer; GL: internal granular layer. Scale bar, 100 mm.
R. Pascual et al. / Neuroscience Letters 272 (1999) 171±174
Fig. 2. Density of calbindin D-28k immunoreactive Purkinje cells (A) and thickness of the molecular layer (B) in vermal sublobule VI of rats reared under social (SC) and isolated (IC) conditions. *P , 0:05; **P , 0:01; ***P , 0:001 (mean ^ SD, unpaired ttest).
was similar between isolated and socially reared rats at both 32 (IC: 61:5 ^ 11:4; SC: 71:3 ^ 7:2; n.s.) and 62 (IC: 263:4 ^ 18:7; SC: 259:7 ^ 17:3; n.s.) postnatal days. The immunocytochemical results found indicate that early social isolation signi®cantly reduced the expression of CAD-28k in vermal Purkinje cells. Moreover, isolatedreared rats submitted to later social interaction during one month showed a signi®cant recovery in the expression of this calcium binding protein. Considering that CAD-28k plays a pivotal role in the regulation of intracellular Ca 21 transients, it is probable that the underexpression observed in Purkinje cells severely impair the physiology of these cells and, consequently, the information processing in the cerebellar cortex. Taking into account that the cerebellum is involved mainly in the modulation of motor functions, it is expected that isolated rats show measurable motor behavior abnormalities. By examining the motor behavior displayed by IC rats in the open ®eld test, we found a signi®cant locomotor hyperactivity, but no signs of tremor or ataxia (unpublished observations). This ®nding is consistent with that described by Airaksinen et al. [1], who evaluated the physiological role of CAD-28k by using mutant mice lacking CAD-28k (Cb 2/2). These rodents exhibited a normal locomotor behavior in standard environments, but motor incoordination when subjected to tests of motor coordination. It is not possible to discard that the IC rats evaluated in the present study were similarly ataxic when subjected to a demanding task test. This mismatch between decline in the expression of CAD-28k by Purkinje cells and relatively normal locomotor behavior, may be explained in part by the presence of additional intracellular calcium binding proteins such as parvalbumin, that is normally present in Purkinje cells and may compensate for the CAD-28k failure. The relatively short period of isolation (2 weeks) used in the present study was suf®cient to induce a signi®cant impairment in the CAD-28k expression, indicating that vermal Purkinje cells are highly sensitive to early environmental deprivation. This vulnerability may be related to the
173
fact that, in the rat, the ontogenetic expression of CAD-28k in developing Purkinje cells takes place during the ®rst month of life, increasing progressively from birth to a peak at postnatal week 2, then declining to a steady-state level at postnatal week 4 [11]. However, the diminished CAD-28k expression in isolated-reared rats may be correlated to the structural impairment in dendritic arborization observed in Golgiimpregnated Purkinje cells of socially deprived rats [18]. This ®nding is in agreement to the signi®cant hypotrophy detected in the molecular layer of isolated rats where Purkinje cell dendrite arborization is distributed. Furthermore, studies carried out in some neurodegenerative diseases [2,10,12] indicate that the decreased levels of CAD-28k are highly correlated with neural degeneration. In addition, Goodman et al. [8] showed that immature dentate granule cells are more vulnerable to hypoxic/ ischemic insults than calbindin-rich mature counterparts; and in vitro studies indicate that calbindin-positive hippo-
Fig. 3. Micrographs of the vermal cerebellar cortex of an isolated 32-day-old rat, labeled against calbindin D-28k (A) and stained with hematoxylin-eosin (B). ML: molecular layer; PL: Purkinje cell layer; GL: internal granular layer. Scale bar, 50 mm.
