Age-associated decline in septum neuronal activation during spatial learning in homing pigeons (Columba livia)

Journal Pre-proof Age-associated decline in septum neuronal activation during spatial learning in homing pigeons (Columba livia) Vincent J. Coppola (Conceptualization) (Methodology) (Validation) (Formal analysis) (Investigation) (Resources) (Writing - original draft) (Visualization) (Project administration), Daniele Nardi (Conceptualization)Writing - review and editing) (Supervision), Verner P. Bingman (Conceptualization) (Methodology) (Validation) (Resources) (Writing - review and editing) (Supervision) (Project administration)

PII:

S0166-4328(20)30647-1

DOI:

https://doi.org/10.1016/j.bbr.2020.112948

Reference:

BBR 112948

To appear in:

Behavioural Brain Research

Received Date:

27 May 2020

Revised Date:

11 September 2020

Accepted Date:

26 September 2020

Please cite this article as: Coppola VJ, Nardi D, Bingman VP, Age-associated decline in septum neuronal activation during spatial learning in homing pigeons (Columba livia), Behavioural Brain Research (2020), doi: https://doi.org/10.1016/j.bbr.2020.112948

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Age-associated decline in septum neuronal activation during spatial learning in homing pigeons (Columba livia)

Vincent J. Coppola1,2 [email protected], Daniele Nardi4, & Verner P. Bingman1,2,3

Department of Psychology, University of Findlay, Findlay, OH

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J.P. Scott Center for Neuroscience, Mind, & Behavior, Bowling Green, OH

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Department of Psychology, Bowling Green State University, Bowling Green OH

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Department of Psychological Science, Ball State University, Muncie, IN

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Corresponding author: Vincent J. Coppola, 1000 N Main St, Findlay, OH 45840, 419-601-5504,

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Abstract

The relationship between hippocampal aging and spatial-cognitive decline in birds has

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recently been investigated. However, like its mammalian counterpart, the avian hippocampus does not work in isolation and its relationship to the septum is of particular interest. The current study aimed to investigate the effects of age on septum (medial and lateral) and associated nucleus of the diagonal band (NDB) neuronal activation (as indicated by c-Fos expression) during learning

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of a spatial, delayed non-match-to-sample task conducted in a modified radial arm maze. The results indicated significantly reduced septum, but not NDB, activation during spatial learning in older pigeons. We also preliminarily investigated the effect of age on the number of cholinergic septum and NDB neurons (as indicated by expression of choline acetyltransferase; ChAT). Although underpowered to reveal a statistical effect, the data suggest that older pigeons have

substantially fewer ChAT-expressing cells in the septum compared to younger pigeons. The data support the hypothesis that reduced activation of the septum contributes to the age-related, spatial cognitive impairment in pigeons. Keywords: Avian, spatial cognition, septum, nucleus of the diagonal band, immediate early

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gene, choline acetyltransferase

1. Introduction

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The mammalian hippocampus is often at the forefront of discussions regarding the neural underpinnings of spatial cognition and episodic memory. However, proper hippocampal

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functioning, and by extension spatial cognition and episodic memory, is dependent on the network

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of connections between the hippocampus and other brain regions, including the septum [1]. For example, in rodents, lesions of the septum results in similar spatial-cognitive deficits to that seen

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with hippocampal lesions [e.g., 2-4]. Additionally, Vann et al. [5] reported greater expression of the activity-dependent immediate early gene c-Fos in the rodent septum following performance on

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a spatial-cognitive task compared to a non-spatial control task. Given the importance of the septum in influencing hippocampal control of spatial cognition and memory processes, age-related decline in septal function could potentially contribute to the spatial-cognitive impairments associated with hippocampal aging. Consistent with this hypothesis,

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Sava and Markus [6] found that pharmacological activation of the septum diminished observed deficits in hippocampal place cell remapping in old rats, suggesting that age-related changes in hippocampal function are in part explained by reduced septal activity. Additionally, Nagahara and Handa [7] found an age-related reduction in septal activation (specifically c-Fos expression) in rodents following exposure to a novel spatial environment, while Touzani et al. [8] found an age-

related reduction in septal engagement during the completion of a spatial discrimination task. In addition to the observations of reduced septal activity in older rodents, other studies have found cholinergic neurons in the medial septum to be fewer in number and reduced in size in older, compared to younger, rodents [9, 10]. Taken together, the above findings suggest that age-related, spatial memory impairments in rodents are associated with decreased neuronal activity in the

