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Phase inversion of neural activity in the

olfactory and visual systems of a nightmigratory bird during migration
Article in European Journal of Neuroscience June 2011
DOI: 10.1111/j.1460-9568.2011.07737.x Source: PubMed

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European Journal of Neuroscience

European Journal of Neuroscience, Vol. 34, pp. 99109, 2011

doi:10.1111/j.1460-9568.2011.07737.x

NEUROSYSTEMS

Phase inversion of neural activity in the olfactory and visual

systems of a night-migratory bird during migration
Ashutosh Rastogi,2 Yatinesh Kumari,2 Sangeeta Rani2 and Vinod Kumar1,2
1
2

DST-IRHPA Center for Excellence in Biological Rhythm Research, Department of Zoology, University of Delhi, Delhi, 110 007, India
Department of Zoology, University of Lucknow, Lucknow 226 007, India

Keywords: bunting, migration, olfaction, phase inversion, vision

Abstract
Olfactory and visual sensory mechanisms seem to play a critical role in migratory orientation and navigation. How these two
mechanisms are functionally linked with other migratory processes is unknown. We investigated this, in relation to the profound
behavioural shift that occurs during migration in the night-migratory blackheaded bunting (Emberiza melanocephala). Photosensitive
unstimulated birds singly housed in activity cages were subjected to long days (LD 16 8). The activity of each bird was continuously
monitored. Daily activity pattern defined the nonmigratory phase (no nocturnal activity) and migratory phase (intense nocturnal
activity, Zugunruhe). Body mass and testis size were measured at the beginning and end of the experiment. Long days induced the
migratory phenotype (body fattening and Zugunruhe) and testis maturation. The c-fos (Fos) immunoreactivity, as marker of the neural
activity of the olfactory and visual subsystems, was measured at midday (8 h after lights-on) and midnight (4 h after lights-off) after
the first seven long days (nonmigratory phase) and after seven nights of the Zugunruhe (migratory phase). In the nonmigratory
phase, Fos-like immunoreactive (Fos-lir) cells in the olfactory and visual subsystems were high in the day and low at night. In the
migratory phase, this was reversed; Fos-lir cells were high at night and low in the day. The phase inversion of neural activity in the
olfactory and visual systems in parallel with the behavioral shift suggests a functional coupling between the systems governing
migratory flight (expressed as Zugunruhe) and migratory orientation and navigation.

Introduction
Each year, millions of songbirds undertake two long-distance
journeys. In the autumn, they y from their breeding grounds in the
north to wintering grounds in the south (autumnal migration). In the
spring, they do the opposite (spring or vernal migration). These
migrants must anticipate the timing of their migration in order to get
physiologically prepared, and begin ying such that they arrive at their
destination when conditions are favourable (Gwinner, 1996; Gwinner
& Helm, 2003; Rani et al., 2006). They do so using their endogenous
daily and seasonal clocks which, in synchronization with the day
length (photoperiod), provide birds with information about the time of
day and time of year.
Also, birds need to nd their geographic position and direction en
route to their destination. It is suggested that birds use magnetitemediated magnetic pathways to locate their geographic position
(Walker et al., 1997; Fleissner et al., 2003; Falkenberg et al., 2010;
Heyers et al., 2010). In contrast, it seems that night-migratory
songbirds use a vision-mediated pathway acting via cryptochromes
in the retinal ganglion cells (RGCs) and or photoreceptors to nd their
direction (Ritz et al., 2000; Moller et al., 2004; Mouritsen et al.,
2004; Mouritsen & Ritz, 2005). Recent lesion studies by Zapka et al.

Correspondence: Prof. Vinod Kumar, 1Department of Zoology, as above.

E-mail: drvkumar11@yahoo.com
Received 18 October 2010, revised 7 April 2011, accepted 18 April 2011

(2009) on migratory European robins (Erithacus rubecula) indicate a

key role of the telencephalic Cluster N (cN) in magnetic compass
orientation. The ZENK and c-fos (immediateearly genes) expression
studies also support the functional role of the RGCs and cN in
migration (Mouritsen et al., 2004, 2005; Liedvogel et al., 2007;
Zapka et al., 2010).
The olfactory sensory circuits also appear to be actively involved in
avian navigation. Studies both at the behavioural (Gagliardo et al.,
2006, 2008, 2009; Mehlhorn & Rehkamper, 2009) and cellular
(ZENK expression; Patzke et al., 2010) levels suggest the role of
olfactory cues during navigation in the homing pigeon. Also,
migratory starlings (Sturnus vulgaris) and swifts (Apus apus) deprived
of olfactory perception do not show homeward navigation (Fiaschi
et al., 1974; Wallraff et al., 1995). Similarly, anosmic (individuals
with smell sense loss) catbirds (Dumetella carolinensis) seem to loose
the migratory orientation ability (Holland et al., 2009).
Thus, the olfactory and visual sensory systems seem to play a
critical role in avian migration, in particular in migratory orientation
and navigation. Recent studies on migratory garden warblers (Sylvia
borin; Bartell & Gwinner, 2005) and blackheaded buntings (Emberiza
melanocephala; Rani et al., 2006) also suggest (i) a circadian clock
controlling the nocturnal migratory restlessness (Zugunruhe; a
behavioral phenotype characterizing migration in caged birds), and
(ii) phase inversion of the behavioural rhythm during migration.
Whether the olfactory and visual sensory systems are functionally

