Purkinje cell compartmentation as revealed by Zebrin II expression in the cerebellar cortex of pigeons (Columba livia)

Abstract

Purkinje cells in the cerebellum express the antigen zebrin II (aldolase C) in many vertebrates. In mammals, zebrin is expressed in a parasagittal fashion, with alternating immunopositive and immunonegative stripes. Whether a similar pattern is expressed in birds is unknown. Here we present the first investigation into zebrin II expression in a bird: the adult pigeon (Columba livia). Western blotting of pigeon cerebellar homogenates reveals a single polypeptide with an apparent molecular weight of 36 kDa that is indistinguishable from zebrin II in the mouse. Zebrin II expression in the pigeon cerebellum is prominent in Purkinje cells, including their dendrites, somata, axons, and axon terminals. Parasagittal stripes were apparent with bands of Purkinje cells that strongly expressed zebrin II (+ve) alternating with bands that expressed zebrin II weakly or not at all (-ve). The stripes were most prominent in folium IXcd, where there were seven +ve/-ve stripes, bilaterally. In folia VI-IXab, several thin stripes were observed spanning the mediolateral extent of the folia, including three pairs of +ve/-ve stripes that extended across the lateral surface of the cerebellum. In folium VI the zebrin II expression in Purkinje cells was stronger overall, resulting in less apparent stripes. In folia II-V, four distinct +ve/-ve stripes were apparent. Finally, in folia I (lingula) and X (nodulus) all Purkinje cells strongly expressed zebrin II. These data are compared with studies of zebrin II expression in other species, as well as physiological and neuroanatomical studies that address the parasagittal organization of the pigeon cerebellum.

Zebrin-Immunopositive and -Immunonegative Stripe Pairs Represent Functional Units in the Pigeon Vestibulocerebellum

“…The ZIIϩ/Ϫ pairs from P1ϩ to P5Ϫ are indicated in ventral IXcd as well as P6ϩ. P6Ϫ, P7ϩ, and P7Ϫ are found more rostrally (Pakan et al, 2007). P1Ϫ is divided into medial and lateral portions by a small satellite immunopositive band one to two Purkinje cells wide in the middle of P1Ϫ denoted "?."…”

“…ZII expression was visualized using established immunohistochemical techniques described previously (Pakan et al, 2007). Briefly, sections were rinsed thoroughly in 0.1 M PBS and blocked with 10% normal donkey serum (Jackson ImmunoResearch Laboratories) and 0.4% Triton X-100 in PBS for 1 h. Tissue was then incubated in PBS containing 0.1% Triton X-100 and the primary antibody, mouse monoclonal anti-zebrin II (kindly provided by Richard Figure 2.…”

“…1 B, 5) consisting of seven ZIIϩ/Ϫ stripes. We used the nomenclature from Pakan et al (2007), whereby the most medial positive stripe is designated as P1ϩ and the number increases as the stripes move laterally to P7ϩ (Fig. 1 B) (Brochu et al, 1990;Eisenman and Hawkes, 1993;Ozol et al, 1999;Sillitoe and Hawkes, 2002) (for review, see Sillitoe et al, 2005).…”

“…ZII is expressed in PCs such that there are sagittal stripes of high expression (ZIIϩ) interdigitated with stripes of little expression (ZIIϪ). The spatial pattern and number of ZII stripes is highly conserved in the cerebella of birds and mammals (Pakan et al, 2007;Iwaniuk et al, 2009;Marzban and Hawkes, 2011), indicating that ZII may be critical for cerebellar function.…”

Early development of the nervous system of the eutherian <i>Tupaia belangeri</i>

“…Other groups have subsequently studied zebrin-IIimmunoreactive cerebellar compartments in order to carry out the following investigations: (1) interspecific comparison with the tammar wallaby (Macropus eugenii) (Marzban et al 2012), microchiropteran bats (Kim et al, 2009), hummingbirds (Aves: Trochilidae) (Iwaniuk et al, 2009), chicks (Gallus domesticus) (Marzban et al, 2010), pigeons (Columba livia) (Pakan et al, 2007; for an overview, see Marzban and Hawkes, 2011); (2) visualization of aldolase C with fluorescence through gene manipulation with the help of aldolase CVenus knock-in mice to facilitate studies on cerebellar compartmentalization (Fujita et al, 2014); (3) presentation of parasagittal stripes in the vermis which, complementary to zebrin II, are immunoreactive for neurofilament H (Demilly et al, 2011); (4) identification of links between the olivocerebellar projection and zebrin-immunoreactive compartments in the laboratory mouse (Sugihara and Quy, 2007) and in marmoset (Callithrix jacchus) (Fujita et al, 2010); (5) clarification of the role played by the helix-loop-helix (HLH) transcription factor early B-cell factor 2 (EBF2) (Croci et al, 2006); and (6) evaluation of the cerebellar connectivity in spinocerebellar ataxia type 1 (Solodkin et al, 2011). The second main research area of Wolfgang Knabe and colleagues, whose roots date back to the former anatomical department of Hans-Jürg Kuhn, continued previous projects on the retina, then served as a bridge between the retina and the forebrain, and, thereafter, was successively expanded to include the entire brain, spinal cord, neural crest, and the placodes.…”