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septum, with an associated reduction in the amount/intensity of septal cholinergic signaling. In contrast to the considerable amount of research carried out in mammals, the literature

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on neurocognitive aging in birds is relatively understudied, with the existing studies focusing solely on the hippocampal formation (HF) and spatial cognition. Specifically, there are two reports

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of age-related, spatial memory impairment in homing pigeons [11, 12], as well as two studies that

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found the HF of old homing pigeons to be different from younger birds. Specifically, the HF of old homing pigeons is larger and contains more neurons than that of younger pigeons [13, 14], a

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finding that sharply contrasts with the age-related atrophy typically seen in mammals [15]. More recently, Kosarussavadi et al. [16] reported age-related changes in the activation of the zebra finch

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HF following spatial memory retrieval, while Coppola and Bingman [17] assessed c-Fos expression and found that the HF of older pigeons is less engaged during spatial learning compared to younger pigeons.

In order to more fully understand the relationship between HF aging and spatial-cognitive

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decline in birds, age-related changes to HF afferents are of particular interest. One intuitive candidate to begin such an investigation is the avian septum, which, as in mammals, shares reciprocal connections with the HF [18, 19]. The similarity of avian and mammalian HF-septal connectivity suggests that the avian septum may too play an important role in spatial cognition and that age-related changes to the septum might contribute to hippocampal-dependent, age-related

spatial memory decline [11, 12]. Highlighting the importance of the septum for HF function, septal lesions have been reported to impair spatial working memory in homing pigeons tested on a spatial working memory task [20] and cholinergic agonists readily elicit theta oscillations in the HF of chickens (Gallus gallus) [21]. Additionally, intramuscular injections of scopolamine impaired short-distance (i.e., hippocampal-dependent) homing behavior in pigeons [22], as well as

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performance on a non-spatial working memory task in a laboratory setting [23-25]. Mineau et al. [26] also found scopolamine to disrupt spatial working memory in black-capped chickadees

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(Poecile atricapillus).

A notable difference between mammals and birds is that septal input to the HF is seemingly

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modest in birds compared to that in mammals [27-29]. In fact, the avian nucleus of the diagonal

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band (NDB), which is homologous to the mammalian nucleus of the diagonal band of Broca (NDBb), appears to be more strongly connected to the HF than is the septum proper [27, 28]. While

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little is known about the functional significance of the avian NDB, lesions of the mammalian NDBb have been reported to impair spatial reference memory performance [30, 31]. Furthermore,

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the NDBb displays similar age-related degeneration as the medial septum. Neurons in the NDBb are fewer in number and reduced in size in old rodents [9, 10, 32], and the extent of that reduction correlates with a spatial memory impairment in the Morris water maze [10]. Given its homology to the mammalian NDBb and strong connectivity with the HF, age-related changes to the avian

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NDB, like the main body of the septum, may also contribute to the spatial-cognitive deficits previously reported in pigeons [11, 12] and are also of interest for the present study. The current study aimed to investigate the potential contribution of septum and NDB aging

toward explaining the spatial-cognitive decline known to correlate with an older HF. We were principally interested in determining whether, as previously reported for HF [17], aging was

associated with a reduction in septum and NDB neuronal activation (as indicated by c-Fos expression) during the learning of a spatial, delayed non-match-to-sample task. We also carried out a preliminary analysis of possible differences in the number of cholinergic neurons (as indicated by the expression ChAT).

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2. Methods 2.1. Subjects

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The subjects of the current study were the same homing pigeons used in Coppola and Bingman [17]. The original sample that received behavioral training consisted of 14 pigeons, 7

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young (male = 6, female = 1; 2-3 years post-hatch; M = 2.14, SD = 0.35) and 7 old (male = 5,

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female = 2; 10-15 years post-hatch; M = 12.86, SD = 1.46). Unfortunately, after extraction and processing not all tissue was of good enough quality for the c-Fos analysis (i.e., tissue was lost due

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to poor perfusion, fixation, sectioning, or inadequate labeling). As such, the data on septal c-Fos presented below are from a sample of 12 pigeons, 7 young (2-3 years post-hatch; M = 2.14, SD =