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campal neurons were more resistant to glutamate or calcium ionophore neurotoxicity than were neurons lacking CAD28k [15]. In the current study, comparing images of alternating sections stained by using immunocytochemical and hematoxylin-eosin procedures, it is apparent that the wide cortical areas free of immuno¯uorescent Purkinje cells are not due to cell death but to CAD-28k underexpression. This ®nding is consistent with the restoration of the density of immuno¯uorescent Purkinje cells found in the isolatedreared rats at 62 postnatal days, after 1 month of normal interaction. The bene®cial effects of re-socialization on rats previously reared under social deprivation was evidenced only in the immunoreactive expression of CAD-28k but not in the thickness of the cerebellar molecular layer. This functional recovery is consistent with studies that demonstrated that the reinstatement of a social environment to previously isolated rats may recover some behavioral and neural abnormalities such as motor hyperactivity, greater tailpinch latencies and reduced activity in mesocortico-frontal dopaminergic neurons [4,7]. Furthermore, the lack of recovery of the molecular layer hypotrophy is consistent with the fact that Purkinje cell dendritic arborization of isolatedreared rats remained stunted despite further social stimulation (unpublished observations). It is possible that failure in structural recovery may be due to the fact that the re-socialization timing applied in this study was between 32 and 62 postnatal days, when structural Purkinje cell plasticity progressively declines [3]. In conclusion, the present study demonstrates that vermal Purkinje cells are highly vulnerable to early social deprivation as shown by a marked underregulation of CAD-28k expression, and that this alteration may be recovered by adequate social interaction. This work was partially supported by grant SAF97±0147 from the CICYT (Spain). R.P. was on a leave of absence from the Faculty of Health Sciences, Universidad CatoÂlica del Maule, Talca (Chile). [1] Airaksinen, M.S., Eilers, J., Garaschuk, O., Thoenen, H., Konnerth, A. and Meyer, M., Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene. Proc. Natl. Acad. Sci. USA, 94 (1997) 1488±1493. [2] Alexianu, M.E., Ho, B-K., Mohamed, A.H., La Bella, V., Smith, R.G. and Appel, S.H., The role of calcium-binding proteins in selective motoneuron vulnerability in amyotrophic lateral sclerosis. Ann. Neurol., 36 (1994) 846±858. [3] Anderson, W.A. and Flumerfelt, B.A., Purkinje cell growth beyond the twenty-third postnatal day. Dev. Brain Res., 17 (1985) 195±200. [4] Blanc, G., HerveÂ, D., Simon, H., Lisoprawski, A., Glowinski, J. and Tassin, J.P., Response to stress of mesocortico-frontal dopaminergic neurones in rats after long-term isolation. Nature, 284 (1980) 265±267. [5] Chard, P.S., Bleakman, D., Christakos, S., Fullmer, C.S. and Miller, R.J., Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones. J. Physiol., 472 (1993) 341±357.
[6] Essman, W.B., Differences in locomotor activity and brainserotonin metabolism in differentially housed mice. J. Comp. Physiol. Psychol., 66 (1968) 244±246. [7] Gentsch, C., Lichtsteiner, M. and Feer, H., Individual housing of rats causes divergent changes in spontaneous and reactive activity. Experientia, 37 (1981) 61±62. [8] Goodman, J.H., Wasterlain, C.G., Massarweh, W.F., Dean, E., Sollas, A.L. and Sloviter, R.S., Calbindin-D28k immunoreactivity and selective vulnerability to ischemia in the dentate gyrus of the developing rat. Brain Res., 606 (1993) 309±314. [9] Hilakivi, L.A., Ota, M. and Lister, R.G., Effect of isolation on brain monoamines and the behavior of mice in tests of exploration, locomotion, anxiety and behavioral `despair'. Pharmacol. Biochem. Behav., 33 (1989) 371±374. [10] Iacopino, A.M. and Christakos, S., Speci®c reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases. Proc. Natl. Acad. Sci. USA, 87 (1990) 4078±4082. [11] Iacopino, A.M., Rhoten, W.B. and Christakos, S., Calcium binding protein (calbindin-D28k). gene expression in the developing and aging mouse cerebellum. Mol. Brain Res., 8 (1990) 283±290. [12] Ichimiya, Y., Emson, P.C., Mountjoy, C.Q., Lawson, D.E.M. and Heizmann, C.W., Loss of calbindin-28k immunoreactive neurones from the cortex in Alzheimer-type dementia. Brain Res., 475 (1988) 156±159. [13] Jaffe, E.H., De Frias, V. and Ibarra, C., Changes in basal and stimulated release of endogenous