10 1016@j BBR 2016 07 046

Accepted Manuscript
Title: Differential activation and tyrosine hydroxylase

distribution in the hippocampal, pallial and midbrain brain
regions in response to cognitive performance in Indian house
crows exposed to abrupt light environment
Author: S.K. Tahajjul Taufique Vinod Kumar
PII:
DOI:
Reference:
S0166-4328(16)30481-8
http://dx.doi.org/doi:10.1016/j.bbr.2016.07.046
BBR 10353
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Behavioural Brain Research
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Revised date:
Accepted date:
18-3-2016
24-7-2016
28-7-2016
Please cite this article as: Taufique SK Tahajjul, Kumar Vinod.Differential activation and
tyrosine hydroxylase distribution in the hippocampal, pallial and midbrain brain regions
in response to cognitive performance in Indian house crows exposed to abrupt light
environment.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2016.07.046
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1
Taufique and Kumar, Behavioural Brain Research (2nd revision 24 July 2016)
Differential activation and tyrosine hydroxylase distribution in the hippocampal, pallial

and midbrain brain regions in response to cognitive performance in Indian house crows
exposed to abrupt light environment
Running head: Cognitive deficit and associated neural changes in crows
S. K. Tahajjul Taufique and Vinod Kumar*
Department of Zoology, University of Delhi, Delhi, 110 007, India
*Corresponding author:
Email: drvkumar11@yahoo.com, vkumar@zoology.du.ac.in
2
Highlights
Hippocampus and pallium are involved in spatial and visual learning, respectively.
Both cognition and neuronal activity were declined under constant light (LL).
ZENK activation pattern differs with the light environment and learning task type.
ZENK and tyrosine hydroxylase coexpression show role of dopamine in light-effects.
Dopamine decrease in VTA and SN parallels cognitive deficits under LL in corvids.
Abstract
Disruption of the cyclic feature of the day-night environment can cause negative effects on
daily activity and advanced brain functions such as learning, memory and decision-making
behaviour. These functions in songbirds, including corvids, involve the hippocampus, pallium
and midbrain, as revealed by ZENK (a neuronal activation marker) and tyrosine hydroxylase
(TH) expressions. TH is rate-limiting marker enzyme of the biosynthesis of dopamine, widely
implicated in learning and memory. Here, we measured ZENK and TH immunoreactivity in
the hippocampal, pallial and midbrain regions in response to cognitive performance
(learning-memory retrieval) tests in Indian house crows (Corvus splendens) exposed to
constant light environment (LL) with controls on 12 hour light:12 hour darkness. Along with
the decay of circadian rhythm in activity behaviour, LL caused a significant decline in the
cognitive performance. There was also a decrease under LL in the activity of neurons in the
hippocampus, medial and central caudal nidopallium, and hyperpallium apicale, which are
widely distributed with TH-immunoreactive fibres. Further, under LL, TH- immunoreactive
neurons were reduced in number in midbrain dopamine synthesis sites, the venteral tegmental
area (VTA) and substantia nigra (SN), with a negative correlation of co-localized ZENK/THimmunoreactive cells on errors during the association tasks. These results show decreased
activity of learning and memory neural systems, and underscore the role of dopamine in
reduced cognitive performance of diurnal corvids with disrupted circadian rhythms under an
abrupt light environment.
3
Abbreviation
APH
Area parahippocampus
DA
Dopamine
HA
Hyperpallium apicale
HP
Hippocampus
LL
Constant illumination
LMR
Learning and memory retrieval
LD
Light-dark
NCC
Central caudal nidopallium
NCL
Lateral caudal nidopallium
NCM
Medial caudal nidopallium
RI
Retention interval
SN
Substantia nigra
TH-ir
Tyrosine hydroxylase immunoreactive
VTA
Ventral tegmental area
Keywords: Indian house crow; constant light; learning and memory; ZENK; dopamine;
tyrosine hydroxylase
4
1. Introduction
Temporal partitioning of day-night periods is vital to the organization of behavioural and
physiological adaptations including the advanced brain functions [1]. An abrupt light
environment or exposure to light at night can disturb the cyclic feature of the day-night
environment, and cause widespread negative effects on daily cycles of sleep, activity, energy
homeostasis and brain functions. This mainly occurs due to disruptions of the temporal
organization, causing mismatch of an individuals internal circadian rhythms with its external
light environment [1,2].
Increasing evidence suggests that cognitive abilities are intimately tied with the
circadian rhythms, synchronized by light-dark (LD) cycle [1,2]. Hence, desynchronization of
circadian rhythms conditions can severely impact the cognitive processes and performance.
Experiments have shown that light at night attenuates learning and memory, increases anxiety
and depression [3,4] and decreases neuronal plasticity such as the spine density [5] and
neurogenesis [6] in nocturnal rodents. Mice under constant illumination show circadian
rhythm disruption as well as associated sleep deficits, altered mood and depression-like
behaviours [7]. Also, alterations in the natural LD environment significantly impacts sleep
and singing behaviours in diurnal great tits, Parus major [8,9].
Hippocampus plays a major role in the acquisition of spatial learning and memory in
both birds and mammals [10-12]. Avian hippocampus has been shown to be involved in
homing behaviour (pigeon [13]), spatial orientation (zebra finch, Taeniopygia guttata
[11,14]) and food cache memory (corvids [15]). Similarly, the nidopallium is important for
visual learning [16] and working memory in birds [17]. The expression of ZENK, a neuronal
activation marker, in response to the learning test paradigms has been used to show the role
of the hippocampus and nidopallium regions in the learning and memory in birds [11,14].
5
In mammals, the executive functions including manipulation of the informations in

