Original article
Differential expression of calbindin D28k, calretinin and parvalbumin in the cerebellum of pups of ethanol-treated female rats

Introduction

Calcium (Ca) is a unique ion that is involved in a large number of vital physiological processes and neuronal functions [3,5,13,39,47]. Ca regulates the libe­ration and synthesis of neurotransmitters, hormones, axonal transport, control of enzymatic reactions, gene transcription and other processes throughout life, in­cluding the developmental period [6,42]. During deve­lopment, Ca controls neuronal migration, process extension, formation of neuronal networks, synaptogenesis, cell division and also the process of apoptosis [4,7,20]. A lot of intracellular calcium-responsive proteins are required to control the intraneuronal level of Ca2+. These proteins are members of the so-called “EF-hand” family, also termed calcium-binding proteins (CaBPs) [26,33,42,56]. The EF-hand family of calcium-binding proteins is widely distributed in the brain. Among other well-known members of CaBPs are calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV). Calbindin D28k takes se­veral forms but only the 28 kDa molecule occurs in the neural population. CR molecular weight in the brain is about 29 kDa. PV, a small protein with mole­cular weight of about 12 kDa, occurs in the brain [6,49]. The distribution of these proteins in developing brain is not comparable to that observed in the adult brain, and their immunoreac­ti­vity changes during the developmental process [1,2,56].

Cerebellar development takes an exceptionally long time during the embryonic phase and the first postnatal period [36]. The cerebellum, particularly the cerebellar cortex, with the whole population of its neurons and processes, performs very important motor and autonomic functions, and participates in cognitive and emotional development [12,17,24, 29,48]. The cerebellar cortex contains Purkinje, basket, stellate, Golgi, Lugaro, unipolar brush and gra­nule cells, which have characteristic morphological features [16,29]. The population of these cerebellar neurons demonstrates immunohistochemical locali­zation of three calcium-binding proteins (CB, CR, PV) [44,56]. The intensity of immunoreaction and the number of positive neurons for these proteins are related to the intracellular calcium concentration [2,10,56]. Many pathological factors may change the intracellular Ca concentration and thus damage CaBPs expression.

Ethanol exposure during brain development, intrauterine exposure and/or in the early postnatal period, causes significant neuronal damage to the central nervous system (CNS) [11,15,32,34,55]. Consumption of ethanol by mothers during pregnancy can lead (in humans) to fetal alcohol syndrome (FAS), or less specific fetal alcohol effects (FAE) and fetal alcohol disorders (FAD) in their offspring. These are characterized by specific facial deformation, growth retardation and CNS dysfunction. Experimental animal studies have demonstrated that the cerebellum is among the brain target sites of ethanol CNS toxigenesis [8,23,25,34]. Neuropathological studies indicate that structural and functional deficits of the cerebellum are dependent on ethanol exposure during the developmental period [23,32,34,37]. The pathogenesis of this differential vulnerability has not yet been established. In this study we report on the cerebellar cortex localization of three major calcium-binding proteins (CB, CR, PV) in ten-day-old rat pups of ethanol-treated dams. The evidenced correlation between the duration of ethanol intoxication, the expression of calcium-binding proteins and patholo­gical changes of neurons during the developmental process of rat cerebellar cortex may help us understand the pathogenesis of ethanol teratogenesis.

Material and methods

The investigations were carried out on cerebellum collected from the ten-day-old pup offspring of outbred Wistar female rats. Females were treated with ethanol (12%, 6 g/kg body mass) during pregnancy and/or lactation. The control groups were composed of dams treated with isocaloric dextran solution with maltose, during pregnancy and/or lactation, and also untreated female rats (Table I). Solutions were admini­stered via intragastric intubation once a day.

The animals were fed standard chow and received tap water ad libitum. To assess the cerebellum by light microscope, it was fixed in 4% buffered para­for­maldehyde (pH 7.4) then embedded in paraffin, cut serially into 8 µm sections. Every tenth slide was stained with haematoxylin and eosin, and selected preparations were subjected to immunohistochemical reactions.

The specimens of cerebellum were stained immu­nohistochemically with the antibodies calbindin D-28k (Sigma, clone CB-955) at dilution of 1 : 2500, calretinin (Dako, clone DAK calret 1) at dilution 1 : 75 and parvalbumin (Sigma, clone Parv-19) at dilution 1 : 2000.