serotonin from different nuclei of rats subjected to two models of depression. Neurosci. Lett., 162 (1993) 157±160. [14] Jones, G.H., Hernandez, T.D., Kendall, D.A., Marsden, C.A. and Robbins, T.W., Dopaminergic and serotonergic function following isolation rearing in rats: study of behavioural responses and postmortem and in vivo neurochemistry. Pharmacol. Biochem. Behav., 43 (1992) 17±35. [15] Mattson, M.P., Rychlik, B., Chu, Ch. and Christakos, S., Evidence for calcium-reducing and excito-protective roles for the calcium-binding protein calbindin-D28k in cultured hippocampal neurons. Neuron, 6 (1991) 41±51. [16] Miachon, S., Manchon, M., Fromentin, J.R. and Buda, M., Isolation-induced changes in radioligand binding to benzodiazepine binding sites. Neurosci. Lett., 111 (1990) 246±251. [17] Ojima, K., Matsumoto, K., Tohda, M. and Watanabe, H., Hyperactivity of central noradrenergic and CRF systems is involved in social isolation-induced decrease in pentobarbital sleep. Brain Res., 684 (1995) 87±94. [18] Pascual, R., Hervias, M.C., TohaÂ, M.E., Valero, A. and Figueroa, H.R., Purkinje cell impairment induced by early movement restriction. Biol. Neonate, 73 (1998) 47±51. [19] Prescott, J.W., Early somatosensory deprivation as an ontogenetic process in the abnormal development of the brain and behavior. In E.I. Goldsmith, and J. Moor-Jankowski (Eds.), Medical Primatology, Karger, Basel, 1971, pp. 356± 375. [20] Rogers, J.H., Immunoreactivity for calretinin and other calcium-binding proteins in cerebellum. Neuroscience, 31 (1989) 711±721. [21] Uno, H., Tarara, R., Else, J.G., Suleman, M.A. and Sapolsky, R.M., Hippocampal damage associated with prolonged and fatal stress in primates. J. Neurosci., 9 (1989) 1705±1711. [22] Winterfeld, K.T., Teuchert-Noodt, G. and Dawirs, R.R., Social environment alters both ontogeny of dopamine innervation of the medial prefrontal cortex and maturation of working memory in gerbils (Meriones unguiculatus). J. Neurosci. Res., 52 (1998) 201±209.
Early social isolation decreases the expression of calbindin D-28k in rat cerebellar Purkinje cells Rodrigo Pascual*, Enrique VerduÂ, Antoni Valero, Xavier Navarro Neuroplasticity Group, Department of Cell Biology, Physiology and Immunology, Universitat AutoÁnoma de Barcelona, Barcelona, Spain Received 6 July 1999; received in revised form 12 July 1999; accepted 13 July 1999
Abstract The main objective of this study was to analyze the effects of early social isolation on the expression of calbindin D-28k (CAD-28k) in cerebellar Purkinje cells. Sprague±Dawley rats were reared either in isolation (IC) or socially housed conditions (SC) from postnatal days 18±32. Half of the rats of each group were sacri®ced at 32 days of age and the cerebella processed for immunocytochemical labeling against CAD-28k. The remaining IC animals were housed under standard conditions for re-socialization between postnatal days 32 and 62. The results indicate that Purkinje cells of rats reared under early isolation show a marked loss of CAD-28k immunoreactivity, and that this de®ciency is recovered by later social interaction, although the thickness of the molecular layer remains reduced. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calbindin; Cerebellum; Development; Isolation; Purkinje cells; Rat
Social deprivation imposed during the early postnatal period in laboratory animals is known to cause profound and long-lasting CNS abnormalities. These alterations involve dopaminergic [14], serotonergic [13] and noradrenergic [17] systems. In addition, structural sequelae include a signi®cant decrease in mesolimbic innervation of medial prefrontal cortex [22] and hippocampal neural degeneration [21]. These impairments are associated with behavioral disorders, such as locomotor hyperactivity [6,14], anxiety [9] and de®cits in memory tasks [22]. Some decades ago, Prescott [19] hypothesized that abnormal behaviors observed in animals reared under early social deprivation are related to developmental alterations in the cerebellar cytoarchitecture and function. This hypothesis received support from neurochemical studies indicating that rodents reared in isolation showed alterations in cerebellar noradrenaline and serotonin metabolism [6], associated with a signi®cant decrease in benzodiazepine binding sites [16]. In a recent study [18] we demonstrated that Golgi-impregnated Purkinje cells of rats subjected to early social isolation showed a delayed dendritic arborization; however, it is not known if such structural impairment is correlated with cellular functional abnormalities. Taking * Corresponding author. Tel.: 134-93-581-1966; fax: 134-935812-986. E-mail address: [email protected] (R. Pascual)
