Compartmentation of the cerebellar cortex of hummingbirds (Aves: Trochilidae) revealed by the expression of zebrin II and phospholipase C beta 4.

Abstract

The parasagittal organization of the mammalian cerebellar cortex into zones has been well characterized by immunohistochemical, hodological and physiological studies in recent years. The pattern of these parasagittal bands across the cerebellum is highly conserved across mammals, but whether a similar conservation of immunohistochemically defined parasagittal bands occurs within birds has remained uncertain. Here, we examine the compartmentation of the cerebellar cortex of a group of birds with unique cerebellar morphology-hummingbirds (Trochilidae). Immunohistochemical techniques were used to characterize the expression of zebrin II (aldolase C) and phospholipase C beta 4 (PLC beta 4) in the cerebellar cortex of two hummingbird species. A series of zebrin II immunopositive/immunonegative parasagittal stripes was apparent across most folia representing three major transverse zones: an anterior zone with a central stripe flanked by three lateral stripes on either side; a central zone of high/low immunopositive stripes; and a posterior zone with a central stripe flanked by four to six lateral stripes on either side. In addition, both folia I and X were uniformly immunopositive. The pattern of PLC beta 4 immunoreactivity was largely complementary-PLC beta 4 positive stripes were zebrin II negative and vice versa. The similarity of zebrin II expression between the hummingbirds and the pigeon indicates that the neurochemical compartmentation of the cerebellar cortex in birds is highly conserved, but species differences in the number and width of stripes do occur.

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Compartmentation of the cerebellar cortex of hummingbirds (Aves: Trochilidae)
revealed by the expression of zebrin II and phospholipase Cb4
Andrew N. Iwaniuka, Hassan Marzbanb,c, Janelle M.P. Pakand, Masahiko Watanabee,
Richard Hawkesb,c, Douglas R.W. Wyliea,d,*
aDepartment of Psychology, University of Alberta, Edmonton, Alta., Canada T6G 2E9
bDepartment of Cell Biology and Anatomy, Genes and Development Research Group, University of Calgary, Calgary, Alta., Canada T2N 4N1
cHotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, Alta., Canada T2N 4N1
dCentre for Neuroscience, University of Alberta, Edmonton, Alta., Canada T6G 2E9
eDepartment of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan
1. Introduction
The gross morphology of the cerebellum varies tremendously
both within and among the vertebrate classes. Most of the
variation within classes can be attributed to variations in the size
and morphology of components of the cerebellar vermis (i.e., folia
or lobules) and hemispheres. In mammals, for example, the
paraflocculus of bats and whales is enlarged compared to other
groups (Paulin, 1993) and the foliation pattern of the cerebellar
hemispheres varies widely among primates (Larsell, 1970). This
variation in gross cerebellar morphology does, however, belie a
highly conserved parasagittal organization of the cerebellar
cortex. Using zebrin II/aldolase C (Brochu et al., 1990; Ahn
et al., 1994: hereafter referred to as simply as ZII) as an
immunohistochemical marker, the cerebellar cortex can be
subdivided into a series of parasagittal stripes that correspond
to physiologically and hodologically defined zones in the
cerebellum (Gravel et al., 1987; Gravel and Hawkes, 1990; Ji
and Hawkes, 1994; Hallem et al., 1999; Voogd et al., 2003;
Sugihara and Shinoda, 2004; Voogd and Ruigrok, 2004; Pijpers
et al., 2005; Gao et al., 2006; Pijpers and Ruigrok, 2006). The
pattern of ZII expression is highly conserved among mammalian
taxa that vary in the size and morphology of their cerebella
(reviewed in Sillitoe et al., 2005). Thus, diversity in cerebellar
morphology does not appear to reflect changes in the pattern of
cerebellar compartmentation as revealed by zebrin II immunor-
eactivity. Whether this is also true of other vertebrate groups,
however, is unknown.
Recently, Pakan et al. (2007) showed that ZII immunoreactivity
inthepigeon(Columbalivia)isexpressedinparasagittalstripesina
Journal of Chemical Neuroanatomy 37 (2009) 55–63
A R T I C L EI N F O
Article history:
Received 21 June 2008
Received in revised form 13 September 2008
Accepted 3 October 2008
Available online 18 October 2008
Keywords:
Cerebellum
Purkinje cell
Immunohistochemistry
Evolution
Comparative anatomy
A B S T R A C T
The parasagittal organization of the mammalian cerebellar cortex into zones has been well characterized
by immunohistochemical, hodological and physiological studies in recent years. The pattern of these
parasagittal bands across the cerebellum is highly conserved across mammals, but whether a similar
conservation of immunohistochemically defined parasagittal bands occurs within birds has remained
uncertain. Here, we examine the compartmentation of the cerebellar cortex of a group of birds with
unique cerebellar morphology—hummingbirds (Trochilidae). Immunohistochemical techniques were
used to characterize the expression of zebrin II (aldolase C) and phospholipase Cb4 (PLCb4) in the
cerebellar cortex of two hummingbird species. A series of zebrin II immunopositive/immunonegative
parasagittal stripes was apparent across most folia representing three major transverse zones: an
anterior zone with a central stripe flanked by three lateral stripes on either side; a central zone of high/
low immunopositive stripes; and a posterior zone with a central stripe flanked by four to six lateral
stripes on either side. In addition, both folia I and X were uniformly immunopositive. The pattern of
PLCb4 immunoreactivity was largely complementary—PLCb4 positive stripes were zebrin II negative
and vice versa. The similarity of zebrin II expression between the hummingbirds and the pigeon indicates
that the neurochemical compartmentation of the cerebellar cortex in birds is highly conserved, but
species differences in the number and width of stripes do occur.