Results

The distribution of calcium-binding proteins in the cerebellar cortex in ten-day-old rat pups of control groups (ID, IID, IIID, C) and in ethanol-treated dams of three experimental groups (IA, IIA, IIIA) is summarized in Table I. The number of immunoreactive neurons and the staining intensity for CaBPs (CA, CR, PV) in control groups (ID, IID, IIID, C) were the same.

Calbindin D28k-positive cells in the cerebellar cortex in ten-day-old rats in the control groups were all Purkinje cells with their processes and Golgi cells (Figs. 1, 1A). The staining intensity for CB proteins was very impressive. In the pups of dams treated with ethanol during pregnancy and lactation (group IA), CB immunoreactivity was observed only in a few Purkinje cells, but in Golgi cells it was not visible (Figs. 2, 2A). In dams treated with ethanol during pregnancy (group IIA), positive Purkinje cells were occasionally observed and there was no immunoreactivity in Golgi cells (Fig. 3). In the cerebellar cortex in pups of dams treated with ethanol during lactation (group IIIA), expression of CB proteins in Purkinje and Golgi cells was slightly visible (Figs. 4, 4A).

Calretinin-positive cells in the cerebellar cortex of pups in the control groups were interneurons – Lugaro, Golgi, unipolar brush cells – and some weak Purkinje cells (Figs. 5A-C). In the experimental group IA, the localization of CR expression was visible only in interneurons (Fig. 6). All unipolar brush cells show­ed very positive immunoreactivity (Figs. 6, 6A). Calretinin-positive interneurons showed lower intensity in the remaining two groups (IIA, IIIA) (Figs. 7, 8). Positive Purkinje cells were found in experimental group IIIA (Fig. 8).

Parvalbumin-positive neurons were found in the cerebellar cortex in the control group in some Purkinje cells and in Golgi, basket and stellate cells (Figs. 9A-C). In the IIA and IIIA experimental groups, a PA-posi­tive reaction was visible in a few Purkinje cells and sometimes also in basket cells. (Figs. 10, 11). The slight­est immunoreaction in interneurons and no immu­noreaction in Purkinje cells was observed in group IA (dams treated with ethanol during pregnancy and lactation) (Fig. 12).

In all the experimental groups (IA, IIA, IIIA), a fall in the number of Purkinje cells and their hypoxic/ ischaemic damage and loss of interneurons were also noted (Figs. 2, 7, 8, 10-12).

Discussion

The expression of calbindin D28k, calretinin and parvalbumin, three important calcium-binding proteins, observed in distinct neuronal populations, was examined in ten-day-old neonatal rat pups of both control and experimental groups. Immunoreactivity for these CaBPs has been detected in similar populations of neurons in mammals (including humans) and rodents or birds [18,22,30,53,54]. The distribution of three CaBPs in the rat cerebellar cortex including Purkinje cells, granule cells and interneurons, as well as their processes, has been documented previously [21,46,56]. Our study also showed the distribution of calbindin D28k immunoreactivity in the cerebellar cortex of control pups virtually in all Purkinje cells with their processes and in Golgi cells. All CB immu­noreactive Purkinje cells have a regular surface. Lugaro, Golgi, and unipolar brush cells and some Purkinje cells were positive for calretinin. The majority of Purkinje cells showed a strong PV reactivity. In addition, a PV-positive reaction was observed in Golgi, basket and stellate cells.

The localization of expression of CaBPs in the cerebellum of ten-day-old pups of ethanol-treated dams underwent changes. The ethanol-induced cerebellar degeneration not only in adults but also in children whose mothers have been drinking during pregnancy and lactation has been described previously [15,31,38,41,55]. Unfortunately, ethanol-induced cerebellar degeneration causes neuropsychological deficits observed in children with FAS, FAE and FAD [9,32,40]. A lot of mechanisms underlying ethanol-induced cellular degeneration and functional altera­tions have been documented. Hoffman et al. (1995) suggested that excitotoxicity (excessive stimulation of neurotransmitters) is an important mechanism con­tributing to ethanol-induced neuropathology in the cerebellum [28]. Pathophysiological mechanisms such as glial abnormalities, increase in pro-apoptosis gene expression, decreased mitochondrial enzyme activities, change in growth factors, oxidative stress and many others, cause cerebellar degeneration with cognitive disorders observed in offspring whose mothers have been drinking ethanol [19,27,43,50,51]. Earlier experimental studies demonstrated that Pur­kinje and granule cells and interneurons of the cerebellar cortex are the neurons vulnerable to ethanol exposure [31,40,52]. The specific distribution pattern of calcium-binding proteins in the cerebellar cortex suggests that they may be involved in cell activities, and the alteration of their immunohistological pre­sentation may lead to neurodegenerative conditions. In our study, calbindin D28k-positive immunoreaction was not observed in Golgi cells and only sometimes in Purkinje cells in the experimental IA and IIA groups. The staining intensity of CB decreased in all experimental groups. CB-expressing neurons are resistant to migration, differentiation and excitotoxic insults; thus the decrease in CB reaction may cause developmental and functional disturbances [7,36,56].