into account that the intracellular protein calbindin D-28k (CAD-28k) regulates Ca 21-dependent aspects of neuronal function [5] and that Purkinje cells highly express CAD28k [20], the main objective of the present study was to evaluate the effect of early social isolation on the expression of CAD-28k by using an immunocytochemical approach. Twenty-eight Sprague±Dawley female rats born from multiparous mothers were weaned at 18 days and divided in two groups counterbalanced by weight: isolated rats (IC, n 14), housed singly in small opaque plastic cages (10 £ 10 £ 10 cm), and socially-reared rats (SC, n 14) maintained in groups of three to four animals per cage (50 £ 28 £ 20 cm). Both groups had food and water available ad libitum and were maintained in the same room with regulated environmental conditions (temperature: 21 ^ 28C, humidity: 65±70%, 12:12 h light/dark cycle). Half of the IC and SC rats were sacri®ced at 32 postnatal days for histological evaluation, while the remaining IC rats were rehoused socially (three to four animals per cage) until 62 postnatal days. At either 32 or 62 postnatal days the rats were weighed, deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused transcardially with cold 0.9% saline for 1 min followed by 4% paraformaldehyde in 0.1 M phosphate buffer solution (PBS; pH 7.4) for 6 min. The cerebella were removed from the skull and post®xed in 4% paraformaldehyde for 24 h and then cryoprotected in 0.1 M PBS containing 20% sucrose and stored at 48C. Parasa-
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 60 8- 4
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R. Pascual et al. / Neuroscience Letters 272 (1999) 171±174
gital vermal sections, cut at 40 mm thickness with a freezing sliding microtome, were collected and washed free-¯oating in 0.1 M PBS with 0.3% Triton X-100 (Fluka, Buchs, Switzerland) and 1% fetal calf serum (FCS) (ICN, Costa Mesa, USA) for 1 h and then incubated for 48 h with primary polyclonal antibody generated against calbindin D-28k (rabbit; 1:2000; Chemicon). After washes with the same diluted solution, sections were incubated in secondary antisera goat anti-rabbit cyanine 3.18 labeled IgG (1:2000; Jackson Immunoresearch, West Grove, USA) overnight at 48C. Following additional washes, sections were mounted on gelatin-coated slides, dehydrated in ethanol, cleared in methylsalicylate, and coverslipped with DPX (Fluka). CAD-28k immunoreactivity was analyzed under an Olympus BX-40 microscope equipped for epi¯uorescence. Selected images (10£ magni®cation) were taken with the aid of a digital camera (Olympus DP20) attached to the microscope and analyzed using NIH Image software (freely distributed from NIH). One hundred and sixty-six parasagital vermal sections (IC: 87 sections; SC: 79 sections) were evaluated, measuring both the density of Purkinje cells (cells/mm 2) immunoreactivity to CAD-28k and the thickness of the molecular layer (where Purkinje cell dendrite arborization is distributed). Measurements were performed in the subvermal lobule VId (according to Larsell's terminology) in all the samples, because morphology is clearly distinguishable and varies little from section to section in
the vermis. Furthermore, hematoxylin-eosin alternate sections were obtained in order to evaluate possible cytoarchitectonic abnormalities. All measurements were performed on coded slides in a blind manner. Data are expressed as mean ^ SD and were compared by unpaired t-test (Stat View 4.5) and differences considered signi®cant when P , 0:05. Low power magni®cation (10£) images of cerebellar sections immunostained against CAD-28k show that 32 days old IC rats exhibit a heterogeneous pattern of Purkinje cell ¯uorescence with wide patches lacking immunoreactivity (Fig. 1A). In contrast, a highly ordered pattern of CAD28k expression was observed in age-matched SC rats (Fig. 1B). On the other hand, IC rats submitted to 30 days of socially normal experiences undergo an almost complete restoration of the CAD-28k de®cit (Fig. 1C). The density of CAD-28k immunoreactive Purkinje cells was markedly reduced in isolated versus socially reared rats by 32 days of age (P , 0:001), a difference not found after 30 days of social housing (Fig. 2A). In addition, the thickness of the molecular layer was signi®cantly reduced in IC rats versus SC rats at both 32 (IC: 165:2 ^ 17:3 mm; SC: 189:1 ^ 14:1 mm; P , 0:001) and 62 (IC: 211:9 ^ 21:9; SC: 222:9 ^ 24:3 mm; P , 0:05) postnatal days (Fig. 2B). The lack of CAD-28k-immunoreactive Purkinje cells was not due to cell death, as evidenced in cerebellar alternate sections stained with hematoxylin-eosin (Fig. 3). Body weight gain
