Variation in hippocampal morphology along an environmental gradient: controlling for the effects of day length.

Abstract

Environmental conditions may create increased demands for memory, which in turn may affect specific brain regions responsible for memory function. This may occur either via phenotypic plasticity or selection for individuals with enhanced cognitive abilities. For food-caching animals, in particular, spatial memory appears to be important because it may have a direct effect on fitness via their ability to accurately retrieve food caches. Our previous studies have shown that caching animals living in more harsh environments (characterized by low temperatures, high snow cover and short day lengths) possess more neurons within a larger hippocampus (Hp), a part of the brain involved in spatial memory. However, the relative role of each of these environmental features in the relationship is unknown. Here, we dissociate the effects of one theoretically important factor (day length) within the environmental severity/Hp relationship by examining food-caching birds (black-capped chickadee, Poecile atricapillus) selected at locations along the same latitude, but with very different climatic regimes. There was a significant difference in Hp attributes among populations along the same latitude with very different climatic features. Birds from the climatically mild location had significantly smaller Hp volumes and fewer Hp neurons than birds from the more harsh populations, even though all populations experienced similar day lengths. These results suggest that variables such as temperature and snow cover seem to be important even without the compounding effect of reduced day length at higher latitudes and suggest that low temperature and snow cover alone may be sufficient to generate high demands for memory and the hippocampus. Our data further confirmed that the association between harsh environment and the hippocampus in food-caching animals is robust across a large geographical area and across years.

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doi: 10.1098/rspb.2010.2585
published online 2 February 2011
Proc. R. Soc. B
Timothy C. Roth II, Lara D. LaDage and Vladimir V. Pravosudov
gradient: controlling for the effects of day length
Variation in hippocampal morphology along an environmental


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Variation in hippocampal morphology along
an environmental gradient: controlling for
the effects of day length
Timothy C. Roth II*, Lara D. LaDage and Vladimir V. Pravosudov
Department of Biology, University of Nevada, 1664 North Virginia Street, MS 314, Reno, NV 89557, USA
Environmental conditions may create increased demands for memory, which in turn may affect specific
brain regions responsible for memory function. This may occur either via phenotypic plasticity or selec-
tion for individuals with enhanced cognitive abilities. For food-caching animals, in particular, spatial
memory appears to be important because it may have a direct effect on fitness via their ability to accu-
rately retrieve food caches. Our previous studies have shown that caching animals living in more harsh
environments (characterized by low temperatures, high snow cover and short day lengths) possess
more neurons within a larger hippocampus (Hp), a part of the brain involved in spatial memory. However,
the relative role of each of these environmental features in the relationship is unknown. Here, we dis-
sociate the effects of one theoretically important factor (day length) within the environmental severity/
Hp relationship by examining food-caching birds (black-capped chickadee, Poecile atricapillus) selected
at locations along the same latitude, but with very different climatic regimes. There was a significant
difference in Hp attributes among populations along the same latitude with very different climatic fea-
tures. Birds from the climatically mild location had significantly smaller Hp volumes and fewer Hp
neurons than birds from the more harsh populations, even though all populations experienced similar
day lengths. These results suggest that variables such as temperature and snow cover seem to be impor-
tant even without the compounding effect of reduced day length at higher latitudes and suggest that low
temperature and snow cover alone may be sufficient to generate high demands for memory and the hippo-
campus. Our data further confirmed that the association between harsh environment and the
hippocampus in food-caching animals is robust across a large geographical area and across years.
Keywords: hippocampus; neuron; environmental gradient; black-capped chickadee;
caching; food hoarding
1. INTRODUCTION
Memory is an important trait used to gather, retain and
recall information about the environment. As such, it is
probably important for survival. We know that many
environmental factors can produce increased demands
on memory [1], which in turn may potentially affect
memory function. However, it is not clear how variation
in these traits is produced and which aspects of the
environment might be most relevant for changes in the
function and the underlying mechanism(s) of memory.
It is possible that variation in memory is a function of
one or two prevailing factors of the environment. Alterna-
tively, multiple aspects of the environment may affect
memory in an additive or multiplicative fashion, thereby
makingtherelationshipbetween
memory and the brain more complex.