Parvalbumin immunoreactivity was not observed in Purkinje, basket and stellate cells in the experimental IA group. In two other experimental groups (IIA and IIIA), PV-positive weaker staining intensity reaction was visible only in a few Purkinje and basket cells. As has been postulated, PV regulates the liberation and synthesis of the neurotransmitter -amino­butyric acid (GABA), and in the after-hyperpolarization period the lack of or decrease in PV immunore­activity may suggest the alteration of cerebellar function in these mechanisms [1,7,42].

Only the calretinin immunoreaction was more intense in all interneurons, above all, in unipolar brush cells in the experimental groups as compared with the control groups. The highest intensity of CR was visible in group IA. No immunoreactivity of CR in Purkinje cells was observed. Excessively high CR immu­noreactivity in interneurons may cause functional alteration and death of neurons in mechanisms of excessive activity of neuronal metabolism [1,14].

In our experimental groups, the loss of or ischae­mic changes in Purkinje cells and loss of interneurons in the cerebellar cortex suggest that the disturbed immunoreactivity of CaBPs after ethanol intoxication may induce neuronal death by necro­sis after a period of ischaemia or apoptosis [35,57]. Disturbances of CaBPs are able to prevent the neuronal death by more than one mechanism, as well as mitochondrial dysfunction [35]. CB probably inhibits the activity of caspase-3, while the increase of CR may indicate mitochondrial cal­cium accumulation with enhanced production of reactive oxygen species (ROS), whereas PV may provoke the de­crease in mitochondrial membrane potential [45].

Therefore, too high or too low level of intracellular calcium causes both neuronal and functional disturbances.

The results of our present study showed that ethanol intoxication during pregnancy and/or lactation causes alteration in the immunoreaction of cal­cium-binding proteins (CB, CR, PV) in neurons of the cerebellar cortex of ten-day-old rat pups. Changes of immunoreactivity of these CaBPs may indicate an important role in calcium-dependent ethanol-induced developmental and functional alterations.

References

 1. Alfahel-Kakunda A, Silverman WF. Calcium-binding proteins in the substantia nigra and ventral tegmental area during development: correlation with dopaminergic compartmentalization. Dev Brain Res 1997; 103: 9-20.  

2. Andressen C, Blümcke I, Celio MR. Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res 1993; 271: 181-208.  

3. Berridge MJ, Lipp P, Bootman MD. The versatility and univer­sality of calcium signalling. Nat Rev Mol Cell Biol 2000; 1: 11-21.

4. Bootman MD, Collins TJ. Peppiatt CM, Prothero LS, MacKenzie L, De Sm et P, Travers M, Tovey SC, Seo JT, Berridge MJ, Ciccolini F, Lipp P. Calcium signalling – an overview. Semin. Cell Dev Biol 2001; 12: 3-10.  

5. Braet K, Cabooter L, Paemeleire K, Leybaert L. Calcium signal communication in the central nervous system. Biol Cell 2004; 96: 79-91.  

6. Braun K. Calcium binding Proteins in Avian and Mammalian Central Nervous System: Localization. Development and Possible Function, Prog Histochem Cytochem 1990; 21: 1-64.  

7. Bregestovski P, Spitzer N. Calcium in the function of the nervous system: New implications. Cell Calcium 2005; 37: 371-374.  

8. Byrnes ML, Reynolds JN, Brien JF. Effect of prenatal ethanol exposure during the brain growth spurt of the guinea pig. Neurotoxicol Teratol 2001; 23: 355-364.  

9. Calhoun F, Warren K. Fetal alcohol syndrome: Historical perspectives. Neurosci Biobehav Rev 2007; 31: 168-171.

10. Campbell AK. Intracellular calcium: its universal role as regulator. In: Gutfreund H (ed.). Monographs in Molecular Biophysics and Biochemistry. Wiley, New York 1983; pp. 1-556.