? 2008 Elsevier B.V. All rights reserved.
* Corresponding author at: Department of Psychology and Centre for Neu-
roscience, University of Alberta, Edmonton, Alta., Canada T6G 2E9.
Tel.: +1 780 492 5274; fax: +1 780 492 1768.
E-mail address: dwylie@ualberta.ca (Douglas R.W. Wylie).
Contents lists available at ScienceDirect
Journal of Chemical Neuroanatomy
journal homepage: www.elsevier.com/locate/jchemneu
0891-0618/$ – see front matter ? 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchemneu.2008.10.001

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similar fashion to that of mammals. Cerebellar morphology in
birds, however, varies widely among taxa with significant
differences in the degree of foliation of the cerebellar cortex
(Iwaniuk et al., 2006a) as well as the relative size of the individual
folia (Iwaniuk et al., 2007). The most divergent cerebellar
morphology observed in birds thus, far is the significant reduction
of the anterior lobe in hummingbirds (Trochilidae) and swifts
(Apodidae) (Larsell, 1967; Iwaniuk et al., 2006a,b, 2007). As shown
in Fig. 1, hummingbirds have an anterior lobe that is characterized
by an extreme reduction in the size of folia II and III compared to
other birds (Iwaniuk et al., 2006b, 2007). Whether this significant
evolutionary change in the hummingbird cerebellum has resulted
inachangeintheorganizationofthecerebellumisunknown.Here,
we examine the pattern of cerebellar compartmentation in
hummingbirds as revealed by ZII expression and compare it to
previous results for the pigeon (Pakan et al., 2007). Based upon the
highly conserved pattern of expression in mammals, we predict
that ZII expression will be largely concordant between humming-
birds and the pigeon.
ZII is not, however, the only antigen that reveals parasagittal
bands in the cerebellar cortex. Over 20 different molecules yield a
striped pattern in mammalian cerebella including 50-nucleotidase
(Scott, 1963), acetylcholinesterase (Marani and Voogd, 1977),
corticotropin-releasing factor (King et al., 1997), heat shock
protein 25 (Armstrong et al., 2001), human natural killer cell
antigen (Eisenman and Hawkes, 1993; Marzban et al., 2004)
cadherins (Arndt and Redies, 1998; Vanhalst et al., 2005; Neudert
and Redies, 2008) and phospholipase Cb4 (PLCb4, Sarna et al.,
2006;Marzban etal.,2007). Inorderto gainabetter understanding
of how the cerebellar cortex is organized in birds as well as the
correspondence between ZII and other antigens, we also examined
the pattern of PLCb4 labeling throughout the hummingbird
cerebellum.
2. Materials and methods
The brains from a total of nine adult and sub-adult Anna’s (Calypte anna, n = 5)
and Rufous (Selasphorus rufus, n = 4) hummingbirds that were immersion fixed in
4% paraformaldehyde in 0.1 M phosphate buffer were kindly provided to us by Drs.
Kenneth Welch Jr. and Raul Suarez (University of California, Santa Barbara). All
animal procedures conformed to institutional regulations at the University of
California, Santa Barbara (Institutional Animal Care and Use Committee Approval
#672) and the Guide to the Care and Use of Experimental Animals from the Canadian
Council of Animal Care.
2.1. Immunohistochemistry for serial sections
Following several weeks of fixation, eight of the hummingbird brains were
cryoprotected, embedded in gelatin and sectioned on a freezing stage microtome in
the coronal plane at a thickness of 40 mm. Every section throughout the cerebellum
was collected in 0.1 M phosphate buffered saline (PBS, pH 7.4). The sections were
then divided into four alternate series. One series from each bird was mounted onto
gelatinized slides, stained for thionin, dehydrated through a graded series of
ethanolsand coverslipped with Permount.Theremaining three series were usedfor
immunohistochemistry.
