Journal of Chemical Neuroanatomy 61–62 (2014) 88–93
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Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu
Calcium-binding proteins in the laterodorsal thalamic nucleus during development of the guinea pig Witold Z˙akowski *, Krystyna Bogus-Nowakowska, Barbara Wasilewska, Beata Hermanowicz, Anna Robak Department of Comparative Anatomy, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Pl. Ło´dzki 3, 10-727 Olsztyn, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 May 2014 Received in revised form 8 August 2014 Accepted 8 August 2014 Available online 18 August 2014
The laterodorsal thalamic nucleus (LD) is often treated as a part of the anterior thalamic nuclei (ATN) because of its location and similar connectivity. Our previous studies have shown that distribution of three calcium-binding proteins, i.e. calbindin D28k (CB), calretinin (CR) and parvalbumin (PV), changes within the ATN during development of the guinea pig. The aim of this study is to examine the immunoreactivity pattern of these proteins in the LD in the guinea pig ontogeny. Brains from animals ranging from 40th embryonic day to 80th postnatal day were used in the study. Two methods were applied: a single-labelling immunoenzymatic method and double-labelling immunofluorescence. No changes of the distribution pattern of the substances were observed throughout the examined developmental stages. CB and CR were the most abundantly expressed proteins in perikarya of the LD. Numerous CB- and CR-immunoreactive cell bodies were found throughout the whole extent of the nucleus. In most of these cell bodies both proteins colocalized vastly. The highest immunoreactivity of the perikarya containing CB and CR was observed in the mediodorsal part of the LD and in its rostral portion. In regard to PV, single cell bodies were observed mostly in the dorsal part of the nucleus. PV did not colocalize with the other proteins. In summary, all the studied calcium-binding proteins were already present in the LD at prenatal developmental stages and the pattern of distribution remained virtually constant until adulthood. Thus, the LD differs considerably from the ATN in an aspect of neurochemical cell differentiation. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Laterodorsal thalamic nucleus Calbindin Calretinin Parvalbumin Development Guinea pig
Introduction The laterodorsal thalamic nucleus (LD) is often treated as a part of the anterior thalamic nuclei (ATN) complex, because of its close location and similarities in afferent and efferent connectivity (Jones, 2007; Price, 1995). Likewise the ATN, the LD is involved in learning and memory processes, particularly these related to spatial tasks (Mizumori et al., 1994; Shibata, 2000; Shibata and Naito, 2005; Van Groen et al., 2002; Wilton et al., 2001). However, some studies have suggested that the LD and each nucleus of the anterior thalamus may contribute to different aspects of spatial memory processing (Shibata and Naito, 2005; Van Groen et al.,
Abbreviations: AD, anterodorsal nucleus; AM, anteromedial nucleus; ATN, anterior thalamic nuclei; AV, anteroventral nucleus; CaBPs, calcium-binding proteins; CB, calbindin; CR, calretinin; LD, laterodorsal nucleus; PV, parvalbumin. * Corresponding author. Tel.: +48 89 523 43 01; fax: +48 89 523 43 01. E-mail addresses: [email protected] (W. Z˙akowski), [email protected] (K. Bogus-Nowakowska), [email protected] (B. Wasilewska), [email protected] (B. Hermanowicz), [email protected] (A. Robak). http://dx.doi.org/10.1016/j.jchemneu.2014.08.003 0891-0618/ß 2014 Elsevier B.V. All rights reserved.
2002). Our recent studies have shown that the distribution pattern of three calcium-binding proteins (CaBPs), i.e. calbindin D28k (CB), calretinin (CR) and parvalbumin (PV), differs among the ATN and changes during the development of the guinea pig (Z˙akowski et al., 2013; Z˙akowski and Robak, 2013). These proteins, along with others CaBPs, are essential to control the intracellular level of Ca2+ in a temporal and spatial dimension. CB and CR have been suggested to have similar functions, such as neuroprotection against excitotoxic cell death (D‘Orlando et al., 2001, 2002), while PV, which is mainly expressed in GABA-ergic interneurons, plays a major role in regulating the local inhibitory effects (Schwaller, 2009). Interestingly, our studies have revealed that the pattern of CaBPs immunoreactivity in the ATN of the adult guinea pig differs from that described in the rat, but partially matches the human one, especially in regard to CR (Z˙akowski and Robak, 2013). The distribution of the studied proteins has been described in the LD of the adult rat (Arai et al., 1994; Battaglia et al., 1992; Celio, 1990; ˜ as et al., 1991; Re´sibois and Rogers, 1992; Se´quier et al., Coven 1990; Winsky et al., 1992) and human (Fortin et al., 1998; Mu¨nkle et al., 2000), but only few studies have concerned this matter in the developing thalamus (Frassoni et al., 1991, 1998). Moreover, there
