cerebellum, neurons, development, doublecortin, NeuN.Experiments were performed on the offspring of five mongrel white rats using comparative immunochemical assessments of doublecortin (DCX) and neuronal nuclear antigen (NeuN) expression in neurons in the cortex and nucleus interpositus of the cerebellum in animals during early postnatal ontogeny (days 2-15). DCX expression was seen in postmitotic neurons in the external granular layer and migrating neurons in the cerebellar cortex. DCX expression was greater in the neocerebellum than in the paleocerebellum in rat pups aged two and seven days. NeuN expression was seen in migrating granule neurons, reaching a maximum in more mature neurons in the internal granular layer. DCX expression was not seen in Purkinje cells or in neurons in the nucleus interpositus of the cerebellum. Neurons in the nucleus interpositus showed progressive increases in NeuN from day 2 to day 15 after birth. Thus, comparative immunohistochemical studies of the dynamics of the expression of this pair of molecular markers provide an effective means of evaluating the development of cerebellar granule neurons during early postnatal ontogeny

Karnyushko, O. A.

Zimatkin, S. M.

Repository of Grodno State Medical University

1220097-0549/17/4702-0122 ©2017 Springer Science+Business Media New YorkNeuroscience and Behavioral Physiology, Vol. 47, No. 2, February, 2017 Molecular markers are ever more widely used to assess brain development during ontogeny in animals and humans [2–4]. Doublecortin (DCX) is a protein associated with cyto-skeletal microtubules in vertebrate neurons and expressed by immature neurons before and during migration during brain development. It was subsequently detected in growing neu-ron processes [7]. DCX is also expressed in brush neurons in the vestibulocerebellum in adult rats and plays a particular role in the plasticity of these cells [13]. NeuN (neuronal nu-clear antigen) is a neuronal nuclear antigen which is ex-pressed in the nuclei and perinuclear cytoplasm of most gran-ule cells in the CNS but is not detected in immature nerve cells. NeuN is not expressed in Purkinje cells or neurons in the dentate nucleus of the cerebellum, or in glial cells [12]. The cortex of the developing cerebellum in rats contains, along with the usual, “internal” granular layer (the IGL), an additional external granular layer (the EGL), where cell pro-liferation persists into the postnatal period. The EGL of the cerebellum forms in 17-day-old embryos as a result of the fi rst wave of migration to the outer surface of the developing cerebellum from the dorsal part of the neural tube, i.e., the rhombic lip, located on the boundary of the mesencephalon and telencephalon [16]. The EGL is a germinative zone con-sisting of 3–6 rows of granule neuron precursors [10]. The 1–3 outer layers form proliferating cells which, after their last mitosis, are discharged into the inner rows of the EGL, acquire a fusiform shape, and initiate radial migration to the future IGL [11]. The EGL reaches its maximal development by day 7, and most of the granule neurons making up the IGL form between days 7 and 15 of postnatal development; the EGL in rats disappears by postnatal days 20–21. A num-ber of studies have established that the migratory behavior of granule cells is due to a genetic program [17], and is also infl uenced by the microenvironment, activation of specifi c N-methyl-D-aspartic acid (NMDA) receptors, and the po-lymerization and degradation of microtubules creating pull-ing and pushing forces on the nucleus and cytoplasm [14]. The aim of the present work was to carry out a compar-ative evaluation of the dynamics of DCX and NeuN expres-sion in the cortex of the paleocerebellum and neocerebellum Expression of Doublecortin and NeuN in Developing Neurons in the Rat CerebellumS. M. Zimatkin and O. A. Karnyushko UDC 611.817.1.018:612.65:599.323.4Translated from Morfologiya, Vol. 149, No. 1, pp. 38–42, January–February, 2016. Original article submitted July 12, 2015.Experiments were performed on the offspring of fi ve mongrel white rats using comparative immunochem-ical assessments of doublecortin (DCX) and neuronal nuclear antigen (NeuN) expression in neurons in the cortex and nucleus interpositus of the cerebellum in animals during early postnatal ontogeny (days 2–15). DCX expression was seen in postmitotic neurons in the external granular layer and migrating