working memory and decision making are mainly ascribed to the prefrontal cortex (PFC) and
frontostriatal neural circuits, which heavily rely on neuromodulation, particularly on
dopamine (DA) [18]. The contribution of DA and its receptors in PFC to learning and
memory processes has been shown in several species [18,19]. The functional equivalent of
mammalian PFC is the avian lateral caudal nidopallium, NCL [20,21]. Several recent studies
have confirmed the role of NCL in the working memory and decision-making behaviour
[22,23] including those required during the learning of associations [24], judgements of
numerical ability [25] and executive control of behaviour in corvids [26]. Therefore,
investigations of the role of dopaminerergic (DA-ergic) system could be a vital step in
understanding the learning, memory and decision-making processes in birds.
The DA-ergic system is very similar in anatomy between mammals and birds [27,28].
DA neurons originate in several closely lying midbrain nuclei, in particular the ventral
tegmental area (VTA) and substantia nigra (SN), and project to many brain areas including
hippocampus and pallium [27]. The identity of the DA neurons can easily be ascertained by
immunohistochemical localization of the catecholamine-synthesizing enzymes, namely
tyrosine hydroxylase (TH), aromatic amino acid decarboxylase, and dopamine--hydroxylase
[29,30]. As a rate-limiting enzyme of the pathway [27,29,30], TH has been commonly used
as a reliable marker of the dopamine synthesis [27]. Further, DA synthesis and its functional
roles as shown by DA receptor expressions in target brain areas have been reported to be
under the circadian clock control [31,32].
The consequence of circadian rhythm disruption on neural processes underlying
cognitive processes and performance under abrupt light environment has not been well
understood. We hypothesized that disruption of the circadian rhythms, as evidenced by
activity behaviour, would negatively affect the activity of learning and memory neural
6
systems and dopamine synthesis, and in turn would result in decreased cognitive
performance. We tested this in a diurnal corvid species, the Indian house crows (Corvus
splendens) in which the timing of activity, ingestive and grooming behaviours has been
shown to be under the circadian rhythm control [33]. In general, corvids are ideal avian
model systems for studying the reward-based learning and memory processes, and they are at
par with the mammalian system when it comes to the cognitive abilities [15]. Therefore, we
examined ZENK and TH expressions in hippocampal, pallial and midbrain regions in order to
assess alterations in the activity and DA synthesis in response to cognitive performance
(learning-memory retrieval, LMR) tests in Indian house crows under constant illumination
(LL), with controls on 12 hour light:12 hour darkness. We predicted disruption in circadian
rhythms in activity behaviour as well as decline in the cognitive performance of crows under
LL. We also expected an overall decrease in activity of the hippocampus and pallium regions,
and reduction in number of TH-ir neurons in the VTA and SN of crows under the LL
condition.
2. Materials and methods