2011 The Authors. European Journal of Neuroscience 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd

100 A. Rastogi et al.

coupled with other migratory processes such as migratory ight is,
however, unknown. If the answer is yes, then the activity of the
olfactory and visual sensory circuits should be phase-inverted in
parallel with the behavioral shift occurring with the onset of the
migration. We have investigated this in the PalaearcticIndian
migratory blackheaded bunting (Emberiza melanocephala), using
c-fos expression as a marker of the neural activity of the olfactory and
visual subsystems.

Materials and methods

Animal subjects
The blackheaded bunting (Emberiza melanocephala) is a Palaearctic
Indian latitudinal migrant songbird species. It arrives in India
(approximately 25oN) in the fall, overwinters, and returns to its
breeding grounds situated in west Asia and southeast Europe
(approximately 40N) in the spring (Ali & Ripley, 1974). It is a
photoperiodic species and, under long days (> 12 h light per day),
blackheaded buntings exhibit the migratory phenotype (e.g. body
fattening and Zugunruhe; Rani et al., 2006).
The study was carried out on adult male birds that were caught in
the late February 2009 and acclimated for a week to captive conditions
in the outdoor aviary receiving natural sunlight and temperature
conditions. Then, buntings were brought indoors under laboratory
conditions and maintained on short days (8 h light : 16 h darkness;
LD 8 16) at relatively constant temperature (25.0 2.0 C) until the
beginning of the experiment. In short days, buntings remain unstimulated and maintain their photosensitivity (Misra et al., 2004). All
experimental procedures implemented in this study were approved by
the Institutional Animal Ethics Committee (IAEC).

Experimental setup
Buntings were singly housed in specially designed activity cages [size
(length width height) 60 35 45 cm] individually placed inside the photoperiodic boxes (75 50 70 cm) lit by uorescent
bulbs on a programmed lightdark (LD) cycle (L, 150 5.0 lux; D,
< 1 lux). Each activity cage had food and water cups, and two perches
positioned at different heights. A passive infrared motion sensor
(Haustier PIR-Melder Intellisense XJ 413T with 12 m (40) range; C
& K Systems, Conrad Electronic, Germany) was mounted on to the
cage such that it covered the entire cage area. The sensors, connected
to separate channels of a computerised data recording system,
continuously detected the movement of the bird within its cage. The
movements were counted and recorded in 5-min bins by a software
program from Stanford Software Systems, Stanford, CA, USA.
Activity records (actograms) over the duration of the experiment
were generated as a double plot by plotting 2 days across the paper,
with successive pairs of days underneath (Malik et al., 2004; Rani
et al., 2006). In addition, the activity was quantied and presented in a
bar diagram to better illustrate the activity response and to make
statistical comparisons. For this, activity counts over the selected
number of days were averaged for a specied period (e.g. 24 h, daily;
16 h, day; 8 h, night) for each bird, and from this the group
mean SD was calculated. At the beginning and end of the
experiment, we recorded body mass (g) and testis size (testis volume;
TV) as measures of birds physiological status. We measured body
mass using a top-pan balance to an accuracy of 0.1 g. For testis size,
we recorded dimensions of the left testis dissected out from perfused
birds, and TV was calculated as 4 3 pab2, where a and b denote half
of the long and short axes, respectively. The general husbandry and

experimental conditions were the same as described in Misra et al.

(2004).

Experiment
The experiment involved a total of 23 buntings (group A, n = 11;
group B, n = 12) individually housed in activity cages inside the
photoperiodic boxes. At the beginning, birds had unstimulated (no
visible fat, normal) body mass (group A, 26.25 0.98 g; group B,
27.22 1.89 g; mean SD) and small testes (group A TV,
0.45 0.12 mm3; group B TV, 0.45 0.10 mm3; mean SD). They
were rst exposed for 1 week to short days (LD 8 16) and then the
light period was extended to 16 h per day, i.e. LD 16 8 (long days).
After 7 days of LD 16 8, group A birds were perfused at midday (8 h
after lights-on; n = 7) or midnight (4 h after lights-off; n = 4). At this
time, birds had slight and variable fattening (body mass
30.86 3.40 g; mean SD) and testis size (TV 6.66 2.93 mm3;
mean SD). This was referred to as the nonmigratory phase as birds
had not shown Zugunruhe yet. After 1113 long days, group B birds
started showing Zugunruhe. After a bird had shown 7 nights of
Zugunruhe, this was referred to as the migratory phase; it was perfused
at midday or midnight (n = 6 each time). At this time, birds were fully
fattened (body mass 39.62 2.51 g; mean SD) and had matured
testes (TV 38.19 11.38 mm3; mean SD).