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is a lack of data regarding coexistence of CB, CR and PV in neurons of the LD. Taking into account a crucial role of CaBPs in a proper functioning of nervous cells, it seems quite interesting to reveal their distribution in the LD during prenatal and postnatal development. The present study will allow to compare the expression of the studied substances in the LD and the ATN, showing differences and similarities among these nuclei in an aspect of neurochemical differentiation during development of the guinea pig. Therefore, the aim of the study was to examine the distribution of calbindin, calretinin and parvalbumin as well as their coexistence pattern in the LD of the guinea pig at several developmental stages since 40th foetal day to 80th postnatal day. Materials and methods Tissue preparation All experimental protocols were approved by the Local Ethical Commission for Animal Experimentation at University of Warmia and Mazury in Olsztyn, Poland (in accordance with EU Directive 2010/63/EU for animal experiments). The study was performed on brains of the Dunkin–Hartley guinea pigs (Cavia porcellus) at several developmental stages: E40, E50, E60 (40th, 50th, 60th day of gestation; n = 3 for each stage), P0 and P80 (newborn and 80th day after birth; n = 3). All procedures of the tissue preparation were described in detail in our previous publication (Z˙akowski and Robak, 2013). Immunohistochemical procedures Sections through the LD from the postnatal and foetal brains were processed for two immunohistochemical methods: a single-labelling immunoenzymatic method and double-labelling immunofluorescence. For the first method we used solutions of rabbit anti-calbindin (1:4000, Swant, CH, product no. 6B38a), mouse anti-parvalbumin (1:2000, Sigma, USA, P3088) and mouse anti-calretinin (1:2000, Swant, CH, 6B3). The sections were then incubated with ImmPRESS Reagent (Vector Laboratories, USA) and subsequently with 3,3-diaminobenzidine substrate-chromogen solution
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(DakoCytomytation, USA). For the immunofluorescence, we used various mixtures of rabbit anti-calbindin (1:4000, Swant, CH, 6B38a), mouse anti-parvalbumin (1:2000, Sigma, USA, P3088) and mouse (1:2000, Swant, CH) or rabbit anti-calretinin (1:2000, Swant, CH, 7699/3H). Each mixture composed of antibodies raised in different species. In order to show the binding sites of the primary antibodies, the sections were incubated with mixtures of secondary antibodies: Cy3-conjugated-anti-mouse (1:10,000, Jackson ImmunoLabs, USA, 715-165-150) and FITC-conjugated-anti-rabbit (1:800, Jackson Immunolabs, USA, 711-095-152). Both methods were described in detail by Z˙akowski and Robak (2013). To test antibody specificity various controls were applied. To test primary antibodies specificity, antisera produced in different species and/or provided by different manufacturers were tested on the same sections according to the immunohistochemical methods. The staining patterns were identical for all variants used. The antibodies, which fitted better with various combinations of the others antibodies were chosen. The antibodies and method specificity were also tested in standard controls, i.e. the omission as well as replacement of all primary antisera by non-immune sera. In order to confirm the boundaries of the LD, some coronal sections from each of the studied stages were stained with cresyl violet according to the Nissl method and with mouse anti-NeuN (Millipore, USA, MAB377) – a neurochemical pan-neuronal marker – according to the immunohistochemical protocol. The sections were examined with an Olympus BX51 microscope equipped with a CCD camera connected to a PC. Images were acquired with Cell-F software (Olympus GmbH, Germany).
Results The localization and structure of the LD was maintained throughout all the examined developmental stages, i.e. E40, E50, E60, P0 and P80. Calbindin, calretinin and parvalbumin were already present at E40 and the distribution pattern of all the three proteins was virtually unchanged in the ontogeny of the guinea pig (Fig. 1). Numerous CB- (Figs. 1 and 2A) and CR-immunoreactive (Fig. 2E) perikarya were detected throughout the whole extent of the LD, with the highest concentration in the mediodorsal part of
Fig. 1. Photomicrographs of coronal sections through the middle portion of the laterodorsal thalamic nucleus in the guinea pig stained for calbindin at: (A) E40; (B) E50; (C) P0 and (D) P80. Calbindin, as well as the other studied proteins, was detected in the laterodorsal nucleus already in 40th day of the prenatal development and the distribution pattern was virtually unchanged throughout the examined developmental stages. Scale bars: 500 mm.
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Fig. 2. Photomicrographs of coronal sections through the rostral portion of the laterodorsal thalamic nucleus in the guinea pig brain at E60 stained for: (A and B) calbindin (green) and parvalbumin (red); (C and D) calbindin (green) and calretinin (red); (E and F) parvalbumin (green) and calretinin (red). The strongest immunoreactivity of calbindin and calretinin was present in the mediodorsal part of the nucleus, while parvalbumin was observed mostly in the dorsal part. Notice the abundant colocalization (yellow) of calbindin and calretinin (D) and a lack of parvalbumin colocalization with other proteins (B and F). Scale bars: A, C and E – 500 mm; B, D and F – 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
the nucleus (Fig. 2B and F) and in its rostral portion. In most of these perikarya, both proteins, CB and CR, colocalized abundantly (Figs. 2C, D and 3A). CB and CR were also observed individually in some perikarya of the LD, especially in its lateroventral part. PV was detected in single cell bodies limited mostly to the dorsal part of the nucleus (Fig. 2B and F). PV did not coexist in neurons with the other CaBPs (Figs. 2B, F and 3B). At P80, the perikarya immunopositive for the studied CaBPs were more scattered throughout the nucleus in comparison to the earlier stages, but CB- and CR-immunoreactive cell bodies were still the most numerous (Fig. 4A–C). In all the studied developmental stages, neurons containing CB, CR and PV had oval- or polygonal-shaped
perikarya and gave rise to various-length processes (Fig. 3A and B). The LD displayed strongly stained neuropil for CB (Fig. 4A) and CR (Fig. 4B), especially in the mediodorsal part of the nucleus, as well as weakly stained one for PV (Fig. 4C). Discussion The present study has shown that the studied CaBPs were already present in the LD of the guinea pig at E40 and the pattern of distribution remained virtually unchanged until P80. It is in contrast to the rat LD, in which only CR has been detected prenatally, since E19 (Frassoni et al., 1998). This developmental