2.1 Subjects and experiments
Adult Indian house crows (Corvus splendens) were caught from the wild, and thus were naive
to the experimentation. These crows were brought to the facility and were housed
individually in cages (100 x 80 x 80 cm), each fitted with two perches and mounted with a
passive infrared motion sensor (DSC, LC100 PI Digital PIR detector, Canada) on the top of
the cage. These cages were illuminated by compact florescent lamps (Phillips CFL lamp, 5W,
220-240 V). Temperature of the room was always maintained at 24 2 C. Food (prepared
meal consisting of bread crumbs, egg, fruits and cheese) and water were provided ad libitum.
7
During the training and testing for behavioural assessment crows were deprived of basic food
for 2-3 hours prior to the onset of training and test trials. All the training and testing were
done in the same cage, in which crows were initially housed to avoid any effect of novelty
during the test. The meat pellet, which was found to be preferred diet, was chosen to serve as
the reinforcement (reward) during the training and testing.
Two experiments were performed. Experiment 1(n = 12) tested the spatial learning
and experiment 2 (n = 6) tested the pattern associative learning under periodic and aperiodic
light conditions, as per the protocol summarized in Table 1. Crows were exposed first to
periodic (12 h light: 12 h darkness, 12L:12D) for a week, and then half of them were
maintained on 12L:12D and other half were subjected to constant illumination (LL; Fig. 1a).
This change in light cycle was done to compare the cognitive performance between crows
showing rhythmic (synchronized) and arrhythmic activity behaviour under 12L:12D and LL,
respectively. The general activity of each crow within the cage was continuously monitored
by motion sensor detector and recorded and analyzed by computerized data acquisition
system equipped with The Chronobiology Kit software program of Stanford Software
Systems, Stanford, USA, as earlier described [34]. Activity records over 7 days in each
condition were used to calculate the daily and hourly activity profiles. Hourly activity over 24
h for each crow was averaged over 7 days, and then mean SEM was calculated for the
group (Fig. 2a, b). All experimental procedures were adopted in full compliance with
guidelines of the Institutional Animal Ethical Committee of the University of Delhi, Delhi,
India.
2.2 Apparatus and training
A plastic tray (size = 15 x 23 cm) with six food cups (size = 4.5 x 4.5 cm) arranged in 2 rows
of 3 wells were used as the set-up for training and testing the learning and memory retrieval
8
(LMR). Crows were first habituated to the set-up to avoid neophobia, and thereafter were
trained by experimenter to open lids and search for the reward. For spatial LMR task, white
plastic lids (6 x 6 cm) were taped to the food cups by a double-sided tape (Fig. 1b), whilst for
pattern LMR task, six different patterns for lids were used to cover the food cups during the
training and testing phases.
At the end of each experiment, crows were tested for the cognitive performance each
time in the middle of the light period, in between 11:00 13:00 h. Two cognitive
performance tests were done. The first test involved the spatial learning, in which crows (n =
6) in both LD and LL conditions were trained for 5 trials to learn the position of a rewarded
food cup, randomly assigned to each crow. The task acquisition was measured in the number
of errors that a crow made across test trials and the proportion of crows in the group
successfully completed the test phase. We set out the following criteria to determine the task
perfomance relative to random performance. (1) The proportion of crows participating and
completing the task must be 80%. (2) The error component during test should not exceed 2.
This is because if a crow learnt only the row in which the rewarded cup is located, it can
make a maximum error of 2, and if a crow learns only the column of the rewarded cup
location it can make a maximum error of 1 error. Hence, 0 error would mean learning both
the row and column (exact location) of the rewarded location. The experimental protocol that
we used during the training and testing sessions for spatial learning was adapted from Guigueno
et al. [35], but with specific modifications. For example, we carried out 5 training trials and only
before the first testing session, unlike 5-7 or 10 practice trials carried out before each test by
Guigueno et al. [35]. This was because 5 training trials met our experimental goal. That is, all
crows were able to learn in 5 trials the rewarded food cup in the set-up that we used, as was also
confirmed in the pilot study that we performed before the actual experiment began.
9
The second test involved the pattern learning in which under both LD and LL
conditions, crows (n = 3 each) were trained for discrimination learning between the two
patterns assigned randomly to each crow and introduced before the trials began. Crows were
trained for 10 trials per session per day, to discriminate between the two patterns, of which
only one was rewarded. We set the success criteria of learning at 80% correct responses in
2 consecutive test sessions [36]
2.3 Test for cognitive performance
During the test for spatial memory, each crow was tested for 10 trials after 1h and
subsequently after 24 h of training to test the memory retention (retention interval, RI), with a
10-trial session each time with same rewarded location [35]. After the correct location of
food, the set up was reintroduced after 10 sec for the next trial (Video S1). The test for
pattern learning was similar in the sense that crows were tested for 10 trials after 1 h and
subsequently 24 h RI, but here they were tested for the ability to discriminate previously
rewarded from the 5 unrewarded patterns. The position of covered cups was randomly altered
with each trial to avoid crows to form a spatial association (Video S2). Crows were allowed
to choose until they found the correct location or pattern in a trial. The errors committed and
duration required for the completion of task was calculated to assess learning and memory for
the location. We controlled for inadvertent visual and olfactory cues by preparing trays out of
birds' field of view and keeping meat pellets in all the food cups before the presentation.
Videos were recorded for all behavioural tests and analyzed using Observer XT 10 software,
Noldus Information Technology, Wageningen, The Netherlands.
2.4 Immunohistochemistry of ZENK and TH
We used immunohistochemical procedures as standardized and used in several of our recent
publications [37,38]. Crows were deeply anesthetized with ketamine-xylazine solution (0.003
10
ml/g body weight) after 1 h of the completion of 24 h RI test in both the experiments. Crows
were transcardially perfused with 100 ml saline followed by 100 ml of fixative (4%
paraformaldehyde; PFA in 0.1 M phosphate buffer, pH 7.4). Post perfusion, brain was
removed and again post-fixed overnight in 4% PFA. Thereafter, brain was sectioned 40 m