neurons in the cerebellar cortex. DCX expression was greater in the neocerebellum than in the paleocerebellum in rat pups aged two and seven days. NeuN expression was seen in migrating granule neurons, reaching a maximum in more mature neurons in the internal granular layer. DCX expression was not seen in Purkinje cells or in neurons in the nucleus interpositus of the cerebellum. Neurons in the nucleus interpositus showed progres-sive increases in NeuN from day 2 to day 15 after birth. Thus, comparative immunohistochemical studies of the dynamics of the expression of this pair of molecular markers provide an effective means of evaluating the development of cerebellar granule neurons during early postnatal ontogeny.Keywords: cerebellum, neurons, development, doublecortin, NeuN.Department of Histology, Cytology, and Embryology, Grodno State Medical University, Grodno, Belarus; e-mail: smzimatkin@grsmu.by, karnyushko-olga@mail.ru.DOI 10.1007/s11055-016-0374-y123Expression of Doublecortin and NeuN in Developing Neurons Fig. 1. Expression of doublecortin (DCX) and neuronal nuclear antigen (NeuN) in the cortex of the paleocerebel-lum in rat pups aged two (a, b), seven (c, d), and 15 days (e, f). EGL – external granular layer; IGL – internal granular layer. Immunocytochemical reactions: a, c, e) for DCX; b, d, ƒ) for NeuN. Magnifi cation ×100.124 Zimatkin and Karnyushkoand the nucleus interpositus of the cerebellum in rats in ear-ly postnatal ontogeny. Materials and Methods Experiments were performed on fi ve female mongrel white rats with starting weights of 180 ± 20 g and their off-spring. All experiments were performed in compliance with the “Regulations for Studies Using Experimental Animals” [1]. The study was approved by the Biomedical Ethics Com mittee of Grodno State Medical University (protocol No. 7 of December 12, 2013). Animals were kept on a stan-dard animal-house diet. A single pup was taken from each animal at 2, 7, and 15 days after birth; pups were decapitated. To ensure comparable results, samples of cerebellum from all animals were processed together in identical conditions. Samples were fi xed in zinc-ethanol-formaldehyde at 4°C (overnight) and then embedded in paraffi n by standard meth-ods. Sections of thickness 5 μm were cut with a LeicaRM 2125 RTS microtome (Leica, Germany). Immuno chemical detection of DCX was with primary rabbit polyclonal anti-bodies (Abcam, UK) ab.18723, and NeuN was detected with antibody ab.128886 (antibodies diluted 1:400, 4°C, 20 h, moist chamber). Binding of primary antibodies was detect-TABLE 1. Expression of Doublecortin in Premigratory Neurons in the External Granular Layer of the Cerebellar Cortex in Early Postnatal Ontogeny in Rats (Me ± IQR, OD units)Time after birth, daysPart of cerebellumPaleocerebellum Neocerebellum2 0.25 ± 0.03 0.33 ± 0.05+7 0.20 ± 0.020* 0.24 ± 0.04*+15 0.190 ± 0.010* 0.190 ± 0.020*#Here and in Tables 2–4: signifi cant differences compared with *two-day-old rats; #seven-day-old rats; +the paleocerebellum, p < 0.05.TABLE 2. Characteristics of Cerebellar Neurons in Rats during Postnatal Ontogeny (Me ± IQR)Parameter Period after birth, daysPart of cerebellumPaleocerebellum Neocerebellum Nucleus interpositusNumber of NeuN-positive neurons in the IGL of the cortex per 1000 μm2 section area2 4.4 ± 1.8 3.9 ± 0.5 –7 8.89 ± 0.28* 9 ± 4* –15 14.1 ± 1.6*.# 15.4 ± 2.1*.# –NeuN expression in neurons, OD units: 2 0.14 ± 0.06 0.10 ± 0.10 0.130 ± 0.010    in the IGL of the cortex 7 0.20 ± 0.09 0.18 ± 0.03 0.140 ± 0.010    in the nucleus interpositus 15 0.25 ± 0.04*,# 0.26 ± 0.04*,# 0.170 ± 0.020*,#IGL – internal granular layer of the cortex; NeuN – neuronal nuclear antigen.TABLE 3. Thickness of the Granular Layers of the Cerebellar Cortex in Postnatal Ontogeny in Rats (Me ± IQR, μm)Granular layers Time after birth, daysPart of cerebellumPaleocerebellum NeocerebellumExternal2 14 ± 3 13.8 ± 2.87 41 ± 11* 45 ± 6*15 20.2 ± 1.2*# 26 ± 7*#+Internal2 72 ± 7 60 ± 5+7 62 ± 8* 48 ± 6*+15 125 ± 23*# 107 ± 24*#125Expression of Doublecortin and NeuN in Developing Neurons ed using an Expose Rabbit-Specifi c HRP/DAB detection IHC kit (ab.80437; Abcam, UK). Neighboring sec tions were stained with 0.1% thionine solution by the Nissl meth-od. The thicknesses of the EGL and IGL in the cortex were measured in the paleo- and neocerebellum. Their locations on histological preparations of the developing cerebellum were identifi ed