Two different primary antibodies were used to examine the immunohisto-
chemical pattern of cerebellar compartmentation in the hummingbirds. First, we
used a monoclonal anti-mouse ZII antibody (1:200–400), which was produced by
immunization with a crude cerebellar homogenate from the weakly electric fish
Apteronotus (Brochu et al., 1990). This antibody recognizes a single polypeptide
band in mouse with an apparent molecular weight of 36 kDa that cloning studies
have identified as the isoenzyme aldolase C (Aldoc: Ahn et al., 1994; Hawkes and
Herrup, 1995). Western blot analysis has shown that a single identical band is also
recognized in pigeon cerebellar homogenate (Pakan et al., 2007). Second, we used
an anti-rabbit PLCb4 antibody that is raised against a synthetic peptide
representing amino acids 15–74 of mouse PLCb4 fused to GST protein and
expressed in bacteria (Nakamura et al., 2004; Sarna et al., 2006; Marzban et al.,
2007). Control immunohistochemistry using either antibody pre-absorbed with
antigen polypeptides or cerebellar sections from a PLCb4 knockout mouse yielded
no significant immunostaining (Nakamura et al., 2004). Western blotting of this
antibody in chicken (Gallus gallus f. domesticus) cerebellar homogenates has also
revealed a band with the same molecular weight as murine PLCb4 (Marzban et al.,
unpubl. data).
For ZII immunohistochemistry, free floating sections were washed several times
in PBS and blocked with 10% normal donkey serum (in PBS, Jackson Immunor-
esearch Laboratories, West Grove, PA) and 0.4% Triton X-100 in PBS for 1–2 h at
room temperature followed by incubation in the primary antibody for 48–72 h at
room temperature. The sections were then rinsed several times in PBS and
incubated in CY3- or CY2-conjugated donkey anti-mouse secondary antibody
(Jackson Immunoresearch Laboratories, 1:200 in PBS, 2.5% donkey serum and 0.4%
Triton X-100) for 2–3 h at room temperature. Following several rinses in PBS, the
sections were then mounted onto gelatinized slides.
2.2. Double labeling
Double labeling followed a similar protocol to that of the ZII immunohisto-
chemistry.Tissuesections were washed, blocked inPBScontaining 10%normalgoat
serum (Jackson Immunoresearch Laboratories, West Grove, PA) and incubated in
blocking solution containing a combination of primary antibodies: anti-ZII (spent
culture mediumdiluted 1:200:Brochuetal., 1990)andanti-PLCb4(1:1000) for16–
18 h at 4 8C. Following incubation in primary antibodies, sections were washed and
then left in PBScontaining CY3-conjugatedgoat anti-rabbit secondary antibody and
CY2-conjugated goat anti-mouse secondary antibody (both diluted 1:1000, Jackson
Immunoresearch Laboratories, West Grove, PA) for 2 h at 4 8C. After incubation in
secondary antibody, the sections were washed in 0.1 M PBS buffer, mounted on
chrome-alum gelatin subbed slides.
Fig.1.Cerebellarmidsagittalsectionsstainedwiththionin.(A)Anna’sHummingbird(Calypteanna)and(B)pigeon(Columbalivia).Thefoliaarenumberedrostrocaudallyfrom
I to X following Larsell’s (1967) terminology. Note the lack of foliated cerebellar cortex in the hummingbird corresponding to folia II/III in the pigeon. Scale bars = 800 mm.
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2.3. Whole mount immunohistochemistry
The cerebellum of one male Rufous Hummingbird was removed from the rest of
the brain by cutting through the cerebellar peduncles. It was subsequently
immunostained as a whole mount using a protocol slightly modified from Sillitoe
andHawkes(2002)asusedinPakanetal.(2007).Afterincubatingthecerebellumin
fixative for 24–48 h,it was post-fixed overnight at 4 8C in Dent’s fixative (Dentet al.,
1989). Next, the cerebellum was incubated in Dent’s bleach (Dent et al., 1989) for
?8 h then dehydrated in 2 ? 30 min each 100% MeOH. The tissue was passed
through 4–5 cycles of chilling to ?80 8C and thawing to room temperature in 100%
MeOH followed by overnight incubation in MeOH at ?80 8C. For ZII staining, the
cerebellum was rehydrated for 90 min each in 50% MeOH, 15% MeOH, and PBS then
enzymatically digested in 10 mg/ml proteinase K (>600 units/ml; Boehringer
Mannheim, Inc.) in PBS for 5 min at room temperature. After rinsing 3 ? 10 min in
PBS, the tissue was incubated in blocking buffer (Davis, 1993) for 6–8 h at room
temperature. The tissue was then incubated for 48–96 h in ZII antibody (see details
above), rinsed 3 ? 2 h at 4 8C, and incubated overnight at 4 8C in goat anti-mouse
secondaryantibody(Jackson ImmunoResearchLaboratories). Finally,thetissuewas
rinsed 4 ? 3 h each at 4 8C followed by a final overnight rinse, incubated in 0.2%
bovine serum albumin, 0.1% Triton X-100 in PBS for 2 h at room temperature and
visualized with DAB.