thick sections and collected in 6 successive bins in the phosphate buffer saline (PBS; 10mM,
pH 7.4) and stored at 4 C until processed for the immunohistochemistry. Every sixth section
was used for the immunostaining of ZENK and tyrosine hydroxylase (TH) double-labelling.
Free-floating sections washed thrice in PBS (10 min each) were first treated with 0.3%
hydrogen peroxide (dissolved in methanol, 30 min) to inactivate the endogenous peroxide
activity, and then with 1% bovine serum albumin (BSA) in PBS with 0.3% Triton X-100;
PBSBT (30 min) to block non-specific binding with the antibody. Thereafter, sections were
incubated with primary antibody for ZENK (1:1500; Erg-1/sc-189, Santa Cruz
Biotechnology, Heidelberg) [37,39] first for 2 h at room temperature and then overnight at 4
C. On the following day, sections were washed in PBS and incubated for 2 h each
sequentially with biotin conjugated secondary antibody (1:200; B2770; Invitrogen, Eugene,
USA) and avidin biotin complex (1:55; Elite ABC Kit, Vector Laboratories, Burlingame,
CA). Thereafter, sections were washed in PBS and visualized for antigen-antibody reaction
by diaminobenzidine solution (DAB; D4293, Sigma, prepared in 0.1 M phosphate buffer, pH
7.4) for 4 min to give brown colouration. After the minimal background reaction, PBS
addition stopped DAB reaction. Thoroughly washed sections in PBS were treated with
PBSBT to block non-specific binding and incubated with primary antibody for TH (1:1500;
Cat no. AP08757PU-N, Acris Antibodies, Herford, Germany) [23] and thereafter the
procedure was the same as above except that this time sections were visualized for THimmunoractivity using SG substrate (SK 4700, Vector labs) for 2 min, which gave blue
colour. After PBS washes, sections were rinsed with distilled water, ordered and mounted
11
onto poly-L-lysine coated slides. Air dried sections were then dehydrated in ascending
alcohol grades (70%, 90% and 100%), cleared in xylene and cover slipped with DPX.
Photomicrographs of sections were acquired using Nikon DS-Fi2 microscope. The
quantification of cell numbers, mean cell area and intensity was done by the NIS Element 4.3
software (Nikon instrument Inc.). To quantify the neuronal activity, ZENK likeimmunoreactive (ZENK-lir) cells were counted in the predetermined square windows and
summed across three consecutive sections of the left hemisphere (Fig. 3a) [39]. We
confirmed and delineated the brain areas by comparing them with the brain atlas of largebilled crow, Corvus macrorhynchos [40]. We focused on the brain regions which are
suggested to be involved in the cognition, and these included the hippocampus (HP),
areaparahippocampus (APH), hyperpallium apicale (HA, visual wulst area), central, medial
and lateral caudal nidopalium (CNC, MNC and LNC), substantia nigra (SN) and ventral
tegmental area (VTA). Visible landmarks were taken into account to position square frames
at the same place for all the animals. TH-immunoreactivity was assessed in the VTA and SN
region and quantified for the number, intensity and area of TH-lir cells as well as the number
of co-expressing ZENK/TH-lir cells. All images were labelled, arranged in panels and minor
adjustment of contrast and brightness were performed, if required, using Corel Draw X3
(Toronto, Canada).
2.5 Statistical analysis
One-way analysis of variance with time as repeated measure (1-way RM ANOVA) followed
by Newman-Keuls post hoc test determined the significant daily variation in activity. R
software program version 3.1.3 (R Foundation for statistical Computing, Vienna, Austria) in
glmmADMB and lme4 packages [41,42] was used to anlyze the total errors and duration (per
trial). Generalized linear mixed models (glmm) fitted to negative binomial distribution was
12
used considering over-dispersion of the data with subject as random term. Separate models
were run to test differences in the error numbers and search time and interactions in each
condition. A general linear model (glm) was used to correlate neural activities with learning
paradigm and light condition as variables [39]. Students t-test tested differences between two
conditions or time-points. Pearson correlation coefficient was calculated to show correlation
between two variables. All statistical analyses, except the glmm and glm were done using
GraphPad prism software programme version 5.0 (San Diego, USA).
3. Results
3.1 Behavioural responses
All crows exhibited diurnal pattern in their activity behaviour with a significant rhythm (p <
0.0001; 1-way RM ANOVA), which persisted under 12L:12D but was decayed under the LL
(p = 0.2079; 1-way RM ANOVA, cf. Fig. 2a, b). In spatial LMR task, we consistently found
that by 3rd of the 10 test trials after 1 h RI, the errors ranged from 0-1, and the error component
remained within 0-2 range until the last test trial (see Video S1). Thus, the performance was
better than the chance performance. Further, all crows participated in the training and testing
sessions, except 1 individual under LL during the test after 24 h RI. As evidenced by
increased errors and per trial search time, there was a significant decline in cognitive
performance under LL, as compared to 12L:12D (p < 0.05, GLMM test, Table 2; Fig. 2c).
In pattern LMR task, crows took 2 sessions of 10 trials each under 12L:12D, but 3 - 4
sessions under LL to reach the defined success criteria of 80% correct response in 2
consecutive sessions for discriminating a rewarded from the unrewarded pattern (Fig. 2d).
As with spatial LMR task, we found a significant decline in the cognitive performance with
respect to the pattern LMR task under LL. The errors committed, not search time, during test
trials were significantly increased under LL, as compared to 12L:12D (p < 0.05, GLMM test,
13
Table 2; Fig. 2e). Under both 12L:12D and LL, however, there were significantly reduced
errors in test trials after 24 h RI, as compared to 1 h RI, in the pattern LMR task (p < 0.05,
GLMM test, Table 2; Fig. 2e).
3.2 Effects on neuronal activation and dopamine synthesis
There was a significant effect of the learning paradigm on ZENK-like immunoreactive (-lir)
cells with increased ZENK-lir cells in HP in response to spatial LMR task (Fig. 3c; p < 0.05,
GLM test, Table 3) and in NCC and NCM in response to pattern LMR task (Fig. 3c; p < 0.05,
GLM test, Table 3). In both test paradigms, LL caused a significant decline in ZENK-like
immunoreactive (-lir) cells in HP, HA, NCM and NCC, but not in APH or NCL (Fig. 3c; p <
0.05, GLM test, Table 3). All the forebrain areas were distributed with TH-lir fibres (Fig. 3b).
Next, we assessed the role of DA in cognitive performance in crows with disrupted circadian
rhythms. For this, overall TH-immunoreactivity (-ir) and co-expression of ZENK/ TH-ir were
evaluated in the VTA and SN (Fig. 4a-e). A significantly reduced TH-ir was found in both
the VTA and SN of birds under LL. As compared to 12L:12D, the number (VTA: t12 = 2.417;
p = 0.0325, SN: t12 = 3.261; p = 0.0068, Students t-test), density (VTA: t12 = 3.308; p =