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Fig. 3. Photomicrographs showing perikarya in the laterodorsal thalamic nucleus of the guinea pig brain at E60, which contained: (A) calbindin (green) and calretinin (red); (B) calbindin (green) and parvalbumin (red). Notice the colocalization (yellow) of calbindin and calretinin (A) and a lack of colocalization between calbindin and parvalbumin (B). Scale bars: 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
stage in rats corresponds to approximately E33 in guinea pigs (Workman et al., 2013). CR-positive cell bodies were still present at P0, but during the second postnatal week of the rat development, CR immunoreactivity decreased (Frassoni et al., 1998). In the LD of adult rats, scattered CR-immunoreactive perikarya were observed mostly in the rostral portion of the nucleus (Arai et al., 1994; Winsky et al., 1992). According to Frassoni et al. (1991), CB appeared in the rat LD not before P11 and an adult-like pattern of the CB distribution was displayed by the nucleus at P22. In adult rats, perikarya containing CB were detected throughout the whole extent of the LD (Arai et al., 1994; Battaglia et al., 1992; Celio, 1990). In regard to PV, the rat LD was devoid of any immunoreactivity at birth and the first postnatal days, but until P22, number of PV-positive fibres and punctate structures progressively increased (Frassoni et al., 1991). In adult rats, high PV immunoreactivity of the LD neuropil, in both fibres and terminals, has been found (Arai ˜ as et al., 1991). The results of our et al., 1994; Celio, 1990; Coven study indicate that the expression of CB, CR and PV in the LD of the guinea pig is much higher than in the rat LD. First of all, some neurons of the guinea pig LD contained PV. Secondly, all the studied proteins were already expressed at prenatal stages of the guinea pig development. Some differences between these two species exist also in an adult-like pattern of distribution: CR were observed mostly in perikarya of the rostral portion in the rat LD,
Fig. 4. Photomicrographs of coronal sections through the middle portion of the laterodorsal thalamic nucleus in the guinea pig brain at P80 stained for: (A) calbindin; (B) calretinin and (C) parvalbumin. Notice the strongest immunoreactivity of calbindin and calretinin in the mediodorsal part of the nucleus (A and B, respectively). Scale bars: 500 mm.
whereas in the guinea pig, CR was detected in perikarya throughout the whole extent of the nucleus. Interestingly, our results matches the CaBPs distribution pattern in the adult human LD, in which numerous CB- and CR-immunoreactive cell bodies were evenly distributed throughout the nucleus and few PVpositive perikarya were also detected (Mu¨nkle et al., 2000). The nature of the differences between the guinea pig and rat LD, as well as the similarities between the guinea pig and human LD remains unclear. Our previous studies have shown that high expression of
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CB, CR and PV in the anterior thalamus of the guinea pig was also different to that revealed in the rat ATN, but partially similar to the human one (Z˙akowski et al., 2013; Z˙akowski and Robak, 2013). Apparently, the studied CaBPs play more significant role in the functioning of both the LD and ATN in guinea pigs and humans than in rats. The LD plays, through its interactions with the hippocampus, a key role in spatial learning and memory processes (Mizumori et al., 1994; Shibata, 2000; Shibata and Naito, 2005; Van Groen et al., 2002; Wilton et al., 2001). In rats, the LD receives projections from the cortices implicated in various aspects of spatial memory and in cognitive and behavioural functions, i.e. the prelimbic, anterior cingulate and subicular cortices, secondary motor cortex, visual area 18b and retrosplenial cortices (Shibata and Naito, 2005; Thompson and Robertson, 1987; Van Groen and Wyss, 1990a,b,c, 1992a). The LD receives projections from some subcortical structures, such as the pretectal complex, superior colliculus, geniculate nucleus, thalamic reticular nucleus and zona incerta (Thompson and Robertson, 1987), while Bezdudnaya and Keller (2008) have shown that LD neurons receive direct inputs from the trigeminal nuclei. The main projections from the LD are to the infraradiata, precentral agranular, retrosplenial, visual area 18b, subicular, and entorhinal cortices (Van Groen and Wyss, 1992b). Furthermore, Mizumori and Williams (1993) have demonstrated that the LD contains ‘head direction cells’, which fire in accordance to the directional heading in the horizontal plane, independent of animal location and behaviour. Taking all these anatomical and electrophysiological data together, it has been postulated that the LD is a multisensory nucleus that integrates multimodal information (somatosensory and visual inputs) for spatial orientation and learning tasks (Bezdudnaya and Keller, 2008). It is unknown, whether the functions of the LD may have an influence on the developmental distribution of the CaBPs in the guinea pig. According to the obtained results, all the studied proteins were present in the LD already at 40th day of prenatal development of the guinea pig. At this stage, an existence of any visual stimuli is rather unlikely, since guinea pigs open their eyes between 50th and 60th day of embryonic life (Workman et al., 2013; Z˙akowski and Robak, 2013). More studies are needed to determine the relations between the early presence of the CaBPs in the guinea pig LD and the functions of the nucleus, especially these related to somatosensory stimuli and the head direction system. Undoubtedly, the studied proteins are important for normal brain