Fos immunohistochemistry
Birds were deeply anesthetized with ketaminxylazine solution
(0.003 mL g body weight) and perfused transcardially successively
with 50 mL ice-cold saline (pH 7.4) and 30 mL xative (4%
paraformaldehyde in 0.1 m phosphate buffer, pH 7.4). At night,
perfusions were done under nonstimulatory dim green light at the
intensity (< 1 lux) of the dark phase of the LD cycle. Brains were
quickly dissected out and post-xed overnight in the same xative.
They were then cryoprotected by immersing them at 4 C for 2 h each
in 10% and 20% and overnight in 30% sucrose (Merck) solutions.
Finally, brains were embedded into 15% polyvinylpyrrolidone
(PVP40T; Sigma) blocks and serially sectioned in the coronal plane
at 30 lm thickness using a Leica CM 1850 cryostat. Four successive
sections were separately collected in wells lled with phosphatebuffered saline (PBS; 10 mm, pH 7.4) and the fth section was thawmounted onto a poly-l-lysine-coated slide. Thus, we sectioned a brain
in a total of 5 bins, and in each bin the sections were separated by
150 lm. The rst four bin sections were stored at 4 C until processed
for the Fos immunohistochemistry. The fth bin sections mounted on
slides were dried at the room temperature and processed for Nissl
staining (Kluver & Barrera, 1953). These were used as reference to
conrm the area of interest in the present study (Huber & Crosby,
1929; Stokes et al., 1974; Kuenzel & Masson, 1988; Butler & Hodos,
1996; Medina & Reiner, 2000; Mouritsen et al., 2005; Heyers et al.,
2007).
All sections were processed together for the c-fos (Fos) immunohistochemistry. We used the standard avidinbiotin protocol with
minor modications, as described in Kumar et al. (1997). The xative
was removed by three times washing (10 min each) in PBS (pH 7.4).
The sections were similarly washed in between the steps that were
sequentially carried out as follows. The endogenous peroxidase
activity was blocked by treating sections with 0.3% hydrogen
peroxide solution in methanol (30 min). The nonspecic binding
was blocked with 1% normal bovine serum albumin (BSA) solution in
PBS containing 0.3% Triton X-100 (PBSBT; 30 min). The sections

2011 The Authors. European Journal of Neuroscience 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 99109

Phase inversion in sensory activity 101

were then incubated for 18 h at 4 C with rabbit antichicken Fos
antibody at 1 : 6000 dilution in PBSBT. This Fos antibody, a generous
gift from Professor Dr Lut Arckens, Leuven, Belgium, was generated
in rabbits, and its specicity was demonstrated by Dhondt et al.
(1999). The sections were reincubated for 2 h each sequentially with
biotinylated goat antirabbit secondary antibody (1 : 200; B2770,
Invitrogen, Eugene, USA) and avidinbiotin complex (1:55; Elite
ABC Kit, Vector Laboratories, Burlingame, CA, USA). Finally, the
sections were treated for 23 min with diaminobenzidine solution
(DAB; D4293, Sigma) prepared in 0.1 m phosphate buffer (pH 7.4).
This gave colour to Fos-labelled cells and thus helped in visualization
of the antigenantibody reaction. The color reaction was stopped by
adding the PBS. The sections were rinsed in distilled water, ordered,
mounted onto poly-l-lysine-coated slides, and dried overnight.
Thereafter, they were dehydrated through ascending grades of alcohol,
cleared in xylene and cover-slipped in DPX (Merck). The slides were
stored and observed for analysis.
Microscopy and analysis of Fos immunoreactivity in the
olfactory and visual systems
The images of brain sections were taken via a Leica DM 3000
microscope with a xed Leica DFC 420C digital camera. The
dimensions of the image were 2592 1944 pixel RGB 8 bit. The
magnication numerical aperture (NA) used were as follows:

40 0.1, 100 0.25 or 400 0.65. The desired eld from a

section was photographed using standardized illumination. A minor
contrast and brightness adjustment, if needed, was done using Adobe
Photoshop 7.0 (San Jose, CA, USA). All images were labelled and
arranged in panels using Corel Draw X3 (Toronto, Canada).
The olfactory subsystems examined in this study were similar to
several previous studies (Huber & Crosby, 1929; Kuenzel & Masson,
1988; Butler & Hodos, 1996; Medina & Reiner, 2000). In particular,
these included the olfactory bulb (OB), anterior olfactory nucleus
(OA), prepiriform cortex (CPP), lateral olfactory tract (LOT) and
olfactory cortex (piriform cortex, CPI; lateral olfactory cortex, LC).
The visual sensory circuits included both the thalamofugal and
tectofugal pathways. In the thalamofugal pathway, we examined the
thalamic dorsal geniculate complex (GLd; consisting of nucleus
lateralis dorsalis nuclei optici principalis thalami [LdOPT], lateral
division of nucleus dorsolateralis anterior thalami, pars lateralis
[DLLl] and ventral division of nucleus dorsolateralis anterior thalami,
pars lateralis [DLLv]), telencephalic Wulst (tW) which includes
hyperpallium apicale (HA), nucleus interstitialis hyperpallii apicalis
(IHA), hyperpallium intercalatum (HI), hyperpallium densocellulare
(HD) and the cN, which is a specialized part of the tW. In the
tectofugal pathway we analysed the optic tectum (OT), which has
afferents from the tW (Huber & Crosby, 1929; Kuenzel & Masson,
1988; Butler & Hodos, 1996; Medina & Reiner, 2000; Mouritsen
et al., 2005; Heyers et al., 2007).