0.0063, SN: t12 = 2.325; p = 0.0384, Students t-test) and area (VTA: t12 = 4.216; p = 0.0012,
SN: t12 = 2.105 p = 0.0570, Students t-test) of TH-ir and co-expressing ZENK/TH-ir cells
(t12 = 3.803; p = 0.0025 and t12 = 3.435; p = 0.0049; Students t-test) were signficantly
reduced under LL (Fig. 4e-h). Also, there was a significant negative correlation of ZENK/TH
co-expressing cells in VTA and SN on errors committed during the 24 h RI test, irrespective
of the LMR paradigm (VTA: r = -0.6107; p = 0.0349; SN: r = -0.7116; p = 0.0047; Pearsons
correlation, Fig. 4g, k).
14
4. Discussion
We report concurrent decline of the cognitive performance and activity of learning and
memory neural systems in crows under LL, as evidenced by increased errors/ search-time in
learning-retrieval tasks and decreased ZENK expression, respectively. We interpret this as
the result of LL-induced decay of circadian rhythms in Indian house crows. This
interpretation is consistent with several findings which show LL-induced circadian rhythm
disruptions in the activity, sleep and melatonin secretion patterns in other diurnal vertebrates,
including birds [9,43-46]. However, we do not completely discount a direct influence of an
uninterrupted light in LL on the activity of neural systems involved in cognition. Light is
known to affect simultaneously the hypothalamic, thalamic, striatal, brain stem, limbic and
other brain regions [47,48], and hence might contribute to an overall decline in cognitive
performance.
Interestingly, the reduction in error component by 24 h RI test indicated an improved
cognitive performance in the pattern LMR task. This could be because crows tested at 24 RI
had an additional practice during the preceding 1 h RI test. However, this does not explain the
absence of similar trend in birds undergoing the spatial LMR task although they also had
similar additional chance of practice learning during 1 h RI. We speculate this to do with
differences in storage of memory between the spatial and pattern learning. A bird retains for a
longer period of time the learned spatial location, compared to the learned visual pattern. In
fact, an accurate cache memory lasts for over 10 days in food caching corvids e.g., the pinyon
jay (Gymnorhinus cyanocephalus) and Clarks nutcrackers (Nucifraga columbiana) [49-51].
Also, scrub jays (Aphelocoma coerulescens) have been found to retain episodic memory for
when and what of the cache location, after 120 h RI test [52].
15
LL-induced decline in cognitive performance was more intimately tied with the
depression in activity of HP, HA, NCC and NCM neurons in Indian house crows. Differences
in ZENK expression patterns revealed linkage of neuronal activation with the light
environment crows were exposed to, and/ or the task-type they were presented to. Compared
to 12L:12D, ZENK-lir cells were significantly decreased under LL in all the mapped brain
areas, except NCL and APH, irrespective of the learning paradigm (Fig. 3c). This is
consistent with studies in rats showing the impairment of hippocampal neurogenesis, learning
and memory under LL [6,53]. Increased ZENK-ir cells were also found in HP and NCC (also
NCM under 12L:12D) during spatial and pattern LMR tasks, respectively. Perhaps HP was
more actively involved in the spatial learning in crows, consistent with its suggested role in
the spatial orientation in homing piegon [13], food-storing corvids [15,54] and zebra finches
(Taeniopygia guttata) [11]. NCC and NCM neurons, on the other hand, may be involved in
the pattern LMR task [55]. Several lines of evidence do suggest the role of hippcampal and
pallial regions in the visuospatial processing and memory [55-57]. At the same time, NCL
neurons which have been suggested to encode for visual working memory in corvids [22,23]
were not differentially active between the LMR tasks in Indian house crows. This difference
between corvid species could be due to differences in test paradigms used between the
studies. Further, no difference in HA activity between LMR tasks could indicate its
involvement in providing information only on where about a reward location.
We suggest that decline in cognitive performance in Indian house crows was due to
LL-induced suppression of the activity of the DA-ergic system. This is evidenced in multiple
ways by the co-expression of ZENK and TH in the VTA and SN. First, shown by decreased
ZENK/TH-lir cells, the attenuated neuronal activity and DA synthesis in VTA and SN
paralleled decline in the cognitive performance (Fig. 3f, j). Secondly, there was a negative
correlation of VTA and SN ZENK/TH-lir cells on errors during the LMR tests (Fig. 3h, k).
16
Thirdly, hippocampal and pallial regions which contain DA receptors [58] were found widely
distributed with DA fibres (Fig. 2). Moreover, all these are consistent with the presence of a
dense DA-ergic system [20], activation of DA neurones by reward-based spatial and visual
post-trial memory consolidation and reinforcement [19], and DA-dependent modulation of
the activity and synaptic plasticity of learning and memory neural systems [59] in the corvid
brain.
Finally, the exposure to periodic light (day-night) environment seems critical for an
optimal cognitive performance required for the survival and fitness in crows, as in other
animals. Cognition deficits seem closely associated with the disruption of circadian rhythms,
and this involves the DA-ergic system spread through several hypothalamic and cortical brain
areas. An abrupt light environment as in the case of an increased light illumination at night in
urban habitats may impair both the circadian (daily) and circannual (annual, seasonal)
functions [60-62]. Inhabiting in cities that are lighted 24 hours a day could have an effect on
cognition in birds. It will be interesting to find out how much night is essential for an
optimum cognitive performance. Perhaps, future studies on birds from rural vs. urban habitats
and from habitats that are exposed daily to long light hours (20 h light per day) may provide
answer to some of these questions.
Conflict of interest: Authors have no conflict of interest.
Supplemental information
Supplemental information includes 2 videos and can be found with this article online.
17
Acknowledgments
We thank Dr. Gabrielle Davidson and Abhilash Prabhat for help in the data analysis.
Department of Science and Technology-Cognitive Science Initiative grant supported the
research. SKTT receives a Research Fellowship from the University Grants Commission,
New Delhi, India.
18
References
1. R. Benca, M.J. Duncan, E. Frank, C. McClung, R.J. Nelson, A. Vicentic, Biological
rhythms, higher brain function, and behavior: Gaps, opportunities, and challenges, Brain.
Res. Rev. 62(2009)57-70
2. C.P. Kyriacou, M.H.Hastings, Circadian clocks: genes, sleep, and cognition, Trends Cogn.
Sci. 14 (2010)259-67
3. L.K. Fonken, E. Kitsmiller, L. Smale, R.J. Nelson, Dim nighttime light impairs cognition
and provokes depressive-like responses in a diurnal rodent, J. Biol. Rhythms 27 (2012)