development due to their capacity to buffer calcium ions. The concentration of intracellular calcium plays an essential role in both immature neurons and mature ones, thus it have to be kept at a definite level and any aberration may lead to severe alterations during development. Considering a close location and similar connectivity with the cortex, the LD is often treated as a part of the ATN (Jones, 2007; Price, 1995), which comprises three nuclei: the anteromedial (AM), anteroventral (AV) and anterodorsal (AD). We have previously revealed developmental changes of the CaBPs distribution within the ATN of the guinea pig. Briefly, CR appeared in perikarya for the first time at E50 in the AM and at E60 in the AV, while CB was observed in cell bodies of the AM not before P20; PV was vastly present only in the neuropil of the AD since E40 (Z˙akowski et al., 2013; Z˙akowski and Robak, 2013). Our present and previous studies have shown that the LD and each nucleus of the ATN differ considerably in a neurochemical aspect. The most striking difference between the ATN and LD was the presence of PVpositive cell bodies in the latter one, as well as virtually constant distribution pattern of the studied proteins during development of the LD. Van Groen et al. (2001, 2002) have shown that lesions limited to the LD impair spatial learning and memory, but this effect is significantly magnified when the lesion extends into the
ATN. Results of our studies may indirectly support the idea that although individual nuclei of the anterior thalamus and the LD play an important role in spatial learning and memory, they may contribute to different aspects of these processes. The only common feature between the ATN and LD was the coexistence of CB and CR. Both substances colocalized in perikarya of the LD since E40 (present study) and AM since P20 (Z˙akowski et al., 2013). Coexistence of CB and CR may indicate that these two calciumbinding proteins work synergistically both in the LD and AM. D‘Orlando et al. (2001, 2002) have revealed that these two proteins can help to delay the onset of cell death after excitotoxic stimulation. Taking into account that CB and CR have a similar amino acid sequence (Rogers, 1987), they may act in a similar way. On the other hand, these two proteins show differences in domain organization (Palczewska et al., 2003), so they may complement each other to ensure proper functioning of neurons of the two nuclei. In conclusion, our study has shown that the LD of the guinea pig displayed high level of the CaBPs immunoreactivity, especially CB and CR, during prenatal and postnatal development. Despite similar location, connectivity and functions, the LD differed considerably from each nucleus of the ATN in an aspect of neurochemical cell differentiation. Ethical approval Not required. References Arai, R., Jacobowitz, D.M., Deura, S., 1994. Distribution of calretinin, calbindin-D28k, and parvalbumin in the rat thalamus. Brain Res. Bull. 33, 595–614. Battaglia, G., Colacitti, C., Bentivoglio, M., 1992. The relationship of calbindincontaining neurons with substance P, Leu-enkephalin and cholecystokinin fibres: an immunohistochemical study in the rat thalamus. J. Chem. Neuroanat. 5, 453–464. Bezdudnaya, T., Keller, A., 2008. Laterodorsal nucleus of the thalamus: a processor of somatosensory inputs. J. Comp. Neurol. 507, 1979–1989. Celio, M.R., 1990. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375–475. ˜ as, R., De Leo´n, M., Alonso, J.R., Are´valo, R., Lara, J., Aijo´n, J., 1991. Distribution Coven of parvalbumin-immunoreactivity in the rat thalamus using a monoclonal antibody. Arch. Ital. Biol. 129, 199–210. D‘Orlando, C., Celio, M.R., Schwaller, B., 2002. Calretinin and calbindin D-28k, but not parvalbumin protect against glutamate-induced delayed excitotoxicity in transfected N18-RE 105 neuroblastoma-retina hybrid cells. Brain Res. 945, 181–190. D‘Orlando, C., Fellay, B., Schwaller, B., Salicio, V., Bloc, A., Gotzos, V., Celio, M.R., 2001. Calretinin and calbindin D-28k delay the onset of cell death after excitotoxic stimulation in transfected P19 cells. Brain Res. 909, 145–158. Fortin, M., Asselin, M.C., Gould, P.V., Parent, A., 1998. Calretinin-immunoreactive neurons in the human thalamus. Neuroscience 84, 537–548. Frassoni, C., Arcelli, P., Selvaggio, M., Spreafico, R., 1998. Calretinin immunoreactivity in the developing thalamus of the rat: a marker of early generated thalamic cells. Neuroscience 83, 1203–1214. Frassoni, C., Bentivoglio, M., Spreafico, R., Sa´nchez, M.P., Puelles, L., Fairen, A., 1991. Postnatal development of calbindin and parvalbumin immunoreactivity in the thalamus of the rat. Brain Res. Dev. Brain Res. 58, 243–249. Jones, E.G., 2007. The Thalamus. Cambridge University Press, Cambridge. Mizumori, S.J., Miya, D.Y., Ward, K.E., 1994. Reversible inactivation of the lateral dorsal thalamus disrupts hippocampal place representation and impairs spatial learning. Brain Res. 644, 168–174. Mizumori, S.J., Williams, J.D., 1993. Directionally selective mnemonic properties of neurons in the lateral dorsal nucleus of the thalamus of rats. J. Neurosci. 13, 4015–4028. Mu¨nkle, M.C., Waldvogel, H.J., Faull, R.L., 2000. The distribution of calbindin, calretinin and parvalbumin immunoreactivity in the human thalamus. J. Chem. Neuroanat. 19, 155–173. Palczewska, M., Groves, P., Batta, G., Heize, B., Kuz´nicki, J., 2003. Calretinin and calbindin D28k have different domain organization. Protein Sci. 12, 180–184. Price, J.L., 1995. Thalamus. In: Paxinos, G. (Ed.), The Rat Nervous System. 2nd ed. Academic Press, San Diego, pp. 629–648. Re´sibois, A., Rogers, J.H., 1992. Calretinin in rat brain: an immunohistochemical study. Neuroscience 46, 101–134. Rogers, J.H., 1987. Calretinin: a gene for a novel calcium-binding protein expressed principally in neurons. J. Cell Biol. 105, 1343–1353.