Fig. 1. Immuno¯uorescence staining demonstrating changes in calbindin-D28k levels on representative vermal cerebellar sections of isolated (IC; (A,C)) and socially-reared (SC; (B,D)) rats, at 32 (P32) and 62 (P62) postnatal days. ML: molecular layer; PL: Purkinje cell layer; GL: internal granular layer. Scale bar, 100 mm.
R. Pascual et al. / Neuroscience Letters 272 (1999) 171±174
Fig. 2. Density of calbindin D-28k immunoreactive Purkinje cells (A) and thickness of the molecular layer (B) in vermal sublobule VI of rats reared under social (SC) and isolated (IC) conditions. *P , 0:05; **P , 0:01; ***P , 0:001 (mean ^ SD, unpaired ttest).
was similar between isolated and socially reared rats at both 32 (IC: 61:5 ^ 11:4; SC: 71:3 ^ 7:2; n.s.) and 62 (IC: 263:4 ^ 18:7; SC: 259:7 ^ 17:3; n.s.) postnatal days. The immunocytochemical results found indicate that early social isolation signi®cantly reduced the expression of CAD-28k in vermal Purkinje cells. Moreover, isolatedreared rats submitted to later social interaction during one month showed a signi®cant recovery in the expression of this calcium binding protein. Considering that CAD-28k plays a pivotal role in the regulation of intracellular Ca 21 transients, it is probable that the underexpression observed in Purkinje cells severely impair the physiology of these cells and, consequently, the information processing in the cerebellar cortex. Taking into account that the cerebellum is involved mainly in the modulation of motor functions, it is expected that isolated rats show measurable motor behavior abnormalities. By examining the motor behavior displayed by IC rats in the open ®eld test, we found a signi®cant locomotor hyperactivity, but no signs of tremor or ataxia (unpublished observations). This ®nding is consistent with that described by Airaksinen et al. [1], who evaluated the physiological role of CAD-28k by using mutant mice lacking CAD-28k (Cb 2/2). These rodents exhibited a normal locomotor behavior in standard environments, but motor incoordination when subjected to tests of motor coordination. It is not possible to discard that the IC rats evaluated in the present study were similarly ataxic when subjected to a demanding task test. This mismatch between decline in the expression of CAD-28k by Purkinje cells and relatively normal locomotor behavior, may be explained in part by the presence of additional intracellular calcium binding proteins such as parvalbumin, that is normally present in Purkinje cells and may compensate for the CAD-28k failure. The relatively short period of isolation (2 weeks) used in the present study was suf®cient to induce a signi®cant impairment in the CAD-28k expression, indicating that vermal Purkinje cells are highly sensitive to early environmental deprivation. This vulnerability may be related to the
173
fact that, in the rat, the ontogenetic expression of CAD-28k in developing Purkinje cells takes place during the ®rst month of life, increasing progressively from birth to a peak at postnatal week 2, then declining to a steady-state level at postnatal week 4 [11]. However, the diminished CAD-28k expression in isolated-reared rats may be correlated to the structural impairment in dendritic arborization observed in Golgiimpregnated Purkinje cells of socially deprived rats [18]. This ®nding is in agreement to the signi®cant hypotrophy detected in the molecular layer of isolated rats where Purkinje cell dendrite arborization is distributed. Furthermore, studies carried out in some neurodegenerative diseases [2,10,12] indicate that the decreased levels of CAD-28k are highly correlated with neural degeneration. In addition, Goodman et al. [8] showed that immature dentate granule cells are more vulnerable to hypoxic/ ischemic insults than calbindin-rich mature counterparts; and in vitro studies indicate that calbindin-positive hippo-
Fig. 3. Micrographs of the vermal cerebellar cortex of an isolated 32-day-old rat, labeled against calbindin D-28k (A) and stained with hematoxylin-eosin (B). ML: molecular layer; PL: Purkinje cell layer; GL: internal granular layer. Scale bar, 50 mm.