in terms of Olenev’s descriptions [5]. The optical densities of chromogen precipitates in DCX-immu-nopositive neurons in the EGL and NeuN-im mu no positive neurons in the IGL and nucleus interpositus of the cerebel-lum were determined. The density of NeuN-immu no-positive neurons per 1000 μm2 was determined in the IGL. Examination of histological preparations, micropho-tography, and morphometry were performed using an Axio-scop 2 plus microscope (Zeiss, Germany), a Leica DFC 320 digital video camera (Leica, Germany), and the image anal-ysis program ImageWarp (Bitfl ow, USA). Med ian and in-terquartile ranges (Me ± IQR) were determined for each metric. Differences between values under comparison were eva luated using the Mann–Whitney test and were regarded as signifi cant at p < 0.05. Results On postnatal day 2, the outer 2–3 rows of the EGL of the cerebellar cortex, which contains dividing cells, lacked DCX-immunopositive neurons (Fig. 1, a). Intense DCX ex-pression was seen in the 2–3 inner rows of EGL cells, which contain postmitotic granule neurons ready for radial migra-tion (see Fig. 1, a; Table 1). NeuN expression was absent from these cells, though it was detected in migrating gran-ule neurons, reaching a maximum in more mature neurons in the IGL (see Fig. 1, b). On day 7 of postnatal ontogeny, when the EGL reached its maximal thickness (Table 2), its migration-ready post-mitotic neurons showed decreased immunoreactivity (see Table 1). DCX expression increased in migrating neurons and the growing processes of IGL neurons (see Fig. 1, c). NeuN expression in the IGL was low, but increased in mi-grating neurons to reach a maximum in more mature neu-rons completing migration to the IGL (see Fig. 1, d). On day 15 after birth, the thickness of the EGL de-creased to half that on day 7 (see Table 2). At this time, DCX expression in migration-ready neurons decreased further. Expression was detected in migrating granule cells, but was absent from the bodies of IGL neurons. However, the inten-sity of the DCX reaction in the neuropil (probably growing granule neuron processes) was quite high (see Fig. 1, e). Moderate NeuN expression was detected in migrating gran-ule neurons at this time, and was maximal in the bodies of IGL neurons. NeuN immunoreactivity was not detected in the neuropil of this layer (see Fig. 1, ƒ). Thus, DCX expression in premigratory neurons in the EGL of the cortex of the paleo- and neocerebellum gradual-ly decreased from day 2 to day 15 of postnatal ontogeny. DCX expression in the neocerebellum of rat pups aged two and seven days was greater than that in the paleocerebel-lum, though by day 15 it was no different from that in the paleocerebellum (see Table 1). From day 2 to day 15 of postnatal ontogeny, the density of NeuN-immunopositive neurons in the IGL increased pro-gressively (by factors of 3–4); these cells also showed increas-es (by factors of 2–2.5) in NeuN expression (see Table 2). Comparison of the cortex in the paleo- and neocerebel-lum identifi ed a degree of delay in the increase in NeuN expression in the neocerebellum as compared with the pa-leocerebellum (see Table 2). On day 15 after birth, the EGL in the neocerebellum was signifi cantly thicker, while the IGL was somewhat thinner than that in the paleocerebellum (Table 3). Purkinje cells (PC) in two-day-old rat pups were posi-tioned in several rows; by day 7, they settled to form a monolayer. Expression of DCX and NeuN proteins was not detected on day 2 after birth. DCX expression was not seen in neurons in the nu-cleus interpositus of the cerebellum on days 1, 7, or 15 of postnatal development. This is evidence that by day 2 of postnatal ontogeny, the migration of these neurons was al-ready complete and the cells were undergoing differentia-tion, which was accompanied by a progressive increase in NeuN expression within them (see Table 2). Discussion Simultaneous comparative immunohistochemical stud-ies of the pair of molecular markers DCX and NeuN may provide a convenient approach to assessing the develop-mental dynamics of cerebellar neurons in rats during ear-ly postnatal ontogeny. DCX has been shown to be absent from mitotically dividing cells in the EGL; detection starts in migration-ready postmitotic neurons, with no detection in the bodies of neurons which have completed migration and are differentiating. This is consistent with published data on the appearance of DCX in