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Contents lists available at ScienceDirect
Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu
Calcium-binding proteins in the laterodorsal thalamic nucleus during development of the guinea pig Witold Z˙akowski *, Krystyna Bogus-Nowakowska, Barbara Wasilewska, Beata Hermanowicz, Anna Robak Department of Comparative Anatomy, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Pl. Ło´dzki 3, 10-727 Olsztyn, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 May 2014 Received in revised form 8 August 2014 Accepted 8 August 2014 Available online 18 August 2014
The laterodorsal thalamic nucleus (LD) is often treated as a part of the anterior thalamic nuclei (ATN) because of its location and similar connectivity. Our previous studies have shown that distribution of three calcium-binding proteins, i.e. calbindin D28k (CB), calretinin (CR) and parvalbumin (PV), changes within the ATN during development of the guinea pig. The aim of this study is to examine the immunoreactivity pattern of these proteins in the LD in the guinea pig ontogeny. Brains from animals ranging from 40th embryonic day to 80th postnatal day were used in the study. Two methods were applied: a single-labelling immunoenzymatic method and double-labelling immunofluorescence. No changes of the distribution pattern of the substances were observed throughout the examined developmental stages. CB and CR were the most abundantly expressed proteins in perikarya of the LD. Numerous CB- and CR-immunoreactive cell bodies were found throughout the whole extent of the nucleus. In most of these cell bodies both proteins colocalized vastly. The highest immunoreactivity of the perikarya containing CB and CR was observed in the mediodorsal part of the LD and in its rostral portion. In regard to PV, single cell bodies were observed mostly in the dorsal part of the nucleus. PV did not colocalize with the other proteins. In summary, all the studied calcium-binding proteins were already present in the LD at prenatal developmental stages and the pattern of distribution remained virtually constant until adulthood. Thus, the LD differs considerably from the ATN in an aspect of neurochemical cell differentiation. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Laterodorsal thalamic nucleus Calbindin Calretinin Parvalbumin Development Guinea pig
Introduction The laterodorsal thalamic nucleus (LD) is often treated as a part of the anterior thalamic nuclei (ATN) complex, because of its close location and similarities in afferent and efferent connectivity (Jones, 2007; Price, 1995). Likewise the ATN, the LD is involved in learning and memory processes, particularly these related to spatial tasks (Mizumori et al., 1994; Shibata, 2000; Shibata and Naito, 2005; Van Groen et al., 2002; Wilton et al., 2001). However, some studies have suggested that the LD and each nucleus of the anterior thalamus may contribute to different aspects of spatial memory processing (Shibata and Naito, 2005; Van Groen et al.,
Abbreviations: AD, anterodorsal nucleus; AM, anteromedial nucleus; ATN, anterior thalamic nuclei; AV, anteroventral nucleus; CaBPs, calcium-binding proteins; CB, calbindin; CR, calretinin; LD, laterodorsal nucleus; PV, parvalbumin. * Corresponding author. Tel.: +48 89 523 43 01; fax: +48 89 523 43 01. E-mail addresses: [email protected] (W. Z˙akowski), [email protected] (K. Bogus-Nowakowska), [email protected] (B. Wasilewska), [email protected] (B. Hermanowicz), [email protected] (A. Robak). http://dx.doi.org/10.1016/j.jchemneu.2014.08.003 0891-0618/ß 2014 Elsevier B.V. All rights reserved.
2002). Our recent studies have shown that the distribution pattern of three calcium-binding proteins (CaBPs), i.e. calbindin D28k (CB), calretinin (CR) and parvalbumin (PV), differs among the ATN and changes during the development of the guinea pig (Z˙akowski et al., 2013; Z˙akowski and Robak, 2013). These proteins, along with others CaBPs, are essential to control the intracellular level of Ca2+ in a temporal and spatial dimension. CB and CR have been suggested to have similar functions, such as neuroprotection against excitotoxic cell death (D‘Orlando et al., 2001, 2002), while PV, which is mainly expressed in GABA-ergic interneurons, plays a major role in regulating the local inhibitory effects (Schwaller, 2009). Interestingly, our studies have revealed that the pattern of CaBPs immunoreactivity in the ATN of the adult guinea pig differs from that described in the rat, but partially matches the human one, especially in regard to CR (Z˙akowski and Robak, 2013). The distribution of the studied proteins has been described in the LD of the adult rat (Arai et al., 1994; Battaglia et al., 1992; Celio, 1990; ˜ as et al., 1991; Re´sibois and Rogers, 1992; Se´quier et al., Coven 1990; Winsky et al., 1992) and human (Fortin et al., 1998; Mu¨nkle et al., 2000), but only few studies have concerned this matter in the developing thalamus (Frassoni et al., 1991, 1998). Moreover, there