Environmental severity has been suggested as one poss-
ible factor affecting memory and other cognitive abilities in
a variety of different animals [1–3]. For example, in many
food-storing animals the degree of environmental severity
(traditionally characterized by ambient temperature, snow
cover and day length) may be especially important [4–7].
Many temperate zone food-storing species are non-
migratory and store food in numerous locations (i.e.
theenvironment,
scatter-hoard)
autumn) for retrieval at a later time when resources are
scarce (i.e. winter). This behaviour is thought to be an
adaptation to survive harsh winter conditions when ambi-
ent food is unavailable [4,5,8,9]. The retrieval of these
scattered food stores (or caches) is facilitated in part by
memory, of which spatial memory appears to be particu-
larly important [4,5]. Theory (e.g. [9]) predicts that
selection on cache retrieval ability, and hence spatial
memory, may be a function of environmental severity,
where low ambient temperature, high snow cover and
reduced day length demand more efficient foraging in
order to maintain positive energy balance. Thus, there
are probably strong demands for accurate memory,
especially at high latitudes where environmental severity
is increased and caching and retrieving food becomes
more important for survival. Although it is possible that
some caching species do not use memory for the retrieval
of long-term caches, it is clear that chickadees use memory
for short-term retrieval [10], which should still improve
daily survival in harsh environments (e.g. [9]). In addition,
the ability to retrieve caches should be more important
than caching intensity, unless more caching automatically
results in more successful retrieval.
Enhanced memory in harsh environments may poten-
tially be facilitated via a change in the hippocampus (Hp),
the region of the brain responsible in part for spatial
whenresourcesareabundant(i.e.
* Author for correspondence (tcroth@unr.edu).
Proc. R. Soc. B
doi:10.1098/rspb.2010.2585
Published online
Received 1 December 2010
Accepted 11 January 2011
1
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Page 3

memory [5,11,12]. More demanding situations (e.g.
more harsh environments) seem to result in specialized
phenotypes (e.g. a larger Hp region) that underlie a
specific cognitive task (e.g. memory-based cache retrie-
val). This pattern may be produced by the selection of
enhanced traits in specific environments (e.g. adaptive
specialization) [4,5,13,14] or may be the consequence
of phenotypic plasticity whereby enhancements in the
Hp are produced due to increased caching and increased
memory use itself. In either case, our previous work [7]
provides large-scale support for the association between
environmental harshness and the hippocampus. Black-
cappedchickadeesfrom
possessed significantly larger Hp with more neurons
than those from more mild southern latitudes, a relation-
ship that was also found in a preliminary study by
Pravosudov & Clayton [6]. Thus, the evidence for the
relationship between environmental severity and the Hp
is robust in this species and holds as an ecologically
relevant pattern across a latitudinal gradient composed
of locations with differing climates.
However, the relative contribution of the three primary
theoretical environmental factors (temperature, snow
cover and day length) to this relationship is unknown.
As diurnal birds cannot forage in the dark, day length
may potentially comprise a large part of environmental
severity, as it limits the amount of time available to
accumulate necessary food reserves to survive the night.
Because of this constraint, there may be increased
demands for spatial memory function in birds from
higher latitudes (i.e. shorter day lengths) in order to pro-
cure enough food for overnight survival. This may explain
in part why northern chickadees have larger Hp with
more neurons compared with their more southern
conspecifics. However, northern environments are also
characterized by lower ambient temperatures with more
precipitation (snow) during the winter (see figure 1 and
[7]), which could also contribute to latitudinal differences
in memory and the hippocampus. Low ambient tempera-
tures increase the metabolic needs of animals, while snow
cover may reduce food availability and accessibility. Suc-
cessful cache recovery may be especially crucial in these
conditions in order to meet higher energetic demands.