2.4. Microscopy and image analysis
Theserial sections were viewedwith acompound lightmicroscope(Leica DMRE)
equipped with the appropriate fluorescence filters (rhodamine and FITC). Images
were acquired using a Retiga EXi FAST Cooled mono 12-bit camera (Qimaging,
Burnaby, BC, Canada) and analyzed with OPENLAB imaging software (Improvision,
Lexington, MA). The images were compiled with PTGui v 6.0.3 (Rotterdam,
Netherlands) and manipulated using Adobe Photoshop (San Jose, CA) to
compensate for brightness and contrast.
2.5. Nomenclature
In general, we have adopted the avian cerebellar nomenclature developed by
Larsell(1967)withelevenprimaryfolianumberedinarostrocaudaldirectionfromI
to X, but with a division of IX into IXab and IXcd for a total of eleven folia. Although
Larsell (1967) intended this to reflect homologies between mammalian lobules and
cerebellar folia, such a link has yet to be adequately demonstrated. As a result, we
refer to the divisions of the avian cerebellum as folia instead of lobules to
differentiate between birds and mammals and to prevent the inference that these
subdivisions are indeed one and the same. This same nomenclature has been used
for a wide array of species (Larsell, 1967; Iwaniuk et al., 2006b, 2007). The only
exception in the hummingbirds concerns folia II and III. Because of the lack of a
sulcus demarcating folia II from III in the hummingbirds (Fig. 1), we were unable to
distinguish between the two using cytoarchitectonic criteria and therefore refer to
them simply as folia II/III.
In our discussion of the topography of antigen expression, we use the same
nomenclature for the ZII stripes adopted in mammalian studies (e.g., reviewed in
Sillitoe et al., 2005) and previously applied to pigeon (Pakan et al., 2007). Briefly, the
most medial immunopositive (i.e., strongly immunoreactive) stripe, straddling the
midline, is designated P1+ and the number increases as the stripes move laterally to
P7+. ZII immunonegative stripes are numbered P1? to P5? according to the P+
stripe medially. While this does not necessarily reflect stripe homologies among
avian and mammalian species, it provides some consistency in labeling and
discussing the labeling pattern.
3. Results
Purkinje cells were the only neurons immunoreactive for ZII in
the cerebellar cortex of both hummingbird species and no
differences in immunoreactivity were present between the two
species. In ZII immunopositive Purkinje cells, the somata,
dendrites and axons were all labeled. Also readily apparent was
a pattern of parasagittal stripes, consisting of Purkinje cells that
stronglyexpressZIIalternatingwiththosethatweaklyexpress,or
do not express, ZII (see below, Figs. 2–4). In the wholemount
(Figs. 2A, 3A and B), the contrast between the ZII immunopositive
and immunonegative stripes was not as marked as is typically
seen in rodents (e.g., Brochu et al., 1990; Eisenman and Hawkes,
1993), but was slightly higher than that we observed in pigeons
(Pakan et al., 2007) and reminiscent of what has been reported in
cats (Sillitoe et al., 2003) and primates (Sillitoe et al., 2004). In the
serial sections, the contrast between stripes was much stronger
(Figs. 2–4).
3.1. Zebrin II expression in the anterior lobe
As shown in Fig. 2, the anterior lobe of the hummingbird
cerebellum expresses ZII in an immunopositive/immunonegative
striped pattern across folia II–V. Although the number of stripes is
conserved across these folia, one medial and three lateral stripes,
the width and position of the stripes varies depending upon which
folium is examined. In folia II/III, there is a broad central stripe
flanked by three lateral stripes that are quite narrow (1–2 Purkinje
cells in width, Fig. 2A and D). Four stripes were usually observed in
folia IV and V (Fig. 2A–C), butsometimes a fifth couldbe delineated
(Fig. 4B), and overall, stripes were somewhat broader than those in
foliaII/III.StripesP2+andP3+couldbetracedfromtheP3+andP4+
stripes of folia II/III (Fig. 2A–C). The number of stripes then
increases in the dorsal part of V before transitioning into the
largely positively labeled folium VI (Fig. 2B and E). Folium I (the
lingula)isthesoleexceptiontothestripedpatternofZIIexpression
in the anterior lobe; all Purkinje cells are ZII+ and no stripes are
apparent (Fig. 2A, C and E).
3.2. Zebrin II expression in the posterior lobe and vestibulocerebellum
Fig.3 showstheexpression ofZIIacross theposterior lobeofthe
hummingbird cerebellum. The dorsal part of the posterior lobe
(folia VI and VII) does not have a positive/negative pattern of
stripes in the same fashion as folia II–V of the anterior lobe or the
restofthe posteriorlobe.Instead,mostPurkinje cellsinfolia VIand
VII express ZII with ‘negative’ zones identified by lower levels of
immunoreactivity rather than no immunoreactivity. This parallels
a similar pattern observed in both pigeons (Fig. 3E and F; Pakan
et al., 2007) and mammals (Brochu et al., 1990) whereby a ‘central
zone’ in the cerebellum is defined by the presence of Purkinje cells
that are weakly or strongly immunoreactive for ZII rather than
positive and negative stripes.