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is a lack of data regarding coexistence of CB, CR and PV in neurons of the LD. Taking into account a crucial role of CaBPs in a proper functioning of nervous cells, it seems quite interesting to reveal their distribution in the LD during prenatal and postnatal development. The present study will allow to compare the expression of the studied substances in the LD and the ATN, showing differences and similarities among these nuclei in an aspect of neurochemical differentiation during development of the guinea pig. Therefore, the aim of the study was to examine the distribution of calbindin, calretinin and parvalbumin as well as their coexistence pattern in the LD of the guinea pig at several developmental stages since 40th foetal day to 80th postnatal day. Materials and methods Tissue preparation All experimental protocols were approved by the Local Ethical Commission for Animal Experimentation at University of Warmia and Mazury in Olsztyn, Poland (in accordance with EU Directive 2010/63/EU for animal experiments). The study was performed on brains of the Dunkin–Hartley guinea pigs (Cavia porcellus) at several developmental stages: E40, E50, E60 (40th, 50th, 60th day of gestation; n = 3 for each stage), P0 and P80 (newborn and 80th day after birth; n = 3). All procedures of the tissue preparation were described in detail in our previous publication (Z˙akowski and Robak, 2013). Immunohistochemical procedures Sections through the LD from the postnatal and foetal brains were processed for two immunohistochemical methods: a single-labelling immunoenzymatic method and double-labelling immunofluorescence. For the first method we used solutions of rabbit anti-calbindin (1:4000, Swant, CH, product no. 6B38a), mouse anti-parvalbumin (1:2000, Sigma, USA, P3088) and mouse anti-calretinin (1:2000, Swant, CH, 6B3). The sections were then incubated with ImmPRESS Reagent (Vector Laboratories, USA) and subsequently with 3,3-diaminobenzidine substrate-chromogen solution
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(DakoCytomytation, USA). For the immunofluorescence, we used various mixtures of rabbit anti-calbindin (1:4000, Swant, CH, 6B38a), mouse anti-parvalbumin (1:2000, Sigma, USA, P3088) and mouse (1:2000, Swant, CH) or rabbit anti-calretinin (1:2000, Swant, CH, 7699/3H). Each mixture composed of antibodies raised in different species. In order to show the binding sites of the primary antibodies, the sections were incubated with mixtures of secondary antibodies: Cy3-conjugated-anti-mouse (1:10,000, Jackson ImmunoLabs, USA, 715-165-150) and FITC-conjugated-anti-rabbit (1:800, Jackson Immunolabs, USA, 711-095-152). Both methods were described in detail by Z˙akowski and Robak (2013). To test antibody specificity various controls were applied. To test primary antibodies specificity, antisera produced in different species and/or provided by different manufacturers were tested on the same sections according to the immunohistochemical methods. The staining patterns were identical for all variants used. The antibodies, which fitted better with various combinations of the others antibodies were chosen. The antibodies and method specificity were also tested in standard controls, i.e. the omission as well as replacement of all primary antisera by non-immune sera. In order to confirm the boundaries of the LD, some coronal sections from each of the studied stages were stained with cresyl violet according to the Nissl method and with mouse anti-NeuN (Millipore, USA, MAB377) – a neurochemical pan-neuronal marker – according to the immunohistochemical protocol. The sections were examined with an Olympus BX51 microscope equipped with a CCD camera connected to a PC. Images were acquired with Cell-F software (Olympus GmbH, Germany).
Results The localization and structure of the LD was maintained throughout all the examined developmental stages, i.e. E40, E50, E60, P0 and P80. Calbindin, calretinin and parvalbumin were already present at E40 and the distribution pattern of all the three proteins was virtually unchanged in the ontogeny of the guinea pig (Fig. 1). Numerous CB- (Figs. 1 and 2A) and CR-immunoreactive (Fig. 2E) perikarya were detected throughout the whole extent of the LD, with the highest concentration in the mediodorsal part of
Fig. 1. Photomicrographs of coronal sections through the middle portion of the laterodorsal thalamic nucleus in the guinea pig stained for calbindin at: (A) E40; (B) E50; (C) P0 and (D) P80. Calbindin, as well as the other studied proteins, was detected in the laterodorsal nucleus already in 40th day of the prenatal development and the distribution pattern was virtually unchanged throughout the examined developmental stages. Scale bars: 500 mm.
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Fig. 2. Photomicrographs of coronal sections through the rostral portion of the laterodorsal thalamic nucleus in the guinea pig brain at E60 stained for: (A and B) calbindin (green) and parvalbumin (red); (C and D) calbindin (green) and calretinin (red); (E and F) parvalbumin (green) and calretinin (red). The strongest immunoreactivity of calbindin and calretinin was present in the mediodorsal part of the nucleus, while parvalbumin was observed mostly in the dorsal part. Notice the abundant colocalization (yellow) of calbindin and calretinin (D) and a lack of parvalbumin colocalization with other proteins (B and F). Scale bars: A, C and E – 500 mm; B, D and F – 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
the nucleus (Fig. 2B and F) and in its rostral portion. In most of these perikarya, both proteins, CB and CR, colocalized abundantly (Figs. 2C, D and 3A). CB and CR were also observed individually in some perikarya of the LD, especially in its lateroventral part. PV was detected in single cell bodies limited mostly to the dorsal part of the nucleus (Fig. 2B and F). PV did not coexist in neurons with the other CaBPs (Figs. 2B, F and 3B). At P80, the perikarya immunopositive for the studied CaBPs were more scattered throughout the nucleus in comparison to the earlier stages, but CB- and CR-immunoreactive cell bodies were still the most numerous (Fig. 4A–C). In all the studied developmental stages, neurons containing CB, CR and PV had oval- or polygonal-shaped
perikarya and gave rise to various-length processes (Fig. 3A and B). The LD displayed strongly stained neuropil for CB (Fig. 4A) and CR (Fig. 4B), especially in the mediodorsal part of the nucleus, as well as weakly stained one for PV (Fig. 4C). Discussion The present study has shown that the studied CaBPs were already present in the LD of the guinea pig at E40 and the pattern of distribution remained virtually unchanged until P80. It is in contrast to the rat LD, in which only CR has been detected prenatally, since E19 (Frassoni et al., 1998). This developmental