migration-ready neurons and its absence in the bodies of mature neurons [15]. DCX is in-volved in stabilizing the microtubules required for neuron migration [8] and also for the formation of new processes; it may also be important for synaptogenesis in IGL neurons. Increases in DCX in the neuropil of the granular layer on days 7–15 coincided with the period of intense growth of neuron processes and synaptogenesis in the cerebellar cor-tex [9]. Conversely, NeuN expression in immature postmi-totic EGL neurons is very low, but progressively increases in migrating and especially mature IGL neurons, which is consistent with published data [12]. Changes occurring in PC and, particularly, the fact that they formed a monolayer by day 7 are probably due to me-chanical factors: the pressure of the thickening granular lay-er beneath and the barrier formed by parallel fi bers above, preventing penetration of PC perikarya into the molecular layer. This process is not accompanied by translocation of the nucleus and rearrangements of the cytoskeleton [6]. The fact that expression of DCX protein was not detected in PC even on day 2 after birth is evidence that migration of PC is 126 Zimatkin and Karnyushkocomplete by this time. However, NeuN expression is also not seen in early postnatal ontogeny in cerebellar PC, which is consistent with published data [12]. As maturing granule cells in two-day-old rat pups were distributed diffusely, IGL thickness was even rather great-er than that in seven-day-old pups. A delay in the decrease in thickness of the EGL was noted in the neocerebellum, with decreased DCX expression in these cells on the one hand and the absence of thickening of the IGL and increased NeuN expression in its neurons on the other, as compared with the paleocerebellum. All these points are consistent with the view that the development of the paleocerebellar cortex in early ontogeny occurs earlier. Thus, comparative immunohistochemical studies of changes in the expression of a pair of molecular markers – DCX and NeuN – is an effective method of assessing the development of cerebellar granule neurons in early post-natal ontogeny. This approach can also be used in studies of the effects of various experimental interventions on the postnatal morphogenesis of the cerebellum, as well as pathological states.REFERENCES1. N. N. Karkishchenko and S. V. Gracheva, Guidelines on Laboratory Animals and Alternative Models in Biomedical Research, Profi l-2S, Moscow (2010).2. D. E. Korzhevskii, E. G. Gilerovich, N. N. Zin’kova, et al., “Immuno-cytochemical detection of neurons in the brain using the selective marker NeuN,” Morfologiya, 128, No. 5, 76–78 (2005).3. D. E. Korzhevskii, M. N. Karpenko, and O. V. Kirik, “Microtubule-associated proteins as indicators of the differentiation of functional status in nerve cells,” Morfologiya, 139, No. 1, 13–21 (2011).  4. D. E. Korzhevskii, E. S. Petrova, O. V. Kirik, et al., “Assessment of neuron differentiation in rat embryos by immunocytochemical de-tection of doublecortin,” Morfologiya, 133, No. 4, 7–10 (2008).  5. S. N. Olenev, The Developing Brain, Nauka, Leningrad (1978).  6. J. Altman and A. T. Winfree, “Spatial organization of Purkinje cell perikarya,” J. Comp. Neurol., 171, No. 1, 1–16 (1977).  7. G. Friocourt, A. Koulakoff, P. 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H. Hayes, et al., “Dissociation of dou-blecortin expression and neurogenesis in unipolar brush cells in the vestibulocerebellum and dorsal cochlear nucleus of the adult rat,” Neuroscience, 265, 323–331 (2014).14. P. Rakis and E. Knyihar-Csillik, “Polarity of microtubule assemblies during neuronal cell migration,” Proc. Natl. Acad. Sci. USA, 93, No. 17, 9218–9222 (1996).15. J. Takács, R. Zaninetti, J. Vig, et al., “Postnatal expression of Doub-lecortin (DCX) in the developing cerebellar cortex of mouse,” Acta Biol., 59, No. 2, 147–161 (2008).16. R. J. Wingate, “The rhombic lip and early cerebellar development,” Curr. Opin. Neurobiol., 11, No. 1, 82–88 (2001).17. E. Yacubova and H. Komuro, “Intrinsic program for migration of cer-ebellar granule cells in vitro,” J. Neurosci., 22, No. 14, 5966–5981 (2002).

Expression of Doublecortin and NeuN in Developing Neurons in the Rat Cerebellum

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Expression of Doublecortin and NeuN in Developing Neurons in the Rat Cerebellum

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