In contrast to the central zone, folia VIII-IXcd comprise an
expression domain with alternating ZII positive/negative Purkinje
cell stripes. As with the anterior lobe (Fig. 2), one central and three
lateral stripes are ZII-positive in folia VIII, IXa and IXb (Fig. 3A–D).
The number of stripes within folium IXcd is increased by two
additionalstripesbilaterally(Fig.3Cand D).Moremedially,several
weakly immunopositive stripes (‘?’) are interspersed between the
more prominent and broader P+ stripes (Fig. 3B–D). These narrow,
faintly immunoreactive stripes in IXcd were typically only 1–2
Purkinje cells in width, but were nevertheless apparent in both
serial sections (Fig. 3C) and in wholemounts (Fig. 3B).
Finally, the most caudal folium of the posterior lobe, folium X,
was generally uniformly immunopositive in the hummingbirds
(Fig. 5A and B), the exception being the lateral margin where folia
IXcd and X merge to form the auricle (Au), which was ZII
immunonegative.
3.3. PLCb4 expression
Anti-PLCb4 immunoreactivity was expressed in the somata of
the Purkinje cells, but not in the axonal processes (Fig. 4A). In a
similar fashion to ZII, PLCb4 revealed alternating immunopositive/
immunonegative stripes in Purkinje cells of the cerebellar cortex
(Fig. 4B–H). Generally, the pattern of PLCb4 immunoreactivity was
opposite to that of ZII; PLCb4 positive stripes were negative for ZII
andviceversa(Figs.4and5).Similarly,Thiscouldbeobservedmost
clearly in the anterior lobe (Fig. 4B, D and F) and in folia IXcd
(Fig. 5). Folium X is almost entirely negative for PLCb4 and positive
for ZII except for the lateral edge of X, where it joins with IXcd to
form the auricle, in which a stripe of Purkinje cells is clearly
positive for PLCb4 and negative for ZII (Fig. 5). Within the ‘central
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Fig. 2. The expression of zebrin II (ZII) in the anterior lobe of Anna’s Hummingbird (Calypte anna) and pigeon (Columba livia) cerebella as revealed by immunohistochemistry.
(A) A photo of the anterior lobe of a hummingbird cerebellum prepared as a wholemount, with folia I and II/III indicated by Roman numerals. Scale bar = 1 mm. (B) A coronal
section taken through folia IV and V of a hummingbird cerebellum showing the striped pattern of ZII expression with labeling of the somata and dendritic arbors of Purkinje
cells. Four stripes are shown: a medial positive stripe (1) flanked by three lateral stripes (2–4). Scale bar = 100 mm. (C) A coronal section taken through the anterior lobe of a
hummingbird cerebellum that shows the striped pattern of ZII expression across folia IV–VI. Note that the ventral (bottom) half of folium V corresponds more closely to the
stripes of folium IV, whereas the dorsal (top) half of V is more similar to that of folium VI. In addition, folium VI is largely immunopositive with weakly delineated stripes of
low/high immunoreactivity as opposed to the immunonegative/immunopositive stripes of folia IV and V. Scale bar = 200 mm. (D) Zebrin II expression in folia II/III of a
hummingbird is shown. As with folia IV and V, a central stripe (1) is flanked by three lateral stripes (2–4), but in folia II/III they are only 1 or 2 Purkinje cells wide. Scale
bar = 100 mm.(E)Schematic ofZII expressionacrosstheentire anteriorlobe ofthehummingbird withimmunopositivestripes/regionsindicatedin redand theauricleby ‘Au’.
The pale pink stripes in folium VI represent the weakly immunopositive stripes characteristic of this folium. Scale bar = 1 mm. (F) A coronal section through the anterior lobe
of the pigeon cerebellum showing a similar striped pattern of ZII immunoreactivity with stripes labeled 1–4. Scale bar = 500 mm. (G) Schematic of ZII expression across the
entire anterior lobe of the pigeon with immunopositive stripes/regions indicated in red and the weakly immunopositive stripes of folia V and VI in pink. Scale bar = 2.5 mm.
A.N. Iwaniuk et al./Journal of Chemical Neuroanatomy 37 (2009) 55–63
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Fig. 3. The expression of zebrin II (ZII) in the posterior lobe of the Anna’s Hummingbird (Calypte anna) and pigeon (Columba livia) cerebella as revealed by
immunohistochemistry. (A) A photo of the posterior lobe of a hummingbird cerebellum prepared as a wholemount with folia VII–IXcd and the auricle (Au) indicated. As with
the anterior lobe, a parasagittal series of immunpositive/immunonegative ZII stripes are clearly present. Scale bar = 1 mm. (B) A magnified view of folia IXab and IXcd of the
wholemount showing the faint, narrow ZII immunopositive stripes that are interspersed between the strongly ZII immunopositive and broader 1–3 stripes. Scale bar = 1 mm.