C
D

Fig. 1. Changes in (A) body mass and (B) testis size of two groups (group A, n = 11; group B, n = 12) of photosensitive blackheaded buntings exposed to long
days (16 h light: 8 h dark; 16L : 8D). (C) Daily activity pattern of a representative individual. Birds were sampled at midday (8 h after lights-on) and midnight (4 h
after lights-off), as indicated by an asterisk (*) in the middle of the double-plotted representative actogram (C), rst after seven long days (nonmigratory phase,
group A) and then after a bird had seven nights of the Zugunruhe (migratory phase, group B). (D and E) Mean SD activity ( 100 counts) over 24 h (L + D), 16 h
(L, day) or 8 h (D, night) in long day during both the nonmigratory and migratory phases. Signicance at P < 0.05 is indicated at the top of the bar: * compares
initial vs. nal observation of the same group; + compares similar observations of nonmigratory and migratory phases.
2011 The Authors. European Journal of Neuroscience 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 99109

102 A. Rastogi et al.

Fig. 2. Olfactory system in daytime: neural activity in the olfactory system in blackheaded buntings exposed to LD 16 8 and measured in the middle of the day
(8 h after lights-on). (A) Schematic drawing of images shown in (C)(H). They do not represent real anatomical positions, but show structures usually not visible in a
single plane. (B) Total Fos-lir cells (mean SD per grid area, 40 000 lm2) during the nonmigratory and migratory phases of the experiment. (CH) Images showing
Fos expression in nonmigratory (C, E, G; left panel) and migratory (D, F, H; right panel) phases. CI and DI, OB; CII and DII, OA; CIII and DIII, CPP; EI and FI,
CPI; GI and HI, LOT; GII and HII, LC. (IK) Fos-lir cell density (mean SD) in OB, OA, CPP, CPI, LOT and LC. Abbreviations: CPI, piriform cortex; CPP,
prepiriform cortex; LC, lateral olfactory cortex; LOT, lateral olfactory tract; OA, anterior olfactory nucleus; OB, olfactory bulb. Asterisk (*) indicates the signicance
at P < 0.05. Scale bars, 1 mm (A); 500 lm (CH); and 50 lm in magnied views (I, II, III).

The morphometric analyses of the Fos-like immunoreactive (Foslir) cells in different neuronal groups were done as described for the
suprachiasmatic nucleus (SCN) in rodents (Kumar et al., 1997).
Briey, an image (100 ) of the specied region was captured by a
Nikon E400 microscope xed with a DS-Fi1 Nikon digital camera.
The dimensions of the image were 2560 1920 pixel RGB 8 bit. The
magnication numerical aperture (NA) used was 100 0.25. The
microscope setting was standardized and kept constant. The counting
was done using the Nikon NIS-elements BIS program (version 2.3).
For each bird, we counted Fos-lir cells in all sections of the specied
region. For this, Fos-lir cells of the entire specied region were
mapped applying a 200 200 lm grid (olfactory system) or
100 100 lm grid (visual system and medial SCN; mSCN). This
gave the average number of Fos-lir cells per grid area (areas for
olfactory system, 40 000 lm2; for visual system and mSCN,
10 000 lm2) for each bird. We counted both strongly (bright) and

weakly (faint) stained cells in order to avoid a staining intensity-based

bias in quantication (Gentner et al., 2001; Shimizu et al., 2004;
Patzke et al., 2010). As described in the studies of Gentner et al.
(2001) and Shimizu et al. (2004), a threshold optical density was
dened manually based on background staining, and cells which had
an optical density higher than the threshold were counted as Fos-lir
cells. To minimize error, two independent observers with no
knowledge of the actual experimental protocol repeated the counting
procedure. Their numbers were averaged for each neuronal group.
Finally, mean SD for the group was calculated.

Statistical analysis
The data are presented as mean SD. We used Students t-test in
comparing the data of the same region from two groups or conditions.

2011 The Authors. European Journal of Neuroscience 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 99109

Phase inversion in sensory activity 103

Fig. 3. Olfactory system at night: Neural activity in the olfactory system in blackheaded buntings exposed to LD 16 8 and measured in the middle of the night (4 h
after lights-off). A) The schematic drawing of images is shown in CH. It does not represent real anatomical positions, but shows structures usually not visible in a
single plane. B) Total Fos-lir cells (mean SD per grid area, 40 000 lm2) during the nonmigratory and migratory phases of the experiment. CH) Images
showing Fos expression in nonmigratory (C, E, G; left panel) and migratory phase (D, F, H; right panel). C I and D I OB; C II and D II OA; C III and D III CPP;
E I and F I CPI; G I and H I LOT; G II and H II LC. I, J, K) Fos-lir cell density (mean SD) in OB, OA, CPP, CPI, LOT and LC. Asterisk (*) indicates the
signicance at P < 0.05. Scale bar in schematic drawing (A) = 1 mm; general views (CH) = 500 lm; and magnied views (I, II, III) = 50 lm. For abbreviations,
see Fig. 2.