11. Chaudhuri Joydeep D. Alcohol and the developing fetus – a review. Med Sci Monit 2000; 6: 1031-1041.

12. Cheron G, Servais L, Dan B. Cerebellar network plasticity: from genes to fast oscillaltion. Neuroscience 2008; 153: 1-19.

13. Clapham DE. Calcium signalling. Cell 2007; 131: 1047-1058.

14. Crook J, Hendrickson A, Robinson FR. Co-localization of glycine and gaba immunoreactivity in interneurons in Macaca monkey cerebellar cortex. Neuroscience 2006; 141: 1951-1959.

15. Dikranian K, Qin Yue-Qin, Labruyere J, Nemmers B, Olney JW. Ethanol-induced neuroapoptosis in the developing rodent cerebellum and related brain stem structures. Dev Brain Res 2005; 155: 1-13.

16. Dino MR, Perachio AA, Mugnaini E. Cerebellar unipolar brush cell are targets of primary vestibular afferents: an experimental study in the gerbil. Exp Brain Res 2001; 140: 162-170.

17. Eccles JC, Ito M, Szentagothai J. The cerebellum as a neuronal machine. Springer-Verlag, Berlin 1967.

18. Fortin M, Marchand R, Parent A. Calcium-binding proteins in primate cerebellum. Neurosci Res 1998; 30: 155-168.

19. Freund G. Apoptosis and gene expression: Perspectives on alcohol-induced brain damage. Alcohol 1994; 11: 385-387.

20. Gabellini N. Transcriptional regulation by cAMP and Ca2+ links the Na+/Ca2+ exchangers 3 to memory and sensory pathways. Mol Neurobiol 2004; 30: 91-116.

21. Gall D, Roussel C, Susa I, E'Angelo E, Rossi P, Bearzatto B, Ga­las MCh, Blum D, Schurmans S, Schiffmann SN. Altered Neuronal Excitability in Cerebellar Granule Cells of Mice Lacking Calretinin. J Neurosci 2003; 23: 9320-9327.

22. Garcia-Segura LM, Baetens D, Roth J, Norman AW, Orci L. Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system. Brain Res 1984; 296: 75-86.

23. Goodlett ChR, Peaarlman AD, Lundahl KR. Binge neonatal alcohol intubations induce dose-dependent loss of Purkinje cells. Neurotoxicology and Teratology 1998; 20: 285-292.

24. Gordon N. The cerebellum and cognition. Eur J Paediatr Neurol 2007; 11: 232-234.

25. Guerri C. Neuroanatomical and neurophysiological mechanisms involved in central nervous system dysfunctions induced by prenatal alcohol exposure. Alcohol Clin Exp Res 1998; 22: 304-312.

26. Heizmann CW, Braun K. Calcium regulation by calcium-binding proteins in neurodegerative disorders. Neuroscience Intelligence Unit, RG Landes Company, Austin; Springer Verlag, Heidelberg 1995.

27. Henderson CE. Role of neurotrophic factors in neuronal development. Curr Opin Neurobiol 1996; 6: 64-70.

28. Hoffman PL, Iorio KR, Snell LD, Tabakoff B. Attenuation of glutamate-induced neurotoxicity in chronically ethanol-exposed cerebellar granule cells by NMDA receptor antagonists and ganglioside GM1. Alcohol Clin Exp Res 1995; 19: 721-726.

29. Ito M. Cerebellar circuitry as a neuronal machine. Prog Neuro-biol 2006; 78: 272-303.

30. Jande SS, Tolnai S, Lawson DEM. Immunohistochemical localization of vitamin D-dependent calcium-binding protein in duodenum, kidney, uterus and cerebellum of chickens. Histochemistry 1981; 71: 99-116.

31. Jiang Y, Kumada T, Cameron DB, Komuro H. Cerebellar granule cell migration and the effects of alcohol. Dev Neurobiol 2008; 30: 7-23.

32. Jaatiten P, Rintala J. Mechanisms of ethanol induced degeneration in the developing, mature, and aging cerebellum. Cerebellum 2008; 7: 332-347.

33. Kadowaki K, Mc Gowan E, Mock G, Chandler S, Emson PC. Distribution of calcium-binding protein mRNAs in rat cerebellar cortex. Neurosci Lett 1993; 153: 80-84.

34. Kane CJM, Chang JY, Roberson PK, Garg TK, Han L. Ethanol exposure of neonatal rats does not increase biomarkers of oxidative stress in isolated cerebellar granule neurons. Alcohol 2008; 42: 29-36.