To dissociate the importance of the compounding
effects of day length on the relationship between environ-
mental severity and the Hp, we compared the Hp volume
and total number of Hp neurons in the same species, the
black-capped chickadee (Poecile atricapillus), on a large
geographical scale at five locations across its range in
North America (figure 1). We specifically chose three of
theselocations(Seattle,Washington
Minnesota (MN); Presque Isle, Maine (ME)) at approxi-
mately the same latitude that experience similar day
lengths but very different climates. The other two
locations (Anchorage, Alaska (AK); Mt. Vernon, Iowa
(IA)) were used as a latitudinal comparison to ensure
the robustness of our previous work [7]. According to
our hypothesis, better spatial memory and increased Hp
attributes should be more pronounced in populations
living in more harsh, energetically demanding environ-
ments in which dependence on food caches is likely to
be especially important. Thus, if day length itself is an
important constraint in the relationship between environ-
mental severity and the Hp, then we might expect no
harshnorthernlatitudes
(WA);Grant,
significant differences among the populations at the
same latitude that experience differences in temperature
and snow cover. If, however, other climatic variables
(e.g. temperature and snow cover) are important indepen-
dent of day length, then we should see differences among
the populations along the same latitude that follow the
same pattern as previously reported [7] of increased Hp
volume and neuron number in more severe environments.
We do not imply that birds use any of these factors as
direct cues for the production of Hp attributes. Instead,
we are dissociating the importance of the constraint on
the time available to forage (day length) from the con-
straints of energetic demands (temperature) and access
to food (snow cover). Because the MN and ME locations
north/south
comparison
daylength control
(AK)(IA) (ME)(MN) (WA)
average snow depth (mm)
0
100
200
300
400(a)
(b)
(c)
day length (min)
200
300
400
500
600
average temperature (°C)
–8
–6
–4
–2
0
2
4
6
8
Figure 1. Collection locations sorted by the north/south
comparison (left panels) and the latitudinal/day length con-
trol (right panels). Within each panel, the locations are
ranked by environmental severity, as determined by (a) aver-
age snow cover (mm) and (b) average temperature (8C)
during the winter period (see text for details). Day length
(min) at the winter solstice (c) differs for the locations in
the left panel, but is similar in those on the right.
2 T. C. Roth et al.Hippocampal morphology and environment
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experience colder temperatures and greater snow cover
than the WA location, we expect bigger Hp with more
neurons in the MN and ME locations when compared
with the WA location. Finally, we expect the relationship
between AK and IA to be consistent with our previous
examination of populations on a latitudinal gradient [7],
that is birds from AK should have much larger Hp
volume and more neurons than those from IA.
2. METHODS
(a) Collection sites
We compared the hippocampal (Hp) volume and total
number of Hp neurons of black-capped chickadees (Poecile
atricapillus) at five locations across North America: Ancho-
rage, AK (618070N 1498440W); Presque Isle, ME (468390N
688000W); Grant, MN (458050N 928570W); Seattle, WA
(478400N 1228180W); and Mount Vernon, IA (418510N
918250W). These sites were chosen specifically (i) to compare
populations with different climates at approximately the same
latitude/day lengths (WA, MN, and ME; figure 1) and (ii) to
make a comparison with the previous study by Roth & Pravo-
sudov [7] on a latitudinal gradient (AK and IA). At each site,
12 birds were captured at feeders. While it would be best to
collect birds at the peak of caching at each location to maintain
consistency and to maximize the potential effects of food cach-
ing on the brain, due to logistics, we could not determine
specifically when that peak occurred at each site. Thus, we
chose to collect chickadees during the period of intensive
autumn caching from mid September to late October based
on the literature [15,16] and our previous work [7].
(b) Tissue preparation
Tissue preparation was performed exclusively by T.C.R. and
followed Roth & Pravosudov [7], as necessary for proper com-
parison (see [17]). Brains were extracted immediately after
capture. Birds were anaesthetized (0.07 ml of 50 mgml21
Nembutal) and perfused transcardially with 0.1 M phosphate
buffered saline for 10 min followed by 15–20 min of 10 per
cent methanol-free formalin (from paraformaldehyde) made
with 0.1 M phosphate buffer. Brains were post-fixed in the
10 per cent methanol-free formalin solution for 7 days, cryo-
protected in 15 per cent and then 30 per cent sucrose, and
then frozen at 2808C for storage. Tissue was cut into 40 mm
coronal sections on a Leica CM 3050S cryostat at 2208C.