Two-way anova tested the signicance of difference at two factor

levels: factor 1, migratory nonmigratory; factor 2, day night. Bonferronis post hoc test was employed for group comparisons if anova
indicated a signicant difference. A statistically signicant difference
was considered at the P < 0.05 level. All data analyses were done
using Graph Pad Prism software version 5.0 (San Diego, CA, USA).

Results
Changes in physiological status
At the beginning of the experiment, buntings had normal body mass
(2529 g) and small immature testes (TV 0.45 mm3), and were dayactive (Fig. 1AC). After the rst seven long days, birds had initiated
body fattening (D body mass = 4.60 3.81 g, n = 11; P = 0.0025,
paired t-test, group A) and testis recrudescence (Fig. 1A,B). However,
they still had the nonmigratory phenotype, i.e. birds were day-active

and did not exhibit Zugunruhe (Fig. 1C,D). After 1113 long days,
birds began showing Zugunruhe. At this time, the total daily activity
was signicantly increased (P = 0.0120, Students t-test), and birds
were predominantly night-active (Fig. 1C,E). Thus, buntings had
developed the migratory phenotype. After seven nights of Zugunruhe,
all birds were fully fattened (D body mass 12.40 3.25 g; n = 12,
P < 0.0001, paired t-test, group B) and had matured testes (Fig. 1).
Thus, in between the rst and second observations, there was a
signicant increase (P < 0.05, unpaired t-test) in body mass, testis size
and total activity (Fig. 1).

Changes in Fos expression in the sensory systems

Olfactory system
Fos-lir cells were high in the day and low at night during the
nonmigratory phase (Figs 2 and 3). The reverse was found during the

2011 The Authors. European Journal of Neuroscience 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 99109

104 A. Rastogi et al.

Fig. 4. Visual system in daytime. Neural activity of the visual system in blackheaded buntings exposed to LD 16 8 and measured in the middle of the day (8 h after
lights-on). (A) Schematic drawing of images shown in (C)(J). It does not represent real anatomical positions, but shows structures usually not visible in a single
plane. (B) Total Fos-lir cells (mean SD per grid area, 10 000 lm2) during the nonmigratory and migratory phases of the experiment. (CJ) Images showing Fos
expression in nonmigratory (C, E, G and I; left panel) and migratory (D, F, H and J; right panel) phases. CI and DI, LdOPT; CII and DII, DLLv; CIII and DIII, DLLl;
EI and FI, OT; GI and HI, HA; GII and HII, IHA; GIII and HIII, HI; GIV and HIV, HD; II and JI, cN. (KN) Fos-lir cell density (mean SD) in GLd complex
(LdOPT, DLLv and DLLl), OT, tW (HA, IHA, HI and HD) and cN. Abbreviations: cN, Cluster N; DLLl, lateral division of nucleus dorsolateralis anterior thalami,
pars lateralis; DLLv, ventral division of nucleus dorsolateralis anterior thalami, pars lateralis; GLd, dorsal geniculate complex; GLv, nucleus geniculatus lateralis,
pars ventralis; HA, hyperpallium apicale; HD, hyperpallium densocellulare; HI, hyperpallium intercalatum; IHA, nucleus interstitialis hyperpallii apicalis; III, third
ventricle; LdOPT, nucleus lateralis dorsalis nuclei optici principalis thalami; OT, optic tectum; ROT, nucleus rotundus; SPC, nervus supercialis parvocellularis; TrO,
optic tract; TSM, septopalliomesencephalic tract; tW, telecephalic Wulst. *P < 0.05. Scale bars, 1 mm (a), 200 lm (CF), 500 lm (GJ); 50 lm (IIV).

migratory phase: Fos-lir cells were signicantly reduced in the day

(P < 0.0001; Fig. 2B) and increased at night (P = 0.0003; Fig. 3B).
This suggested phase inversion in the neural activity with the onset of
the migration. This was true for most olfactory subsystems that were
individually mapped. During the day the OB, OA, CPP, CPI, LOT and

LC had signicantly lower Fos expression in the migratory phase than

in the nonmigratory phase (P < 0.05; Fig. 2CK). During the night,
this pattern was reversed in all these areas (P < 0.05; Fig. 3CK)
except in the OB (Fig. 3, CI, DI and I). Two-way anova revealed a
signicant difference in the effects of the physiological status