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Fig. 3. Photomicrographs showing perikarya in the laterodorsal thalamic nucleus of the guinea pig brain at E60, which contained: (A) calbindin (green) and calretinin (red); (B) calbindin (green) and parvalbumin (red). Notice the colocalization (yellow) of calbindin and calretinin (A) and a lack of colocalization between calbindin and parvalbumin (B). Scale bars: 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
stage in rats corresponds to approximately E33 in guinea pigs (Workman et al., 2013). CR-positive cell bodies were still present at P0, but during the second postnatal week of the rat development, CR immunoreactivity decreased (Frassoni et al., 1998). In the LD of adult rats, scattered CR-immunoreactive perikarya were observed mostly in the rostral portion of the nucleus (Arai et al., 1994; Winsky et al., 1992). According to Frassoni et al. (1991), CB appeared in the rat LD not before P11 and an adult-like pattern of the CB distribution was displayed by the nucleus at P22. In adult rats, perikarya containing CB were detected throughout the whole extent of the LD (Arai et al., 1994; Battaglia et al., 1992; Celio, 1990). In regard to PV, the rat LD was devoid of any immunoreactivity at birth and the first postnatal days, but until P22, number of PV-positive fibres and punctate structures progressively increased (Frassoni et al., 1991). In adult rats, high PV immunoreactivity of the LD neuropil, in both fibres and terminals, has been found (Arai ˜ as et al., 1991). The results of our et al., 1994; Celio, 1990; Coven study indicate that the expression of CB, CR and PV in the LD of the guinea pig is much higher than in the rat LD. First of all, some neurons of the guinea pig LD contained PV. Secondly, all the studied proteins were already expressed at prenatal stages of the guinea pig development. Some differences between these two species exist also in an adult-like pattern of distribution: CR were observed mostly in perikarya of the rostral portion in the rat LD,
Fig. 4. Photomicrographs of coronal sections through the middle portion of the laterodorsal thalamic nucleus in the guinea pig brain at P80 stained for: (A) calbindin; (B) calretinin and (C) parvalbumin. Notice the strongest immunoreactivity of calbindin and calretinin in the mediodorsal part of the nucleus (A and B, respectively). Scale bars: 500 mm.
whereas in the guinea pig, CR was detected in perikarya throughout the whole extent of the nucleus. Interestingly, our results matches the CaBPs distribution pattern in the adult human LD, in which numerous CB- and CR-immunoreactive cell bodies were evenly distributed throughout the nucleus and few PVpositive perikarya were also detected (Mu¨nkle et al., 2000). The nature of the differences between the guinea pig and rat LD, as well as the similarities between the guinea pig and human LD remains unclear. Our previous studies have shown that high expression of
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CB, CR and PV in the anterior thalamus of the guinea pig was also different to that revealed in the rat ATN, but partially similar to the human one (Z˙akowski et al., 2013; Z˙akowski and Robak, 2013). Apparently, the studied CaBPs play more significant role in the functioning of both the LD and ATN in guinea pigs and humans than in rats. The LD plays, through its interactions with the hippocampus, a key role in spatial learning and memory processes (Mizumori et al., 1994; Shibata, 2000; Shibata and Naito, 2005; Van Groen et al., 2002; Wilton et al., 2001). In rats, the LD receives projections from the cortices implicated in various aspects of spatial memory and in cognitive and behavioural functions, i.e. the prelimbic, anterior cingulate and subicular cortices, secondary motor cortex, visual area 18b and retrosplenial cortices (Shibata and Naito, 2005; Thompson and Robertson, 1987; Van Groen and Wyss, 1990a,b,c, 1992a). The LD receives projections from some subcortical structures, such as the pretectal complex, superior colliculus, geniculate nucleus, thalamic reticular nucleus and zona incerta (Thompson and Robertson, 1987), while Bezdudnaya and Keller (2008) have shown that LD neurons receive direct inputs from the trigeminal nuclei. The main projections from the LD are to the infraradiata, precentral agranular, retrosplenial, visual area 18b, subicular, and entorhinal cortices (Van Groen and Wyss, 1992b). Furthermore, Mizumori and Williams (1993) have demonstrated that the LD contains ‘head direction cells’, which fire in accordance to the directional heading in the horizontal plane, independent of animal location and behaviour. Taking all these anatomical and electrophysiological data together, it has been postulated that the LD is a multisensory nucleus that integrates multimodal information (somatosensory and visual inputs) for spatial orientation and learning tasks (Bezdudnaya and Keller, 2008). It is unknown, whether the functions of the LD may have an influence on the developmental distribution of the CaBPs in the guinea pig. According to the obtained results, all the studied proteins were present in the LD already at 40th day of prenatal development of the guinea pig. At this stage, an existence of any visual stimuli is rather unlikely, since guinea pigs open their eyes between 50th and 60th day of embryonic life (Workman et al., 2013; Z˙akowski and Robak, 2013). More studies are needed to determine the relations between the early presence of the CaBPs in the guinea pig LD and the functions of the nucleus, especially these related to somatosensory stimuli and the head direction system. Undoubtedly, the studied proteins are important for normal brain development due to their capacity to buffer