319-27
4. K.J. Navara, R.J. Nelson, The dark side of light at night: physiological, epidemiological,
and ecological consequences, J Pineal Res. 4 (2007) 215-24
5. T.A. Bedrosian, L.K. Fonken, J.C. Walton, A. Haim,R.J. Nelson, Dim light at night
provokes depression-like behaviours and reduces CA1 dendritic spine density in female
hamsters, Psychoneuroendocrinol. 36 (2011) 1062-9
6. A. Fujioka, T. Fujioka, R. Tsuruta, T. Izumi, S. Kasaoka, T. Maekawa, Effects of a
constant light environment on hippocampal neurogenesis and memory in mice, Neurosci.
Lett. 488(2011) 41-4
7. L.K. Fonken, M.S. Finy, J.C. Walton, Z.M. Weil, J.L. Workman, J. Ross, R.J. Nelson,
Influence of light at night on murine anxiety-and depressive-like responses, Behav. Brain
Res. 205(2009) 349-354.
8. A. Da Silva, J.M. Samplonius, E. Schlicht, M. Valcu, B. Kempenaers, Artificial night
lighting rather than traffic noise affects the daily timing of dawn and dusk singing in
common European songbirds, Behav. Ecol. 25(2014)10371047
9. T. Raap, R. Pinxten, M. Eens, Light pollution disrupts sleep in free-living animals, Sci.
Rep. 5 (2015) 13557
19
10. V.P. Bingman, A. Gagliardo, G.E. Hough, P. Ioale, M.C. Kahn, J.J. Siegel, The avian
hippocampus, homing in pigeons and the memory representation of large-scale space,
Integr. Comp. Biol. 45 (2005) 555-64
11. U. Mayer, S. Watanabe, H.J. Bischof, Spatial memory and the avian hippocampus:
Research in zebra finches, J. Physiol. Paris 107 (2013) 2-12
12. L. Nadel, The hippocampus and space revisited, Hippocampus 1 (1991) 21-9
13. V.P. Bingman, Hough II, M.C. Kahn, J.J. Siegel, The homing pigeon hippocampus and
space: in search of adaptive specialization, Brain Behav. Evol. 62 (2003) 117-27
14. U. Mayer, H.J. Bischof, Brain activation pattern depends on the strategy chosen by zebra
finches to solve an orientation task, J. Exp. Biol. 215 (2012) 426-34
15. N.S. Clayton, N.J. Emery, Avian models for human cognitive neuroscience: a proposal,
Neuron 86 (2015) 1330-42
16. T.B. Patton, S.A. Husband, T. Shimizu, Female stimuli trigger gene expression in male
pigeons, Soc. Neurosci. 4 (2009) 28-39.
17. B. Diekamp, T. Kalt, O. Gunturkun, Working memory neurons in pigeons, J. Neurosci. 22
(2002) RC210
18. M.V. Puig, J. Rose, R. Schmidt, N. Freund, Dopamine modulation of learning and
memory in the prefrontal cortex: insights from studies in primates, rodents, and birds,
Front. Neural Circuits 8 (2014) 93
19. W. Schultz, P. Dayan, P.R. Montague, A neural substrate of prediction and reward,
Science 275 (1997) 1593-9
20. O. Gunturkun, The avian prefrontal cortex and cognition, Cur. Opin. Neurobiol. 15
(2005) 686-93
21. O. Gunturkun, T. Bugnyar, Cognition without cortex, Trends Cogn. Sci. 20 (2016) 291303
20
22. L. Veit, A. Nieder, Abstract rule neurons in the endbrain support intelligent behaviour in
corvid songbirds, Nat. Commun. 4 (2013) 2878
23. L. Veit, K. Hartmann, A. Nieder, Neuronal correlates of visual working memory in the
corvid endbrain, J. Neurosci. 34 (2014) 7778-86
24. L. Veit, G. Pidpruzhnykova, A. Nieder, Associative learning rapidly establishes neuronal
representations of upcoming behavioral choices in crows, Proc. Natl. Acad. Sci. U.S.A.
112 (2015) 15208-13
25. H.M. Ditz, A. Nieder, Neurons selective to the number of visual items in the corvid
songbird endbrain, Proc. Natl. Acad. Sci. U.S.A. 112 (2015) 7827-32
26. F.W. Moll, A. Nieder, Cross-modal associative mnemonic signals in crow endbrain
neurons, Curr. Biol. 25(2015) 2196-201
27. D. Durstewitz, S. Krner, O. Gunturkun, The dopaminergic innervation of the avian
telencephalon, Prog. Neurobiol. 59 (1998) 161-95
28. A. Bjorklund, S.B. Dunnett, Dopamine neuron systems in the brain: an update, Trends
Neurosci. 30 (2007) 194-202
29. P.B. Molinoff, J.U. Axelrod, Biochemistry of catecholamines, Annu. Rev. Biochem. 40
(1971) 465-500
30. S.C. Daubner, T. Le, S. Wang, Tyrosine hydroxylase and regulation of dopamine
synthesis, Arch. Biochem. Biophys. 508 (2011) 1-2
31. M. Weber, T. Lauterburg, I. Tobler, J.M. Burgunder, Circadian patterns of
neurotransmitter related gene expression in motor regions of the rat brain, Neurosci. Lett.
358 (2004) 17-20
32. M.S. Kafka, P.J. Marangos, R.Y. Moore, Suprachiasmatic nucleus ablation abolishes
circadian rhythms in rat brain neurotransmitter receptors, Brain Res. 327 (1985) 344-347.
21
33. S.K.T. Taufique, N.A. Jha, V. Kumar, Circadian rhythm determines the timing of activity,
and ingestive and grooming behaviours in Indian house crows, Corvus splendens, Curr.
Sci. 110 (2016) 897-901.
34. S. Malik, S. Rani, V. Kumar, Wavelength dependency of light-induced effects on
photoperiodic clock in the migratory blackheaded bunting (Emberiza melanocephala),
Chronobiol. Int. 21 (2004) 367-84
35. M.F. Guigueno, D.A. Snow, S.A. MacDougall-Shackleton, D.F. Sherry, Female cowbirds
have more accurate spatial memory than males, Biol. Lett. 10 (2014) 20140026
36. B.A. Bogale, S. Sugawara, K. Sakano, S. Tsuda, S. Sugita, Long-term memory of color
stimuli in the jungle crow (Corvus macrorhynchos), Anim. Cogn. 15 (2012) 285-91
37. A. Rastogi, Surbhi, S. Malik, S. Rani, V. Kumar, Annual life-history dependent seasonal
differences in neural activity of the olfactory system between non-migratory and migratory
songbirds, Behavi. Brain Res. 296 (2016) 233-9
38. Surbhi, A. Rastogi, S. Rani, V. Kumar, Seasonal plasticity in the peptide neuronal
systems: potential roles of gonadotrophin-releasing hormone, gonadotrophin-inhibiting
hormone, neuropeptide Y and vasoactive intestinal peptide in the regulation of the
reproductive axis in subtropical Indian weaver birds, J. Neuroendocrinol. 27 (2015) 35769
39. K. Nishizawa, E.I. Izawa, S. Watanabe, Neural-activity mapping of memory-based
dominance in the crow: Neural networks integrating individual discrimination and social
behaviour control, Neurosci. 197 (2011) 307-19
40. E.I. Izawa, S. Watanabe, A stereotaxic atlas of the brain of the jungle crow (Corvus
macrorhynchos), Integration of comparative neuroanatomy and cognition (2007) pp. 21573, Tokyo: Keio University Press.
22
41. A.J. Carter, H.H. Marshall, R. Heinsohn, G. Cowlishaw, Personality predicts decision
making only when information is unreliable, Anim. Behav. 86 (2013) 633-9
42. Davidson GL, Clayton NS, Thornton, A Wild jackdaws, Corvus monedula, recognize
individual humans and may respond to gaze direction with defensive behaviour, Anim.
Behav. 108 (2015) 17-24
43. J. Aschoff, Handbook of behavioural neurobiology Biological Rhythms, 4 (1981) pp. 545
Plenum Press, New York
44. S.A. Binkley, The clockwork sparrow: time, clocks, and calendars in biological organisms,
(1990) Prentice Hall
45. J. Singh, S. Rani, V. Kumar, Functional similarity in relation to the external environment
between circadian behavioural and melatonin rhythms in the subtropical Indian weaver
bird, Horm. Behav. 61(2012) 527-34
46. Surbhi, Y. Kumari, S. Rani, K. Tsutsui, V. Kumar, Duration of melatonin regulates
seasonal plasticity in subtropical Indian weaver bird, Ploceus philippinus, Gen. Comp.
Endocrinol. 220 (2015) 46-54