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0.35) and 5 old (12-14 years post-hatch; M = 13.00, SD = 0.63), while the septal ChAT data are from a sample of 9 pigeons, 4 young (2-3 years post-hatch; M = 2.25, SD = 0.43) and 5 old (12-14 years post-hatch; M = 13.00, SD = 0.63). For the NDB, the c-Fos data presented below are from a sample of 13 pigeons, 7 young (2-3 years post-hatch; M = 2.14, SD = 0.35) and 6 old (10-14 years

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post-hatch; M = 12.50, SD = 1.25), while the ChAT data are from a sample of 10 pigeons, 5 young (2-3 years post-hatch; M = 2.20, SD = 0.40) and 5 old (12-14 years post-hatch; M = 13.00, SD = 0.63).

All pigeons had prior homing experience but had been in an enclosed, indoor aviary for at least 6 months prior to testing. Importantly, from a behavioral perspective, all of the old pigeons

were of an age when cognitive deficits have been previously reported [11, 12]. During testing, birds were housed individually in wire mesh cages (26.7 × 29.8 × 28.6 cm) in a temperature and humidity-controlled room with a 13/11-hour light-dark cycle, and were food-deprived to no less than 80% of their free-feed body weight. All procedures were conducted in accordance with National Institute of Health guidelines and were approved by Bowling Green State University's

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Institutional Animal Care and Use Committee. 2.2. Behavioral training

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The pigeons of the current study were the same subjects used in Coppola and Bingman [17]. As the full details of the behavioral procedures employed were previously reported [see 17],

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they are only briefly presented here. Pigeons were trained on a spatial, delayed non-match-to-

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sample task conducted in an 8-arm radial maze adapted for birds [33] (see Figure 1). A trial consisted of a sample phase and a test phase, separated by a 10 min interphase interval. In the

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sample phase, pigeons ate food from four of the eight arms (feeders); access to the other four arms was restricted by removing the feeders from the room. In the test phase, pigeons needed to learn

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to avoid the four arms (feeders) sampled in the preceding sample phase and eat from the four, newly baited arms (restored feeders). Pigeons completed one such trial a day for 12 days. The total number of choices to eat from all four newly baited feeders during the test phase (TOT-TEST; four being a perfect score) was recorded and used as the dependent measure of task performance.

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2.3. Perfusion and immunohistochemistry Eighty minutes after the end of the sample phase of the final (12th) training trial, pigeons

were deeply anesthetized with sodium pentobarbital (0.5 mL; intramuscular) and then perfused with 250 mL of phosphate buffered saline (PBS; pH 7.4) followed by 250 mL of 4% paraformaldehyde in PBS (4% PFA). This eighty-minute delay between the approximate midpoint

of the final trial and sacrifice was chosen in order to fix the brain around the time of expected peak c-Fos expression (i.e., greatest quantity of c-Fos protein), which is approximately 90 min following neuronal activation [U. Mayer, personal communication; also see 34]. Following extraction, brains were post-fixed in 20% sucrose in 4% PFA for 24 hr, at which point they were moved to 30% sucrose in PBS for 72 hr for cryoprotection. Next, brains were encased in a block of gelatin (30%

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sucrose and 15% gelatin in PBS) and then placed on a freezing microtome and cut at 50 μm. Labeling for c-Fos- and ChAT-positive neurons was conducted in free-floating brain

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sections, blind to the condition of age, using the ABC method [e.g., 34]. Between all of the following steps, tissue was washed 3 times for 5 min each in 0.1M PBS, except for between the

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blocking of non-specific binding and primary antibody incubation steps. Tissue was first incubated

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in 0.3% hydrogen peroxide in 0.1M PBS for 30 min to block endogenous peroxidase activity. Nonspecific binding was blocked by incubating tissue in 3% normal goat serum in 0.1M PBS for 30

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min. Next, tissue was incubated overnight at room temperature in either a primary antibody against c-Fos produced in rabbit (diluted to 1:2000 in 0.1M PBS; K-25, Santa Cruz Biotechnology) or a

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primary antibody against ChAT produced in rabbit (diluted to 1:1500 in PBS; Millipore, AB5042). The following day the tissue was incubated for 60 min in a biotinylated secondary anti-rabbit produced in goat (diluted to 1:200 in 0.1M PBS; BA-1000, Vector Laboratories). Tissue was then incubated for 60 min in the avidin-biotin complex of the VECTASTAIN ABC HRP Kit (PK-4000,