2011 The Authors. European Journal of Neuroscience 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 99109

Phase inversion in sensory activity 105

Fig. 5. Visual system at night: Neural activity of the visual system in blackheaded buntings exposed to LD 16 8 and measured in the middle of the night (4 h after
lights-off). (A) Schematic drawing of images shown in (C)(J). It does not represent real anatomical positions, but shows structures usually not visible in a single
plane. (B) Total Fos-lir cells (mean SD per grid area, 10 000 lm2) during the nonmigratory and migratory phases of the experiment. (CJ) Images showing Fos
expression in nonmigratory (C, E, G, I; left panel) and migratory (D, F, H, J; right panel) phases. CI and DI, LdOPT; CII and DII, DLLv; CIII and DII, DLLl; EI and FI,
OT; GI and HI, HA; GII and HII, IHA; GIII and HIII, HI; GIV and HIV, HD; II and JI, cN. (KN) Fos-lir cell density (mean SD) in GLd complex (LdOPT, DLLv
and DLLl), OT, tW (HA, IHA, HI and HD) and cN. For abbreviations, see Fig. 4. *P < 0.05. Scale bars, 1 mm (A); 200 lm (CF), 500 lm (GJ), 50 lm (IIV).

(migratory vs. nonmigratory, factor 1: F1,19 = 6.77, P = 0.0175) and

condition (day vs. night, factor 2: F1,19 = 19.74, P = 0.0003) as well
as the interaction between two factors (F1,19 = 145.65, P < 0.0001;
see Figs 2 and 3).
Visual system
There was a clear daynight change in the neural activity with
the change in the physiological status (Figs 4 and 5). During the

migratory phase, the Fos expression was signicantly reduced in

the day (P < 0.0001, unpaired t-test; Fig. 4B) and increased at night
(P = 0.0009, unpaired t-test; Fig. 5B). This pattern persisted at the
level of most individual subsystems. For example, during the
migratory phase, the Fos-lir cells were signicantly lower in the day
in the OT (P < 0.0001; Fig. 4E, F and L) and tW (P < 0.05; Fig. 4G
J,M,N) but not in the GLd (Fig. 4C,D,K). They were increased at
night in all these subsystems (P < 0.05; Fig. 5), except in DLLv of the
GLd and HA, and cN of the tW (Fig. 5). Two-way anova revealed a

2011 The Authors. European Journal of Neuroscience 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 99109

106 A. Rastogi et al.

Fig. 6. Relationships between behavioural and neural activity responses measured at midday (8 h after lights-on) and midnight (4 h after lights-off) in blackheaded
buntings exposed to long days (LD 16 8). (AD) Scatter plots of Fos-lir cells in the olfactory (A and B) and visual (C and D) systems in day (A and C) and night
(B and D) of the long day as function of the behavioral activity of the preceding day (16 h light) and night (8 h dark), respectively, during the nonmigratory and
migratory phases. (E) Total (mean SD) activity and Fos-lir cells over 24 h during the nonmigratory and migratory phases. Activity but not Fos expression was
signicantly increased during the migratory phase. (F) Values on activity and Fos-lir cells are plotted from Figs 1 and 25, respectively, to show the relationship
between behavioural and neural activity responses. Note a signicant increase in activity responses at night during the migratory phase.

signicant difference in the effects of the physiological status

(migratory vs. nonmigratory, factor 1: F1,19 = 4.78, P = 0.0416) and
condition (day vs. night, factor 2: F1,19 = 6.58, P = 0.0189) as well as
the interaction between two factors (F1,19 = 70.57, P < 0.0001; see
Figs 4 and 5).
Figure 6 shows relationships between the neural (Fos-lir cells)
activity responses in the day (Fig. 6A,C) or night (Fig. 6B,D) both
during the nonmigratory (group A) and migratory (group B) phases.
The two activity responses lacked a signicant correlation (cf.
Fig. 6AD). This was further evident when the neural and behavioural
activity responses over 24 h (daily, day + night), 16 h (day) or 8 h
(night) were compared (Fig. 6E,F). A signicant increase (P = 0.0120,
unpaired t-test) in total daily activity but not in the Fos-lir cell density
occurred during the migratory phase.
The Fos expression was also examined in the SCN. The neural
activity of the mSCN, which is thought to be the site of the circadian
pacemaker in song birds (Kumar et al., 2004), was not phase-inverted
(Fig. 7).

Discussion
Photoperiodic induction of migration-linked phenologies
Data presented in Fig. 1 conrm our previous ndings (see Misra
et al., 2004; Rani et al., 2006) that long days induce the migratory
phenotype in the blackheaded bunting. Birds fattened and gained in
weight, and developed Zugunruhe (Fig. 1C,E). There was also testis
maturation under long days, indicating the development of the
reproductive phenotype. However, the two phenotypes are not
causally linked. They are separate photoperiodic phenomena although
they share a common functional basis (that is, the circadian
rhythm-mediated photostimulation; Kumar, 1986, 1988). Buntings
appear to have a lower threshold photoperiod for fattening than for
testis maturation, and the suppression of body fattening does not
depress the testis recrudescence (Kumar & Rani, 1999; Kumar et al.,
2006). Also, castration does not affect body fattening in the
blackheaded bunting subjected to long days (Tewary & Kumar,
1981). Therefore, we would argue that the effects on migration-linked