47. Surbhi, V. Kumar, Avian photoreceptors and their role in the regulation of daily and
seasonal physiology, Gen. Comp. Endocrinol. 220 (2015) 13-22
48. S. Hattar, M. Kumar, A. Park, P. Tong, J. Tung, K.W. Yau, D.M. Berson, Central
projections of melanopsinexpressing retinal ganglion cells in the mouse, J. Comp. Neuro.l
497 (2006) 326-49
49. P.A. Bednekoff, R.P. Balda, A.C. Kamil, A.G. Hile, Long-term spatial memory in four
seed-caching corvid species, Anim Behav. 53 (1997) 335-41
50. S.B. Wall, R.P. Balda, Ecology and evolution of foodstorage behaviour in
coniferseedcaching corvids, Z. Tierpsychol. 56 (1981) 217-42.
23
51. J.M. Marzluff, R.P. Balda,, The pinyon jay: behavioural ecology of a colonial and
cooperative corvid. (1992) Poyser: London
52. N.S. Clayton, A. Dickinson, Episodic-like memory during cache recovery by scrub jays,
Nature 395(1998) 272-4
53. W.P. Ma, J. Cao, M. Tian, M.H. Cui, H.L. Han, Y.X. Yang, L. Xu, Exposure to chronic
constant light impairs spatial memory and influences long-term depression in rats,
Neurosci. Res. 59 (2007) 224-30
54. Healy SD, Krebs JR. Food storing and the hippocampus in Corvids amount and volume
are correlated. Proc. Royal Soci. London B: Biol. 248 (1992) 241-5
55. Y. Atoji, J.M. Wild, Afferent and efferent projections of the central caudal nidopallium in
the pigeon (Columba livia), J. Comp. Neurol. 517 (2009) 350-70
56. E.M. Aminoff, K. Kveraga, M. Bar, The role of the parahippocampal cortex in cognition,
Trends Cogn. Sci. 17 (2013) 379-90
57. M.T. Avey, L.S. Phillmore, S.A. MacDougall-Shackleton, Immediate early gene
expression following exposure to acoustic and visual components of courtship in zebra
finches, Behav. Brain. Res. 165 (2005) 247-53
58. L. Kubikova, K. Wada, E.D. Jarvis, Dopamine receptors in a songbird brain, J. Comp.
Neurol. 518 (2010) 741-69
59. K.C. Berridge, The debate over dopamines role in reward: the case for incentive salience,
Psychopharmacology 191 (2007) 391-431
60. D.M. Dominoni, W. Goymann, B. Helm, J. Partecke, Urban-like night illumination
reduces melatonin release in European blackbirds (Turdus merula): implications of city
life for biological time-keeping of songbirds, Front. Zool. 10 (2013) 1-1
61. K.A. Robert, J.A. Lesku, J. Partecke, B. Chambers, Artificial light at night desynchronizes
strictly seasonal reproduction in a wild mammal, Proc. R. Soc. B 282 (2015) 20151745)
24
62. D.M. Dominoni, The effects of light pollution on biological rhythms of birds: an
integrated, mechanistic perspective, J. Ornithol. 156 (2015) 409-18
25
Figure legends
Fig. 1. Shows a flow chart for the experimental protocol (a) and sketch of the test apparatus
used for spatial (b) and pattern (c) learning-memory retrieval (LMR) tasks.
Fig. 2. Performance of Indian house crows (Corvus splendens) subjected to a spatial or
pattern associative learning-memory retrieval (LMR) paradigm under periodic (12 h light: 12
h darkness; 12L:12D) or aperiodic (constant illumination, LL) as indicated by the unshaded
(light, day) and shaded (darkness, night) areas over a period of about 3 weeks. a-b doubleplotted representative activity recording (actogram) where successive days are plotted
sideways and underneath. The curve of white circle shows hours profile of activity behaviour
at the end of the first of 12L:12D (in both a and b) and after 2 weeks of further 12L:12D (a)
or LL (b). c-e: Cognitive performance (c - spatial learning; d,e pattern learning) tests. In
spatial LMR task, crows were initially trained for 5 training trials to learn the rewarded
location, and then were tested after 1 h and 24 h retention intervals (RI). In pattern LMR task,
crows were first trained to learn the rewarded pattern and reach a success criteria of 80%
correct choices in 2 consecutive sessions, and then they were tested after 1 h and 24 h RI. d:
Shows the number of training sessions (S1, S2, S3, S4) for 3 crows (C1, C2 and C3) to attain
the criterion of 80 % correct choices (indicated by vertical broken line) in 2 consecutive
sessions in pattern LMR task under 12L:12D and LL. Note 2 sessions under 12L:12D,
compared with 3-4 sessions under LL. c and e: Errors and search time (mean SEM) after 1h
(white bar) and 24 h (black bar) RI under 12L:12D and LL condition during the spatial (c)
and pattern (e) LMR tests.
Fig. 2. Neuronal activity in the brain of Indian house crows (Corvus splendens) subjected to a
spatial or pattern associative learning-memory retrieval (LMR) paradigm under periodic (12
h light: 12 h darkness; 12L:12D) or aperiodic (constant illumination, LL). a Schematic
26
drawing of the coronal sections of a crow brain, which interposed black squaresindicating
focal areaswhere ZENK immunoreactivity was quantified. These areas are implicating in the
cognitive processes. b Double immunohistochemistry showing the expressions of ZENK
(brown nuclei) and tyrosine hydroxylase (TH; blue fibres) under 12L:12D and LL in different
brain areas of crows subjected to spatial or pattern learning memory retrieval (LMR) task.
Scale bar, 50 m. cPlot of quantified immunoractivity of ZENK expression, as ZENK-like
immunreactive (ZENK-lir) cells (mean SEM) in HP, APH, HA, NCC, NCM and NCL of
crows subjected to the spatial and pattern LMR tasks under 12L:12D and LLconditions.
Abbreviations: A, Acropallium; APH, Area parahippocampus; HA, Hyperpalllium apicale;
HP, Hippocampus; M, Mesopallium; n, Nidopallium; NCC, Central caudal nidopallium;
NCL, Lateral caudal nidopallium; NCM, Medial caudal nidopallium; SN, Substantia nigra;
TeO, Optic tectum; VTA, Ventral tegmental area; X, Area X.Details of experiment are given
in fig. 1.
Fig. 3. Double immunohistochemistry of ZENK and tyrosine hydroxylase (TH) in the ventral
tegmental area (VTA) and substantia nigra (SN) in Indian house crows (Corvus splendens)
subjected to a spatial or pattern associative learning-memory retrieval (LMR) paradigm under
periodic (12 h light: 12 h darkness; 12L:12D) or aperiodic (constant illumination, LL). a-e are
the photomicrographs of coronal sections showing VTA (a-c) and/ or SN (a, d, e) regions of
the midbrain. aTH immunoreactivity in the VTA and SN in a scale bar of 5 m. band c
ZENK/TH co-expression in the VTA under 12L:12D (b) or LL (c). d and e ZENK/TH coexpression in the SN under 12L:12D (d) or LL (e), each with images at 10 X and 40 X (inset)
magnification shown in the scale bar of 100 or 50 m, respectively. Inset images show coexpression of the ZENK (brown, nucleus) and TH (blue, cytoplasm), indicated by an arrow
head. F-J: Mean ( SEM) TH-immunoreactivity, measured in cell number (f), area (g) and
intensity (i), ZENK/TH-lir co-expressing cells under12L:12D and LL. h and k Correlation
27
plot of number of errors committed during the LMR tasks on ZENK/TH-lir cells in the VTA
(h) and SN (k). The plots (f-k) show pooled data from both spatial and pattern learning
memory retrieval (LMR) tasks in each light condition. Details of experiment are given in fig.
1.
28
29
30
31
32
Table 1: A summary of the experimental protocol for cognitive performance tests.