calcium ions. The concentration of intracellular calcium plays an essential role in both immature neurons and mature ones, thus it have to be kept at a definite level and any aberration may lead to severe alterations during development. Considering a close location and similar connectivity with the cortex, the LD is often treated as a part of the ATN (Jones, 2007; Price, 1995), which comprises three nuclei: the anteromedial (AM), anteroventral (AV) and anterodorsal (AD). We have previously revealed developmental changes of the CaBPs distribution within the ATN of the guinea pig. Briefly, CR appeared in perikarya for the first time at E50 in the AM and at E60 in the AV, while CB was observed in cell bodies of the AM not before P20; PV was vastly present only in the neuropil of the AD since E40 (Z˙akowski et al., 2013; Z˙akowski and Robak, 2013). Our present and previous studies have shown that the LD and each nucleus of the ATN differ considerably in a neurochemical aspect. The most striking difference between the ATN and LD was the presence of PVpositive cell bodies in the latter one, as well as virtually constant distribution pattern of the studied proteins during development of the LD. Van Groen et al. (2001, 2002) have shown that lesions limited to the LD impair spatial learning and memory, but this effect is significantly magnified when the lesion extends into the
ATN. Results of our studies may indirectly support the idea that although individual nuclei of the anterior thalamus and the LD play an important role in spatial learning and memory, they may contribute to different aspects of these processes. The only common feature between the ATN and LD was the coexistence of CB and CR. Both substances colocalized in perikarya of the LD since E40 (present study) and AM since P20 (Z˙akowski et al., 2013). Coexistence of CB and CR may indicate that these two calciumbinding proteins work synergistically both in the LD and AM. D‘Orlando et al. (2001, 2002) have revealed that these two proteins can help to delay the onset of cell death after excitotoxic stimulation. Taking into account that CB and CR have a similar amino acid sequence (Rogers, 1987), they may act in a similar way. On the other hand, these two proteins show differences in domain organization (Palczewska et al., 2003), so they may complement each other to ensure proper functioning of neurons of the two nuclei. In conclusion, our study has shown that the LD of the guinea pig displayed high level of the CaBPs immunoreactivity, especially CB and CR, during prenatal and postnatal development. Despite similar location, connectivity and functions, the LD differed considerably from each nucleus of the ATN in an aspect of neurochemical cell differentiation. Ethical approval Not required. References Arai, R., Jacobowitz, D.M., Deura, S., 1994. Distribution of calretinin, calbindin-D28k, and parvalbumin in the rat thalamus. Brain Res. Bull. 33, 595–614. Battaglia, G., Colacitti, C., Bentivoglio, M., 1992. The relationship of calbindincontaining neurons with substance P, Leu-enkephalin and cholecystokinin fibres: an immunohistochemical study in the rat thalamus. J. Chem. Neuroanat. 5, 453–464. Bezdudnaya, T., Keller, A., 2008. Laterodorsal nucleus of the thalamus: a processor of somatosensory inputs. J. Comp. Neurol. 507, 1979–1989. Celio, M.R., 1990. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375–475. ˜ as, R., De Leo´n, M., Alonso, J.R., Are´valo, R., Lara, J., Aijo´n, J., 1991. Distribution Coven of parvalbumin-immunoreactivity in the rat thalamus using a monoclonal antibody. Arch. Ital. Biol. 129, 199–210. D‘Orlando, C., Celio, M.R., Schwaller, B., 2002. Calretinin and calbindin D-28k, but not parvalbumin protect against glutamate-induced delayed excitotoxicity in transfected N18-RE 105 neuroblastoma-retina hybrid cells. Brain Res. 945, 181–190. D‘Orlando, C., Fellay, B., Schwaller, B., Salicio, V., Bloc, A., Gotzos, V., Celio, M.R., 2001. Calretinin and calbindin D-28k delay the onset of cell death after excitotoxic stimulation in transfected P19 cells. Brain Res. 909, 145–158. Fortin, M., Asselin, M.C., Gould, P.V., Parent, A., 1998. Calretinin-immunoreactive neurons in the human thalamus. Neuroscience 84, 537–548. Frassoni, C., Arcelli, P., Selvaggio, M., Spreafico, R., 1998. Calretinin immunoreactivity in the developing thalamus of the rat: a marker of early generated thalamic cells. Neuroscience 83, 1203–1214. Frassoni, C., Bentivoglio, M., Spreafico, R., Sa´nchez, M.P., Puelles, L., Fairen, A., 1991. Postnatal development of calbindin and parvalbumin immunoreactivity in the thalamus of the rat. Brain Res. Dev. Brain Res. 58, 243–249. Jones, E.G., 2007. The Thalamus. Cambridge University Press, Cambridge. Mizumori, S.J., Miya, D.Y., Ward, K.E., 1994. Reversible inactivation of the lateral dorsal thalamus disrupts hippocampal place representation and impairs spatial learning. Brain Res. 644, 168–174. Mizumori, S.J., Williams, J.D., 1993. Directionally selective mnemonic properties of neurons in the lateral dorsal nucleus of the thalamus of rats. J. Neurosci. 13, 4015–4028. Mu¨nkle, M.C., Waldvogel, H.J., Faull, R.L., 2000. The distribution of calbindin, calretinin and parvalbumin immunoreactivity in the human thalamus. J. Chem. Neuroanat. 19, 155–173. Palczewska, M., Groves, P., Batta, G., Heize, B., Kuz´nicki, J., 2003. Calretinin and calbindin D28k have different domain organization. Protein Sci. 12, 180–184. Price, J.L., 1995. Thalamus. In: Paxinos, G. (Ed.), The Rat Nervous System. 2nd ed. Academic Press, San Diego, pp. 629–648. Re´sibois, A., Rogers, J.H., 1992. Calretinin in rat brain: an immunohistochemical study. Neuroscience 46, 101–134. Rogers, J.H., 1987. Calretinin: a gene for a novel calcium-binding protein expressed principally in neurons. J. Cell Biol. 105, 1343–1353.
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