2011 The Authors. European Journal of Neuroscience 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 99109

Phase inversion in sensory activity 107

Fig. 7. mSCN in day and night. Neural activity of the mSCN in blackheaded buntings exposed to LD 16 8 and measured in the middle of the day (8 h after lightson) and middle of the night (4 h after lights-off). (AD) Images showing Fos expression in the nonmigratory phase (A, day; C, night) and migratory phase (B, day;
D, night). (E and F) Fos-lir cell density (mean SD) in day (E) and night (F). Abbreviations: CO, optic chiasma; III, third ventricle; LHA, lateral hypothalamic area;
mSCN, medial suprachiasmatic nucleus; TSM, septopalliomesencephalic tract. Scale bars, 500 lm (AD), 50 lm (I).

events in the current experiment should be considered independent of

the gonadal status.
Phase inversion in the neural activity: involvement of sensory
systems in migration
The present study for the rst time demonstrates that, in parallel with
the behavioral rhythm, there was a shift in the neural activity of the
olfactory and visual sensory circuits that seem to be playing a critical
role in the migration. In fact, the activity of the neuronal groups in
these sensory circuits appears to have undergone phase inversion (see
Figs 25). High and low neural activity in the day and night,
respectively, during the nonmigratory phase were reversed during the
migratory phase (see Figs 25). Interestingly, daily neural activity of
the sensory subsystems had not signicantly changed as revealed
by the comparison of the total number of Fos-lir cells over 24 h
between the nonmigratory and migratory phases (Fig. 6E). One
explanation could be that the change in activity level in the visual
system simply reects the fact that more activity leads to more visual
input (see Feenders et al., 2008). However, given that the changes
were observed in both the olfactory and visual sensory circuits, we
speculate that the phase reversal of the neural activity in parallel with
the behavioral shift suggest a functional coupling between the systems
governing the migratory ight (expressed as Zugunruhe) and migration-linked olfactory and visual tasks. This appears physiologically
adaptive. By phase inversions, the olfactory and visual systems seem
to synchronize their neural activity with the migratory ight when
orientation and navigation are much needed.
There was an increased night activity during the migratory phase
in all major olfactory subsystems including the OA, CPP, LOT, CPI
and LC (Fig. 3CK). The OB did not show a change in Fos-lir cells
(Fig. 3CI,DI,I), probably because being the primary olfaction unit
for several other vital functions (e.g. nding food, mate etc.) it
needed to be active irrespective of the physiological state. A
signicantly high number of Fos-lir cells at night during the
migratory phase (Fig. 3) may suggest an increased olfactory
perception during the migration. Considered together with the high
daytime Fos expression during the nonmigratory phase (Fig. 2BK),

we would suggest that an increased olfactory perception is linked to

daily active state in the blackheaded buntings, as suggested in
rodents (Granados-Fuentes et al., 2006).
Several studies have suggested that olfaction plays a role in
migration. Migratory starlings (Wallraff et al., 1995) and swifts
(Fiaschi et al., 1974) deprived of olfactory perception fail to home to
their nest. Holland et al. (2009) from their experiments on catbirds
suggest the role of olfactory cues in the migratory orientation.
Experiments on pigeons show that olfactory cues are required for the
development of navigational abilities (Gagliardo et al., 2008, 2009).
Jorge et al. (2009) further report that odours play a signicant role in
activation rather than navigation in homing pigeons. It may be
cautioned, however, that these studies involved sensory manipulations, which might have disturbed the receptive ability of the
physiological systems (Fiaschi et al., 1974; Wallraff et al., 1995;
Gagliardo et al., 2006, 2008, 2009; Holland et al., 2009; Patzke et al.,
2010). Our study had no such sensory manipulation, and so the results
on activity responses seem to suggest a linkage between olfaction and
migration.
Also, we observed a phase inversion in the activity of visionrelated sensory circuits during the migratory phase (Figs 4 and 5).
However, GLd did not show phase inversion, although during the
migratory phase it had signicantly higher Fos expression at night
(Fig. 5, CI and III, D I and III, and K). GLd had a similar daytime
activity during both the migratory and nonmigratory phases
(Fig. 4C,D,K). This is probably because being the rst relay of
the avian thalamofugal pathway, with afferents from the retina and
efferents to tW, the GLd needs to remain active in order to sense
the predictable or unpredictable changes in the environment (see
Mouritsen et al., 2005; Heyers et al., 2007; Valencia-Alfonso et al.,
2009).
In the tW, which is comparable to the primary visual cortex of
mammals (Medina & Reiner, 2000), cN had similar high Fos
expression at night during both the nonmigratory and migratory
phases (Fig. 5I,J,N), as reported in migratory garden warblers and
European robins (Mouritsen et al., 2005; Liedvogel et al., 2007). cN
is clearly involved in magnetic compass orientation, as revealed by a
lesion study on migratory European robins (Zapka et al., 2009).