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Vector Laboratories). Finally, visualization was conducted using the VIP substrate kit for peroxidase (SK‐ 4600, Vector Laboratories) before mounting the tissue on gelatin-coated slides, dehydrating, and cover-slipping. 2.4. Quantification of c-Fos and ChAT labels

As with immunohistochemical processing, quantification of labels was done blind to the condition of age. Stained sections were projected onto a computer monitor using a digital, cameraequipped stereomicroscope (Wild Heerbrugg, M7A; camera: Omax A355OU). Live images were analyzed using ToupView image processing software (v.3.7; Omax). Specifically, anatomical landmarks (e.g., the ventral extent of the lateral ventricle) were used to position sampling boxes

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(see below). The entire section was focused through and all labels within a sampling box or in contact with its boarder were included in the count.

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Quantification of c-Fos labels in the septum was conducted for the lateral (SL) and medial (SM) septum separately. To quantify c-Fos expression in the SL, the number of c-Fos labels in one

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sampling box (0.30 × 0.30 × 0.05 mm) was counted at level A9.50, two sampling boxes at levels

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A9.00, A8.00, and A7.50, and three sampling boxes at level A8.50 (Figure 2; all anterior coordinates are based on the pigeon brain atlas of Karten & Hodos [35]). For the SM, two sampling

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boxes were counted at levels A8.00 and A7.50. To obtain total number of counted septal c-Fos labels in a given hemisphere, the number of labels counted in the SL and SM of a hemisphere were

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summed. The total number of septal c-Fos labels was obtained by summing the counts in the two hemispheres. To quantify c-Fos expression in the NDB, the number of c-Fos labels in three sampling boxes (0.20 × 0.20 × 0.05 mm) were counted at level A9.00 (Figure 2). The total number of NDB c-Fos labels was obtained by summing the number of c-Fos labels counted in the two

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hemispheres.

To quantify the number of ChAT-positive neurons in the SL, the number of ChAT labels

were counted in one sampling box (0.30 × 0.30 × 0.05 mm) at A-P level A9.50 and two sampling boxes at level A7.50 (Figure 2). For the SM, two sampling boxes were counted at A-P level A7.50 (Figure 2). To obtain total hemispheric numbers of ChAT, counts from the SL and SM subregions

of a hemisphere were summed. The total number of ChAT labels counted was then calculated by summing the hemispheric counts. For quantification of the number of ChAT labels in the NDB, three sampling boxes were counted at A-P level A9.00 (Figure 2). The number of ChAT labels counted in each hemisphere was summed to produce a total number of ChAT labels counted in the NDB.

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2.5. Data analysis The 12 behavioral trials were separated into four blocks of three trials. The mean total

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number of choices to complete the test phase (TOT-TEST) was analyzed across the four trial blocks using a 2 (age: young, old) X 4 (block: block 1, block 2, block 3, block 4) mixed factors

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ANOVA. Additionally, age-related differences in TOT-TEST on trial 12 only (i.e., the last trial

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before sacrifice) was analyzed using an independent samples t-tests (this trial 12 analysis was particularly important for the c-Fos-behavior correlations; see below).

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Age-related differences in the number of c-Fos and ChAT labels in the septum and NDB were analyzed separately. For the septum, the number of each label counted was analyzed using

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two MANOVAs, one of which included the left and right SM and SL subregions, and the other of which included the total left and right hemisphere. Additionally, a univariate ANOVA was used to analyze the total septum. For the NDB, the left and right hemispheres were analyzed using a MANOVA, while total NDB was analyzed using a univariate ANOVA. Age group was entered as

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a fixed variable for all of the above analyses. A final goal of the current study was to determine if septum activity (i.e., the number of c-

Fos labels counted) correlated with TOT-TEST. As such, the TOT-TEST values from the last trial only (which the c-Fos activation reflected) and septum c-Fos measures we entered into two, twotailed Pearson’s correlation matrices, one for each age group.

All of the above analyses were performed using SPSS Statistics for Windows, version 25.0 and the criterion for statistical significance was p < .05. Where the assumption of homogeneity was violated (i.e., significant Levene’s test), degrees of freedom and p-values were adjusted. Figures were made using GraphPad Prism for Windows, version 8.0.0 (GraphPad Software, San

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Diego, California USA).