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campal neurons were more resistant to glutamate or calcium ionophore neurotoxicity than were neurons lacking CAD28k [15]. In the current study, comparing images of alternating sections stained by using immunocytochemical and hematoxylin-eosin procedures, it is apparent that the wide cortical areas free of immuno¯uorescent Purkinje cells are not due to cell death but to CAD-28k underexpression. This ®nding is consistent with the restoration of the density of immuno¯uorescent Purkinje cells found in the isolatedreared rats at 62 postnatal days, after 1 month of normal interaction. The bene®cial effects of re-socialization on rats previously reared under social deprivation was evidenced only in the immunoreactive expression of CAD-28k but not in the thickness of the cerebellar molecular layer. This functional recovery is consistent with studies that demonstrated that the reinstatement of a social environment to previously isolated rats may recover some behavioral and neural abnormalities such as motor hyperactivity, greater tailpinch latencies and reduced activity in mesocortico-frontal dopaminergic neurons [4,7]. Furthermore, the lack of recovery of the molecular layer hypotrophy is consistent with the fact that Purkinje cell dendritic arborization of isolatedreared rats remained stunted despite further social stimulation (unpublished observations). It is possible that failure in structural recovery may be due to the fact that the re-socialization timing applied in this study was between 32 and 62 postnatal days, when structural Purkinje cell plasticity progressively declines [3]. In conclusion, the present study demonstrates that vermal Purkinje cells are highly vulnerable to early social deprivation as shown by a marked underregulation of CAD-28k expression, and that this alteration may be recovered by adequate social interaction. This work was partially supported by grant SAF97±0147 from the CICYT (Spain). R.P. was on a leave of absence from the Faculty of Health Sciences, Universidad CatoÂlica del Maule, Talca (Chile). [1] Airaksinen, M.S., Eilers, J., Garaschuk, O., Thoenen, H., Konnerth, A. and Meyer, M., Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene. Proc. Natl. Acad. Sci. USA, 94 (1997) 1488±1493. [2] Alexianu, M.E., Ho, B-K., Mohamed, A.H., La Bella, V., Smith, R.G. and Appel, S.H., The role of calcium-binding proteins in selective motoneuron vulnerability in amyotrophic lateral sclerosis. Ann. Neurol., 36 (1994) 846±858. [3] Anderson, W.A. and Flumerfelt, B.A., Purkinje cell growth beyond the twenty-third postnatal day. Dev. Brain Res., 17 (1985) 195±200. [4] Blanc, G., HerveÂ, D., Simon, H., Lisoprawski, A., Glowinski, J. and Tassin, J.P., Response to stress of mesocortico-frontal dopaminergic neurones in rats after long-term isolation. Nature, 284 (1980) 265±267. [5] Chard, P.S., Bleakman, D., Christakos, S., Fullmer, C.S. and Miller, R.J., Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones. J. Physiol., 472 (1993) 341±357.
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