(C) A coronal section taken through the posterior lobe of a hummingbird cerebellum. As with the anterior lobe, ZII stripes are clearly present across the folia. Note that folium
VIII is largely immunopositive with stripes of low immunoreactivity rather than immunonegative stripes. Several stripes are indicated (1–6) as well as several narrow and
faintly immunopositive stripes in folium IXcd (indicated by ‘?’). Scale bar = 200 mm. (D) Schematic of ZII expression across the entire posterior lobe of the hummingbird with
immunopositive stripes (1–6) indicated in red, the weakly immunopositive stripes of folia VI–VIII in pink, and the auricle by ‘Au’. The ‘?’ refers to narrow and faint
immunopositive stripes characteristic of folium IXcd. Scale bar = 1 mm. (E) A coronal section through the posterior lobe of the pigeon cerebellum (folia VIII–IXcd) showing a
similar striped pattern of ZII immunoreactivity with stripes labeled 1–7. Scale bar = 500 mm. (F) Schematic of ZII expression across the entire posterior lobe of the pigeon
cerebellumwithimmunopositivestripes(1–7) indicatedinred,theweakly immunopositivestripesoffoliaVIandVII inpinkandtheauricleby‘Au’.Aswiththehummingbird
in (D), the ‘?’ refers to the narrow and weakly immunopositive stripes characteristic of folium IXcd. Scale bar = 2.5 mm.
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Fig. 4. The expression of phospholipase Cb4 (PLCb4) in the Rufous Hummingbird (Selasphorus rufus) cerebellum as revealed by immunohistochemistry. (A) The pattern of
PLCb4 expression in the cerebellar cortex of the hummingbird; the somata, but not the axonal processes, are labeled. Scale bar = 50 mm. (B) A coronal section taken through
the anterior lobe of a hummingbird cerebellum showing the topography of PLCb4 (green) and zebrin II (ZII, red) immunolabeling across folia IV–VI. In general, the PLCb4
stripes are complementary to those of ZII. Scale bar = 250 mm. (C) A magnified view of PLCb4 and ZII immunolabeling in folium VI (from Fig. 3B). The arrows indicate double-
labeled cells.Scale bar = 50 mm.(D)Amagnified viewof foliumIV(fromFig.4B).Scale bar = 50 mm.(E)Amagnifiedviewof PLCb4and ZIIimmunolabeling inlateralfoliumVI
(from Fig. 4B). The arrows indicate double-labeled cells. Scale bar = 50 mm. (F) A magnified view of lateral folium IV (from Fig. 4B). Scale bar = 50 mm. (G) A coronal section
taken through folium VI and II/III showing the topography of PLCb4 and ZII. Note that the majority of folium II/III is immunopositive for PLCb4 with only narrow ZII
immunopositivestripes.FoliumVIhasamixofbothPLCb4andZIIimmunopositivecells.Scalebar = 250 mm.(H)Acoronalsectiontakenthroughtheposteriorlobe(foliaVII–
IXcd) of the hummingbird cerebellum showing the topography of PLCb4 and ZII. Scale bar = 250 mm.
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zone’ of folia VI and VII, cells expressed PLCb4 in a series of weak
and strong stripes (Fig. 4B, G and H). Most of these weak/strong
stripes were opposite to that of the ZII immunolabeling, but some
the cells appeared to be double-labeled for both PLCb4 and ZII
(Fig. 4C and E). The remainder of the posterior lobe was heavily
stripedinalargelycomplementaryfashiontothatofZII,with some
Purkinje cells double-labeled for PLCb4 and ZII, particularly at the
borders of the stripes. Finally, the lingula (folium I) was entirely
negative for PLCb4.
4. Discussion
Overall, ZII is expressed in the cerebellar cortex of humming-
birds in a striped pattern of areas of low/none and high
immunoreactivity. These stripes are grossly similar to those of
the pigeon, but with some variations. Furthermore, this pattern of
ZII immunolabeling is largely complementary to that of PLCb4; ZII
immunopositive zones tend to be immunonegative for PLCb4. This
complementary labeling is not, however, exact and there are
populations of Purkinje cells that are double-labeled for both ZII
and PLCb4. These results have significant implications for under-
standing cerebellar evolution in birds by providing insight into the
neurochemical compartmentalization of the cerebellar cortex.