3. Results

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3.1. Behavior

As described in Coppola and Bingman [17], the intent of the behavioral training was not

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to investigate a spatial-learning deficit in the older pigeons, which was already demonstrated in

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[11, 12], but to have the performance of the pigeons still improving, i.e., continuing active learning, at the time of sacrifice with as little difference as possible between the two age groups. This was

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important as the goal of Coppola & Bingman [17], as well as the current study, was to compare the brain activation patterns during spatial learning at a point where performance differences could

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not explain any differential c-Fos labeling.

As designed, TOT-TEST did not significantly differ between the two age groups across the 4 training blocks, f(1, 12) = 0.31, p = .59. Furthermore, an independent samples t-tests confirmed that there was no significant effect of age on TOT-TEST at the end of training (i.e., day/trial 12

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data only), t(12) = 1.51, p = .16. Thus, we are confident that any differences in c-Fos activity (see below) can be primarily attributed to age-related changes in septal and NDB engagement during active spatial learning and not to differences in task performance. 3.2. Septum activation

The effect of age on the number of c-Fos labels counted in the total septum was significant, f(1,8.29) = 6.00, p < .04, ηp2 = .32 (Figure 3A). Impressively, the septum of younger pigeons (M = 96.00, SE = 28.42) had on average nearly five times the number of c-Fos labels as older pigeons (M = 19.20, SE = 13.21). Additionally, the number of c-Fos labels in the right septum significantly differed between the two age groups, f(1,6.54) = 11.31, p < .02, ηp2 = .45 (Figure 3C; young: M =

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44.43, SE = 11.24; old: M = 5.80, SE = 2.40), but not the left septum, f(1,10) = 2.00, p = .19, ηp2 = .17 (Figure 3B; young: M = 51.57, SE = 21.11; old: M = 13.40, SE = 11.20). Regarding the SL

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subregion, the number of c-Fos labels significantly differed between the two age groups in both the left, f(1,10) = 6.46, p < .03, ηp2 = .39 (Figure 3D; young: M = 23.29, SE = 6.28; old: M = 3.60,

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SE = 2.16), and right, f(1,6.77) = 22.98, p < .01, ηp2 = .62 (Figure 3E; young: M = 17.00, SE =

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3.03; old: M = 2.00, SE = 0.78) hemispheres. Qualitatively similar differences were found in the SM, however, those differences in the two age groups did not significantly differ in the left, f(1,10)

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= 0.79, p = .40, ηp2 = .07 (Figure 3F; young: M = 28.29, SE = 16.09; old: M = 9.80, SE = 9.30), nor right, f(1,6.47) = 4.33, p < .08, ηp2 = .24 (Figure 3G; young: M = 27.43, SE = 11.13; old: M =

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3.80, SE = 2.22) SM, although the difference in the right hemisphere did approach significance. In conclusion, younger pigeons displayed much stronger septal activation when faced with the spatial learning demands of the task compared to older pigeons. Given the effect of age on septum activation, we expected significant correlations between

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septum c-Fos and TOT-TEST. Indeed, Coppola and Bingman [17] found that similar age-related differences in HF activity were accompanied by significant correlations between HF activity and TOT-TEST in young, but not old, pigeons. In the current study, however, no septum c-Fos measure correlated with TOT-TEST on the last trial in either age group (see Table 1 and Discussion), 3.3. Septum ChAT

In both mammals and birds, the septum’s contribution to spatial cognition is believed to be modulated by its cholinergic projections to the hippocampus (see Introduction). Therefore, a loss of cholinergic neurons in the avian septum might be a partial explanation for age-related spatialcognitive decline in homing pigeons [21, 22] as it is in mammals [16, 17]. Although statistically underpowered by the small sample size and therefore preliminary, examination of Figure 4

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nonetheless reveals generally twice as many ChAT labels in the septum of the young compared to old pigeons; however, mean differences did not reach statistical significance with respect to the

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total septum, f(1, 7) = 1.54, p = .26, ηp2 = .18 (Figure 4A; young: M = 130.00, SE = 67.12; old: M = 52.20, SE = 19.09), left septum, f(1, 7) = 1.51, p = .26, ηp2 = .18 (Figure 4B; young: M = 73.75,