35. Kristian T. Siesjő BK. Calcium in ischemic cell death. Stroke 1998; 29: 705-718.

36. Laure-Kamionowska M, Maślińska D. Calbidin positive Purkinje cells in the pathology of human cerebellum occurring at the time of its development. Folia Neuropathol 2009; 47: 300-305.

37. Lee Y, Rowe J, Eskue K, West JR, Maier SE. Alcohol exposure on postnatal day 5 induces Purkinje cell loss and evidence of Purkinje cell degradation in lobule I of rat cerebellum. Alcohol 2008; 42: 295-302.

38. Lewandowska E, Kujawa M, Jędrzejewska A. Ethanol-induced changes in Purkinje cells of the rat cerebellum I. The ultrastructural changes following chronic ethanol intoxication (qualitative study). Folia Neuropathol 1994; 32: 51-59.

39. Lledo PM, Somasundaram B, Morton AJ, Emson PC, Mason WT. Stable transfection of calbindin-D28k into the GH3 cell line alters calcium currents and intracellular calcium homeostasis. Neuron 1992; 9: 943-954.

40. Maier SE, West JR. Regional differences in cell loss associated with binge-like alcohol exposure during the first two trimesters equivalent in the rat. Alcohol 2001; 23: 49-57.

41. Oyama LM, Couto RC, Couto GEC, Damaso AR, Oller do Nascimento CM. Ethanol intake during lactation I. Effects on dams' metabolism and pups' body weight gain. Alcohol 2000; 21: 195-200.

42. Parent A, Fortin M, Côté P-Y, Cicchetti F. Calcium-binding proteins in primate basal ganglia. Neurosci Res 1996; 25: 309-334.

43. Ramachandran V, Perez A, Chen J, Senthil D, Schenker S, Henderson GI. In utero ethanol exposure causes mitochondrial dysfunction, which can result in apoptotic cell death in fetal brain: A potential role for 4-hydroxynonenal. Alcohol Clin Exp Res 2001; 25: 862-871.

44. Resibois A, Rogers JH. Calretinin in rat brain: An immunohistochemical study. Neuroscience 1992; 46: 101-134.

45. Richter C. Pro-oxidants and mitochondrial Ca2+: their relationship to apoptosis and oncogenesis. FEBS Lett 1993; 325: 104-107.

46. Rogers JH. Immunoreactivity for calretinin and other calcium-binding proteins in cerebellum. Neuroscience 1989; 31: 711-721.

47. Rottingen J, Iversen JG. Ruled by waves? Intracellular and intercellular calcium spignalling. Acta Physiol Scand 2000; 169: 203-219.

48. Schmahmann JD, Caplan D. Cognition, emotion and the cerebellum. Brain 2006; 129: 290-292.

49. Seto-Ohsima A, Emson PC, Lawson E, Mountjoy CQ, Carasco LH. Loss of matrix calcium-binding protein-containing neurons in Huntington’s disease. Lancet 1988; 1: 1252-1255.

50. Snyder AK. Responses of glia to alcohol. In: The role of glia in neurotoxicity. Aschner M, Kimelberg HK (eds). CRC Press, Boca Raton 1996; pp. 111-135.

51. Sun AY, Sun GY. Ethanol and oxidative mechanisms in the brain. J Biomed Sci 2001; 8: 37-43.

52. Tavares MA, Paula-Barbosa MM, Cadete-Leite A. Chronic alcohol consumption reduces the cortical layer volumes and the number of neurons of the rat cerebellar cortex. Alcohol Clin Exp Res 1987; 11: 315-319.

53. Timurkaan S, Karan M, Atalar O. Distribution of Calbindin-D28k in the cerebellum of Martes foina. Veterinarski Arhiv 2006; 766: 275-279.

54. Ulfig N, Chan WY. Differential expression of calcium-binding proteins in the red nucleus of the developing and adult human brain. Anat Embryol 2001; 203: 95-108.

55. Wierzba-Bobrowicz T, Lewandowska E, Kosno-Kruszewska E, Lechowicz W, Skórzewska A, Gwiazda E, Pasennik E. Dendritic and microglial cells in pups of alcohol-treated female rats. Folia Neuropathol 2003; 41: 131-137.

56. Yew DT, Luo CB, Heizmann CW, Chan WY. Differential expression of calretinin, calbindin D28k and parvalbumin in the developing human cerebellum. Dev Brain Res 1997; 103: 37-45.

57. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin S, Petit P, Mignotte B, Kroemer G. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 1995; 182: 367-377.