4.1. Zebrin II expression in birds and other vertebrates
The overall topography of ZII expression in the hummingbird
cerebellum is similar to that observed in the pigeon (Pakan et al.,
2007). In both taxa, five main transverse zones can be identified: a
lingula zone restricted to folium I, in which all Purkinje cells are
ZII+; an anterior zone with one large medial stripe flanked by
several narrow stripes; a central zone that is largely immunopo-
sitive but within which numerous stripes displaying higher and
lower immunoreactivity can be discerned; a posterior zone with
prominent stripes; and a nodular zone in which almost all Purkinje
cells are ZII immunopositive.
Within each of the zones, the pattern of ZII expression in
pigeons(Pakanetal.,2007) andhummingbirdsisgenerallysimilar.
The lingular zone (folium I), which is unique to birds (Pakan et al.,
2007), is entirely immunopositive. In the anterior zone, a large
medial stripe extends from folium II to IV and is flanked by three
lateral stripes on either side. Folium V then represents an area of
transition between the anterior and central zones; its ventral
aspect is similar to that of folium IV, but the dorsal aspect is more
similarto foliumVI andthe restof the centralzone.Folia VIand VII,
the central zone, are largely immunopositive with numerous thin
stripes of low/high immunoreactivity. Folium VIII is the second
areaoftransitionwherebythepatternofexpressionindorsalVIIIis
similar to that of folium VII, but the ventral VIII is similar to that of
IXab. Thus, ventral VIII has a central stripe flanked by several broad
positive stripes on either side. Folia IXcd even expresses thin
weakly positive stripes adjacent to the medial stripe in humming-
birds and the pigeon. Finally, the nodular zone (folium X) was
entirely immunopositive.
Despite this similarity in overall pattern, several differences
between the hummingbirds and the pigeon are worth noting. First,
the lateral stripes in the anterior zone of the hummingbirds are far
narrowerthan those of the pigeon; in most cases, they are only 1–2
Purkinje cells wide (Fig. 2D). These narrow stripes are also far more
medial in the hummingbirds than in the pigeon, at least in folia II/
III. Second, there are fewer immunopositive stripes in folia VIII-
IXab of hummingbirds compared to the pigeon. In the pigeon, the
central stripe is followed by up to five lateral stripes (Fig. 3E and F),
whereas only three such lateral stripes were identified in the
hummingbird (Fig. 3C and D). This sort of variation in the
orientation, width and number of ZII stripes in the cerebellar
cortex also occurs in mammals. For example, the central zone of
theRhesusmacaque(Macacamulatta,Sillitoeetal.,2004)isstriped
whereas in rodents it is largely immunopositive (Eisenman and
Hawkes, 1993; Sillitoe and Hawkes, 2002). Similarly, there are
other subtle variations in the number and width of stripes across
mammals (reviewed in Sillitoe et al., 2005). The functional
implications of having fewer or more stripes or thinner/wider
stripes is unknown, but given the correspondence between ZII
immunolabeling and physiology and hodology (e.g., Gravel et al.,
1987; Gravel and Hawkes, 1990; Ji and Hawkes, 1994; Hallem
et al., 1999; Voogd et al., 2003; Sugihara and Shinoda, 2004; Voogd
and Ruigrok, 2004; Pijpers et al., 2005; Gao et al., 2006; Pijpers and
Fig. 5. Immunolabeling for zebrin II (ZII, red, left) and phospholipase Cb4 (PLCb4, green, centre) through two sections of folia IXcd and X of the posterior lobe of Rufous
Hummingbird(Selasphorusrufus).Theoverlayisshownontheright.FoliaIXcdandXmergelaterallytoformtheauricle(‘Au’).Thesectionin(A)isposteriortothatin(B).Scale
bars = 500 mm.
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Ruigrok, 2006), these variations could reflect species differences in
the strength of cerebellar connections or the role of the cerebellum
in different behaviours. Thus, the observed differences between
the hummingbirds and the pigeon, particularly in folia II/III, could
reflect species differences in cerebellar afferents and/or behaviour.
Folia II/III are much smaller in hummingbirds (and swifts) than
other birds (Iwaniuk et al., 2006b, 2007) and Larsell (1967)
postulated that this reflected the poor hindlimb musculature of
hummingbirds, so it is possible that this reflects weak connections
with nerves innervating the hindlimbs.