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SE = 33.77; old: M = 31.80, SE = 14.83), or right septum, f(1, 3.25) = 1.06, p = .37, ηp2 = .16

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(Figure 4C; young: M = 58.25, SE = 34.08; old: M = 20.40, SE = 6.92). Additionally, and again despite the notable qualitative differences, younger and older pigeons did not differ in the number

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of ChAT labels in any septal subregion: left SL, f(1, 7) = 1.71, p = .23, ηp2 = .20 (Figure 4D; young: M = 49.00, SE = 26.21; old: M = 16.80, SE = 7.66), right SL, f(1, 3.13) = 0.85, p = .42, ηp2 = .14

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(Figure 4E; young: M = 35.75, SE = 26.53; old: M = 11.00, SE = 3.83), left SM, f(1, 7) = 0.81, p = .40, ηp2 = .10 (Figure 4F; young: M = 24.75, SE = 7.99; old: M = 15.00, SE = 7.26), or right SM, f(1, 7) = 1.73, p = .23, ηp2 = .20 (Figure 4G; young: M = 20.50, SE = 8.11; old: M = 9.40, SE = 4.01). The qualitative contrasts of this preliminary analysis nurture the hypothesis that associated

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with our major finding of reduced septum neuronal activation in older pigeons is a reduction in the number of cholinergic neurons in the septum. 3.4. NDB activation In contrast to the septum proper, there was no significant effect of age on the number of cFos labels in the total NDB, f(1, 11) = 0.03, p = .87, ηp2 = .00 (Figure 5A; young: M = 45.43, SE

= 16.73; old: M = 50.50, SE = 27.28), left NDB, f(1, 11) = 0.00, p = .97; ηp2 = .00 (Figure 5B; young: M = 28.71, SE = 12.59; old: M = 28.00, SE = 14.66), or right NDB, f(1, 11) = 0.16, p = .70, ηp2 = .01 (Figure 5C; young: M = 16.71, SE = 5.27; old: M = 22.50, SE = 14.41) NDB. Thus, unlike the septum (above) and HF [27], activation of the NDB was not reduced in the older compared to younger, pigeons. Importantly, these data suggest that a reduction in c-Fos expression in older

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pigeons does not generalize to all regions of the brain that potentially influence HF control of spatial cognition.

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3.5. NDB ChAT

The preliminary analysis of cholinergic neurons in the NDB revealed no statistical

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difference in the number of ChAT labels in the total NDB between younger and older pigeons, f(1,

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8) = 0.20, p = .67, ηp2 = .02 (Figure 5D; young: M = 63.60, SE = 27.07; old: M = 51.40, SE = 4.73). Furthermore, there was no effect of age on the left NDB, f(1, 8) = 0.61, p = .48, ηp2 = .07 (Figure

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5E; young: M = 35.00, SE = 15.89; old: M = 22.40, SE = 2.77), nor right NDB, f(1, 8) = 0.00, p = .97, ηp2 = .00 (Figure 5F; young: M = 28.60, SE = 11.70; old: M = 29.00, SE = 3.05).

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Acknowledging that a negative result from a preliminary, underpowered analysis should be treated with caution, it is noteworthy that in contrast to the septum (see above) there was no hint of any age-related change in the number of NDB cholinergic neurons.

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4. Discussion

Inspired by the effects of septal and cholinergic aging on spatial cognition in mammals

[see 36], the current study set out to investigate the potential contribution of septum aging to spatial-cognitive decline in birds. Specifically, young and old pigeons were trained on a spatial, delayed non-match-to-sample task in a modified radial arm maze. Similar to what has been

reported in mammals [7, 8], older pigeons had significantly fewer c-Fos-positive septal cells compared to younger pigeons when faced with learning the spatial working memory challenge. Given the role of the avian septum in spatial cognition [21, 21, 26], the reduced engagement of the septum of older pigeons during the learning of a spatial working memory task may in part explain the age-related, spatial-cognitive decline in homing pigeons [11, 12]. Our preliminary analysis also

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revealed a reduced number of cholinergic neurons were also found in the older pigeons of the current study, a difference however, that was not statistically significant. It should be noted,

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however, that due to the loss of tissue during processing, the ChAT data were obtained from a very small sample of pigeons (4 young and 5 old) resulting in an underpowered statistical contrast. In

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fact, a post-hoc power analysis (G* Power, v. 3.1.9.2) on the effect of age on total septal ChAT

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(two-tailed t-test), with the obtained input parameters (Cohen’s d = 0.83, p = .26), revealed an underpowered statistical analysis and only a 53% probability (about chance) of detecting a

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difference between the two age groups.