Every 4th section was mounted and Nissl stained with thionin.
(c) Tissue analysis
Tissue analysis was performed exclusively by T.C.R. and
followed Roth & Pravosudov [7], as necessary for proper
comparison (see [17]). Hp volume and neuron numbers
were estimated with stereological methods using Stereo-
Investigator software (Microbrightfield, Inc.) and Leica
microscope (M4000B). Both the Hp and telencephalon
were measured in their entirety. We measured the Hp as
per Krebs et al. [4] and our previous work. We measured
the telencephalon as an estimate of the brain region generally
associated with cognitive ability to be used as a covariate to
control for overall size of the brain [4]. Brain region volumes
were estimated with the Cavalieri procedure [18]. Hp volume
was measured with a 200 mm grid; telencephalon volume was
measured with a 1200 mm grid. The optimal grid size and
frequency of sections sampling has been determined pre-
viously [6,19]. Neuron counts were performed with an
optical fractionator procedure [20] at 1000x. A 250 mm
grid with a 30 ? 30 mm counting frame, 5 mm dissector
height, and 2 mm guards was used as in previous studies of
chickadees [19]. We calculated a coefficient of error to esti-
mate precision with the nugget effect for both neuron
counts (CE mean (s.e.) ¼ 0.098 (0.002)) and volume (CE
mean (s.e.) ¼ 0.016 (0.0001)). There were no significant
differences between left and right hippocampal volume
and between the total number of neurons in the right
and left Hp (Repeated-measures GLM: Hp, F1,55¼ 0.401,
p ¼ 0.807; Neuron numbers: F1,55¼ 0.842, p ¼ 0.505),
thus the hemispheres were summed to produce the reported
total values. All brains were measured blind to location.
(d) Climate data
Temperature (8C) and snow depth (mm) are represented as
the average over the winter months (November–March) of
yearly averages from 1971 to 2000. Day length (min) was
measured during the winter solstice. Data were obtained
from the National Oceanic and Atmospheric Administration
climate database [21].
(e) Statistical analysis
We analysed Hp volume and neuron count after controlling
for telencephalon volume within a general linear model; we
report least squares means in the analyses and figures (see
table 1). The inclusion of body mass as a covariate has
beenusedinthepast,butmayleadtoerroneousinterpretations
(Roth et al. in preparation; see also [17]). We note that the
relationships in this analysis are, nevertheless, quite robust,
and the inclusion of either telencephalon volume and/or body
mass does not change the interpretation of the results (see §3
for statistics). In addition, the use of Hp volume as a covariate
in the analysis of neuron number has been used in the past.
Thisanalysisproducesaleastsquaredmeanfunctionallyequiv-
alent to an analysis of density. We include this analysis for
completenessintheresults,butalsonotethattheinterpretation
remains the same. Tukey post hoc analyses were performed for
a comparison between the three locations at the same latitude.
A planned comparison analysis was performed between AK
and IA as we expect a directional difference between them.
There was no significant sex bias in sampling (x2¼ 2.986,
d.f. ¼ 4, p ¼ 0.560) and no significant difference between the
sexes in Hp volume (F1,58¼ 2.081, p ¼ 0.155) or neuron
number (F1,58¼ 0.405, p ¼ 0.527); therefore, data for both
sexes were pooled. The age of our birds was unknown, but
there is no reason to suspect any systematic bias in age across
different locations, as we sampled all populations using the
same methodology.
3. RESULTS
There was a significant difference in both the Hp volume
(F4,55¼ 41.106, p , 0.0001) and total neuron count
(F4,55¼ 28.149, p , 0.0001) in black-capped chickadees
from different locations along the latitudinal gradient
when using Te volume as a covariate (table 1, figure 2).