4.2. Expression of PLCb4 and other antigens
In mammals, several immunohistochemical and histochemical
markers have been used to explore the complex pattern of
cerebellar compartmentation. Recently, PLCb4 was shown to
express a striped pattern that was complementary to that of ZII
(Sarna et al., 2006; Marzban et al., 2007). That is, ZII positive zones
are negative for PLCb4 and vice versa. In the hummingbirds, we
found a similar pattern of expression: ZII positive stripes were
generally PLCb4 negative and ZII negative stripes were PLCb4
positive. PLCb4 immunohistochemistry did, however, differ from
that observed in mammals in two respects. First, PLCb4 labeling
within the cerebellar cortex was markedly different in the
hummingbird compared to the mouse. In the mouse, the somata
of Purkinje cells is only weakly stained and the dendritic arbors are
darkly stained (Sarna et al., 2006). As shown in Fig. 4A, this is not
the case in hummingbirds. In fact, the cell bodies were darkly
stained and the dendritic arbors were only weakly stained. This
more closely resembles the expression of phospholipase Cb3
(PLCb3) in the mouse. PLCb3 is expressed in Purkinje cell bodies in
the mouse, whereas PLCb4 is only expressed in the dendrites
(Sarna etal., 2006). However,the expressionof PLCb3 is coincident
with that of ZII, whereas PLCb4 is complementary. Thus, the
expression of PLCb4 in the hummingbird is similar to that of the
PLCb3inthemouseatthecellularlevel,butitstopographyrelative
to ZII is more similar to that of PLCb4.
The second main difference between the PLCb4 labeling in the
hummingbirds versus that of the mouse is the degree to which it is
complementary to that of ZII. In the mouse, PLCb4 positive stripes
are exclusively ZII negative, but this was not true of the
hummingbirds. Instead, double-labeled Purkinje cells were con-
sistently found at the edges of stripes and many double-labeled
cells in the central zone, which is predominantly ZII immunopo-
sitive. Thus, in general, the PLCb4 positive stripes are comple-
mentary to the ZII positive stripes, but not exclusively so.
4.3. Evolutionary implications
Recent studies by Iwaniuk et al. (2006a,b, 2007) have
demonstrated the degree of variation in cerebellar morphology
among birds. Unlike mammals in which variation can occur both
within the vermis and the cerebellar hemispheres, all of the
interspecific variation in birds is in the vermis. For example,
parrots (Psittaciformes) have highly folded cerebella characterized
bylargeposteriorfolia(IXabandIXcd),whereasthehummingbirds
and swifts have cerebella with extremely small folia II and III. This
variation has been correlated with flying ability and, to a lesser
extent, hindlimbstrength among species (Iwaniuk et al., 2007), but
whether this variation also extends to other features of the
cerebellum, such as neurochemical organization, has remained
unknown until now.
As described above, our results clearly indicate that the overall
pattern of cerebellar compartmentation is largely similar between
the pigeon, which represents an ‘average’ bird, and the humming-
bird, which has one of the most divergent cerebellar morphologies
of any species examined to date (Iwaniuk et al., 2006b, 2007). That
is, both the pigeon and hummingbirds exhibit positive ZII
immunolabeling throughout the lingula, anterior and posterior
zones of positive/negative ZII stripes and a central zone of weak/
strong ZII stripes. Given that the hummingbirds and pigeons are
distantly related to one another (see reviews in Sibley and
Ahlquist, 1990; Johansson et al., 2001; Livezey and Zusi, 2001;
Cracraft et al., 2004; Hackett et al., 2008), we predict that the same
pattern of ZII expression will be true of all birds. Thus, despite
marked differences in the degree of foliation in cerebellar cortex
(Iwaniuk et al., 2006a) and the relative size of individual folia
(Iwaniuk et al., 2007), this overall pattern of ZII expression will
persist. Consistent with this hypothesis, preliminary data for the
chicken has shown that it too shares a similar groundplan
(Marzban et al., unpubl data).
Although the overall pattern of expression may be consistent
across avian species, our study indicates that species differences in
the number and thickness of ZII stripes occur. Thus, within the
confines of a broader pattern, species differences in ZII expression
have evolved. As mentioned previously, differences in the number
of stripes or stripe thickness could reflect differential projection
patterns among species. The difference between hummingbirds
and the pigeon in the size of ZII stripes in folia II/III could reflect
species differences in hindlimb innervation. The examination of
species with well developed hindlimb musculature, such as hawks
and eagles (Accipitridae) or owls (Strigiformes) could aid in
addressing this issue with respect to the anterior lobe. Iwaniuk
et al. (2007) showed that the relative size of folia VI and VII are
significantly expanded in strong fliers, such as waterfowl
(Anseriformes), hawks and seabirds (Procellariiformes). If the size
of ZII positive stripes reflects innervation patterns, then it is
possible that stripes in these folia will be larger in strong fliers as a
result of stronger pectoral muscles. By performing such compar-
isons, it will be possible to determine how conserved the anterior-
central-posterior zone organization is across species as well as the
degree to which stripe width or number of stripes reflects
behavioural differences among species.
Acknowledgements
Theauthorswouldlike tothankDrs.KennethWelchJr.andRaul
Suarez for graciously providing us with hummingbird brains for
this study and two anonymous reviewers for their constructive
criticism.SupportforthisstudywasprovidedbyagranttoRHfrom
the Canadian Institutes for Health Research, scholarships to JMPP
from the Alberta Ingenuity Fund and the Natural Sciences and
Engineering Research Council of Canada (NSERC) and grants from
NSERC and the Canada Research Chairs Program to DRWW.
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