Cognitive
performance
Training
Spatial learning
Pattern learning
5 trials for each crow to learn the 10 trials per day to learn two patterns
rewarded location
(one rewarded and one un-rewarded)
(same location of the reward)
till the crows reach 80% success in 2

consecutive session
location of the pattern was changed in
each trial to avoid spatial association
1 h retention interval (RI) 1 h after completion of training (10 trials)

Testing
24 h retention interval (RI) 24 h after completion of 1 h RI testing (10 trials)
testing included discrimination of the
location of the reward was same for rewarded pattern (during training)
both training and testing trials for each from other five un-rewarded patterns.
crow
location of the pattern was changed in
each trial to avoid spatial association.
33
Table 2: A summary of model output from GLMM of factors affecting (condition, RI and
their interaction) the measure of performance (total number of errors and search time) in the
spatial and pattern associative learning paradigms. An asterisk indicates a significant (p <
0.05) factor.
Spatial learning (n = 12)
Number of errors
Intercept
Condition
RI
Condition X RI
Search time
Intercept
Condition
RI
Condition X RI
Pattern learning (n = 6)
Factors
Number of errors
Intercept
Condition
RI
Condition X RI
Search time
Intercept
Condition
RI
Condition X RI
SE
P-value
2.274 0.251
0.834 0.339
-0.270 0.237
0.281 0.308
9.06
2.46
-1.14
0.91
<0.001 ***
0.014 *
0.254
0.360
1.270 0.247
1.200 0.294
0.121 0.311
-0.318 0.379
5.14
4.09
0.39
-0.84
<0.001 ***
<0.001 ***
0.700
0.400
2.457 0.182
0.470 0.236
-0.847 0.324
0.560 0.395
13.46
1.99
-2.62
1.42
<0.001 ***
0.046 *
0.008 **
0.156
1.099 0.335
0.368 0.436
-0.588 0.559
0.662 0.680
3.28
0.84
-1.05
0.97
0.001 ***
0.398
0.293
0.331
34
Table 3: Model output from GLM Zenk expression in different learning paradigms and effect
of light condition. An asterisk indicates a significant (p < 0.05) values.
Factors
Hippocampus (HP)
Intercept
Learning paradigm
Condition
Learning paradigm X Condition
Area parahippocampus (APH)
Intercept
Learning paradigm
Condition
Hyperpallium apicale (HA)
Intercept
Learning paradigm
Condition
Central caudal nidopallium (NCC)
Intercept
Learning paradigm
Condition
Medial caudal nidopallium (NCM)
Intercept
Learning paradigm
Condition
Lateral caudal nidopallium (NCL)
Intercept
Learning paradigm
Condition
SE
P-value
5.099 0.192
0.712 0.253
-0.637 0.275
0.122 0.361
26.47
2.81
-2.31
0.33
<0.001 ***
0.004 **
0.020 *
0.360
5.685 0.232
0.302 0.307
-0.400 0.329
-0.003 0.435
24.45
0.98
-1.21
-0.01
<0.001 ***
0.325
0.224
0.993
5.594 0.169
0.062 0.223
-0.551 0.240
0.017 0.318
33.09
0.27
-2.28
-0.05
<0.001 ***
0.780
0.022 *
0.955
6.606 0.204
-0.547 0.270
-0.763 0.289
0.268 0.3835
32.37
-2.02
-2.63
0.70
0.001 ***
0.042 *
0.008 **
0.483
6.481 0.117
-1.444 0.159
-0.661 0.167
1.386 0.247
55.14
-9.06
-3.94
6.17
<0.001 ***
<0.001 ***
0.001 ***
0.001 ***
5.525 0.170
0.076 0.224
-0.382 0.239
0.499 0.317
32.49
0.34
1.59
-1.57
<0.001 ***
0.733
0.110
0.115

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Title: Differential activation and tyrosine hydroxylase

Behavioural Brain Research

Differential activation and tyrosine hydroxylase distribution in the hippocampal, pallial

Running head: Cognitive deficit and associated neural changes in crows

S. K. Tahajjul Taufique and Vinod Kumar*

Department of Zoology, University of Delhi, Delhi, 110 007, India

ZENK and tyrosine hydroxylase coexpression show role of dopamine in light-effects.

Dopamine decrease in VTA and SN parallels cognitive deficits under LL in corvids.

Learning and memory retrieval

Central caudal nidopallium

Lateral caudal nidopallium

Medial caudal nidopallium

Tyrosine hydroxylase immunoreactive

Ventral tegmental area

In mammals, the executive functions including manipulation of the informations in

2. Materials and methods

Conflict of interest: Authors have no conflict of interest.

Table 1: A summary of the experimental protocol for cognitive performance tests.

till the crows reach 80% success in 2

1 h retention interval (RI) 1 h after completion of training (10 trials)

Spatial learning (n = 12)

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