The inclusion of Hp volume as a covariate in the analysis
of total neuron numbers did not change the interpretation
(F4,55¼ 18.159,
of the Hp in the model was significant; F1,55¼ 8.085,
p , 0.006).Likewise, the
(Hp: F4,56¼ 6.612, p , 0.0001; Neuron: F4,56¼ 29.383,
p , 0.0001) and the inclusion of body size (Hp: F4,54¼
8.417, p , 0.0001; Neuron: F4,54¼ 27.882, p , 0.0001)
did not change the results.
p , 0.0001, even thoughthe effect
exclusionof covariates
Hippocampal morphology and environment
T. C. Roth et al.
3
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Based on post hoc comparisons of ME/MN/WA and a
planned comparison analysis between AK/IA, there were
large and significant differences among the locations
(see figure 2). Along the same latitude, there was a stark
and significant effect of climate. The Hp volume (ps ,
0.008) and neuron numbers (ps , 0.0001) were signifi-
cantly smaller in birds from the Washington location,
but not different between the Maine and Minnesota
locations (all ps . 0.999). The birds from the northern
most location (Alaska) had significantly larger Hp (p ,
0.0001) and more Hp neurons (p , 0.0001) than those
from the southern most location (Iowa) as predicted.
4. DISCUSSION
There was a significant difference in Hp attributes among
populations along the same latitude with very different
climatic features. Birds from the relatively mild location
of Washington had significantly smaller Hp volumes and
fewer Hp neurons than the birds from the more climati-
cally harsh populations of Maine and Minnesota, even
though all three populations experience similar day
lengths. Maine and Minnesota were not different from
each other (figure 2). These results suggest that day
length may not be the main variable that affects caching
intensity and thus may not be solely responsible for the
previously reported relationship between latitude and
Hp attributes. Rather, the other variables of theoretical
interest (temperature and snow cover) seem to be the
relevant factors in this relationship independent of the
Table 1. Average (s.e.m.) morphological data for black-capped chickadees collected at five locations with varying environmental harshness. Locations ranked by latitude from high to low.
location
(latitude)
body mass
(g)
wing length
(mm)
brain mass
(g)
absolute
hippocampal
volume (mm3)
absolute number of
hippocampal neurons
(?106)
telencephalon
volume (mm3)
relative
hippocampal
volume (mm3)*
relative number of
hippocampal
neurons (?106)
dates
collected
(2008)
Anchorage,
AK (618N)
11.10 (0.16)
66.04 (0.76)
0.80 (0.02)
27.20 (0.64)
2.582 (0.065)
511.41 (18.36)
27.38 (0.73)
2.603 (0.094)
18–20 Sep
Seattle, WA
(478N)
11.19 (0.24)
61.69 (0.63)
0.70 (0.01)
22.65 (0.66)
1.719 (0.105)
430.25 (14.03)
22.33 (0.74)
1.680 (0.095)
22 Oct
Presque Isle,
ME (468N)
11.60 (0.23)
67.50 (0.48)
0.78 (0.02)
26.08 (0.99)
2.537 (0.106)
497.13 (22.85)
26.17 (0.72)
2.548 (0.092)
28 Sep
Grant, MN
(458N)
11.64 (0.25)
65.54 (0.61)
0.78 (0.01)
25.91 (0.72)
2.560 (0.092)
497.20 (13.34)
26.00 (0.72)
2.570 (0.092)
09 Oct
Mt. Vernon,
IA (418N)
12.11 (0.26)
65.08 (0.75)
0.74 (0.02)
24.06 (0.45)
1.609 (0.076)
482.53 (19.88)
24.06 (0.72)
1.609 (0.091)
13–16 Oct
* Least squares means values calculated from a GLM analysis using telencephalon volume as a covariate.
relative hippocampal
volume (mm3)
22
24
26
28
30
relative number of
neurons (¥ 106)
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
(a)
(b)
north/south
comparison
daylength control
(AK)(IA) (ME) (MN) (WA)
Figure 2. (a) Relative hippocampal volume and (b) relative
neuron numbers, (least squares means) across locations con-
trolling for telencephalon volume. Collection locations are
sorted as figure 1 (north/south comparison, left panels; lati-
tudinal/day length control, right panels). Within each
panel, the locations are ranked by environmental severity.
4T. C. Roth et al.Hippocampal morphology and environment
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