2011 The Authors. European Journal of Neuroscience 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 99109

108 A. Rastogi et al.

Fos-lir cell density of the cN in buntings seems to suggest that it was
almost inactivated in day during the migratory phase (Fig. 4J,N),
similar to what has been reported in other migratory songbirds
(Mouritsen et al., 2005; Liedvogel et al., 2007; Zapka et al., 2010).
This result is consistent with the idea that cN is involved in processing
night-time light-dependent magnetic compass information in migratory birds. However, the possibility remains that cN is active during
the day but involves a molecular pathway that does not induce ZENK
or c-fos (Zapka et al., 2010). It should also be mentioned that the
daytime ZENK and c-fos were not measured during the nonmigratory
phase in the studies by Mouritsen et al. (2005) and Zapka et al.
(2010). We have measured Fos-lir cells in day and at night during both
the nonmigratory and migratory phases, and the data show that the cN
was active in day during the nonmigratory phase as well (Fig. 4I,N).
Possibly, the cN as an active component of the visual sensory circuit
also shifts its activity, as do the other visual subsystems. However,
given low Fos-lir cell density in the visual system, in general, we will
not rule out the involvement of additional molecular pathways in the
visual system-mediated migratory navigation, as Zapka et al. (2010)
have argued.
he other tW divisions, namely HD and HI, had high Fos expression
at night only in the migratory phase, which could suggest that they are
actively participating in the processing of visual information during
migratory orientation (Gunturkun, 1991; Shimizu & Bowers, 1999).
Signicantly lower Fos-lir cell density in these regions during daytime
of the migratory phase indicates a reduced activity of the visual system
when probably other sensory mechanisms are used (see Zapka et al.,
2010). Thus, during nocturnal migration, buntings appear to involve
the entire thalamofugal visual pathway including cN (see Feenders
et al., 2008), not just the cN as suggested from other studies
(Mouritsen et al., 2005; Liedvogel et al., 2007).

Are behavioural and neural activity responses directly linked?

An increased behavioural activity during migration per se seems to
have no direct effect on levels of Fos induction in the sensory
subsystems (Fig. 6). The two activity responses lacked a signicant
correlation in all (Fig. 6AD). Furthermore, unlike a signicant
increase in the total daily behavioural activity (Figs 1C,E and 6E), the
total daily Fos-lir cell density of both the olfactory and visual systems
did not signicantly change during the migratory phase (Fig. 6E).
However, there was a signicant change in the Fos expression density
between day and night in response to the change in the physiology of
the blackheaded bunting (Figs 25 and 6F). This could mean that
behavioural and neural activity responses are independent at the
regulatory level, although both the regulatory systems seem to
function in synchrony, and possibly share a common physiological
timing mechanism upstream. A circannual clock is suggested in
regulation of the annual migration in birds (Gwinner, 1996). Recent
studies also provide evidence for the circadian clock-controlled
Zugunruhe in a few migratory species including the blackheaded
bunting (Bartell & Gwinner, 2005; Rani et al., 2006). We therefore
suggest that the activation of the olfactory and visual sensory systems
needed for migratory orientation and navigation is phased such that all
physiological and behavioral changes occur synchronously during the
migration. This would probably explain why, in buntings, the
olfactory and visual perceptions are changed (as seen in differential
Fos induction) in the absence of sensory manipulation and when
environmental conditions such as photoperiod, temperature and food
condition remained unchanged.

Conclusions
The neural activity of the brain nuclei and regions that form the
olfactory and visual sensory systems undergo phase inversions, in
parallel with behavioural activity, with the onset of the migration.
Whether the two activity responses are linked upstream to a common
regulatory pathway is unknown. At this time, it seems that the sensory
systems needed for migratory orientation and navigation in the
blackheaded bunting are regulated in such a way that their activity
remains in synchrony with migratory ight. It is possible that this is
achieved through endogenous circadian and circannual clocks, but this
needs further investigation.

Acknowledgements
We acknowledge with gratitude a very generous gift of Fos antibody from
Professor Dr Lut Arckens, Laboratory of Neuroplasticity and Neuroproteomics,
K.U. Leuven, Belgium. The study was funded by a research grant from the
Department of Science and Technology, New Delhi, India, through IRHPA
research funding (IR SO LU-02 2005) and carried out at the Department of
Zoology, University of Lucknow, Lucknow, India.

Abbreviations
cN, cluster N; CPI, piriform cortex; CPP, prepiriform cortex; DLLl, lateral
division of nucleus dorsolateralis anterior thalami, pars lateralis; DLLv, ventral
division of nucleus dorsolateralis anterior thalami, pars lateralis; Fos-lir, Foslike immunoreactive; GLd, dorsal geniculate complex; HA, hyperpallium
apicale; HD, hyperpallium densocellulare; HI, hyperpallium intercalatum; IHA,
nucleus interstitialis hyperpallii apicalis; LC, lateral olfactory cortex; LD, light
dark; LD 8 16, 8h light : 16h darkness; LdOPT, nucleus lateralis dorsalis
nuclei optici principalis thalami; LOT, lateral olfactory tract; mSCN, medial
SCN; OA, anterior olfactory nucleus; OB, olfactory bulb; OT, optic tectum;
SCN, suprachiasmatic nucleus; TV, testis volume; tW, telencephalic Wulst;
Zugunruhe, nocturnal migratory restlessness (a behavioral phenotype characterizing migration in caged birds).

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