Considering Coppola and Bingman’s [17] finding that many HF c-Fos measures

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significantly correlated with TOT-TEST behavioral performance in young but not old pigeons, it was perhaps surprising that no significant behavioral correlations were found between septum cFos and TOT-TEST in either age group. The lack of a septum-behavior correlation, particularly in the younger pigeons, may seem inconsistent with age-related septum activation differences

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explaining age-related cognitive decline. However, it must be noted that septum c-Fos numbers were less variable than HF c-Fos numbers (e.g., standard deviation of total septum c-Fos labels in young pigeons = 69.62; in young pigeons [17] = 389.99). The modest variation associated with the small standard deviation septum scores, together with the small sample size, would make it difficult to statistically detect any real correlations that might exist. Important too is that it has been

established that the avian septum is indeed involved in spatial cognition, with the most relevant finding being that septum lesions impair spatial working memory in homing pigeons tested on the same spatial working memory task employed in the current study [20]. Furthermore, the septohippocampal cholinergic pathway is considered an evolutionarily conserved brain system known to play a prominent role in learning and memory processes across all tetrapods [37]. Future studies

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should further investigate possible behavioral correlates of septum activity in explaining variation in avian spatial-cognitive performance.

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One assumption of the current study is that animals were sacrificed while still learning the behavioral task (i.e., prior to reaching asymptotic performance) with the goal of having as little

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difference as possible between the two age groups. As such, it is important to consider where on

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the learning curve pigeons were at the end of training. The closest comparable data are from Coppola et al. [11], who trained pigeons on the same spatial working memory task for 24 total

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trials but with three, pseudorandomized delay periods between the sample and test phases (2, 15, and 45 min). On the final 15 min trial (most comparable to the 10 min delay used in the current

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study), young pigeons averaged 6.00 choices to complete the test phase, whereas old pigeons averaged 7.67 choices. By contrast, the young pigeons of the current study averaged 9.00 choices to complete the final (trial 12) test phase, whereas older pigeons averaged 8.86 choices. Thus, we are confident that neither the young nor old pigeons of the current study had reached asymptotic

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performance by the time of sacrifice. The comparison between the trial 12 data of the current study and the final 15 min trial of Coppola et al. [11] suggests that the pigeons of the current study were still actively learning the behavioral task. Therefore, the age-related differences in septum activation patterns reported here likely reflect active, spatial learning and only to a lesser extent memory performance.

Although septal activity was greatly reduced in both hemispheres of old pigeons, the effect of age only reached statistical significance in the right hemisphere. The data are therefore suggestive of a potential lateralized effect of age on septal activation. Curiously, however, the reduction in septal activation appears more pronounced in the right hemisphere, contrasting with the more pronounced left-lateralized age-related reduction in HF activity reported by Coppola and

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Bingman [17]. Given the considerable literature on functional lateralization of the HF, which has generally suggested a left-dominance for many spatial-cognitive tasks [see 38, 39], Coppola and

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Bingman [17] proposed the working hypothesis that the cognitive deficits seen in older pigeons [11, 12] might be due to a diminished ability of left HF to render spatially distinct the locations of

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the radial maze’s eight similar feeders. By contrast, speculating on the importance of a greater age-

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related, right-lateralized reduction in septum activity is compromised by the lack of research on functional lateralization of the avian septum. What we can offer from the current data is that the

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pigeon right septum may more strongly control spatial working memory processes compared to the left septum. However, more research on the relationship between septum function and spatial

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cognition is needed before possible differential contributions of each septum can be understood. The current study adds to the growing comparative literature on neurocognitive aging as it is the first to investigate the relationship between aging and septal functioning in an avian species. The comparative value is highlighted by the apparent differences in septal aging between mammals

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and birds. Specifically, it appears that aging affects the mammalian and avian septum similarly (at least with respect to activation during a spatial-cognitive challenge), but that the avian NDB may be less susceptible to change during aging compared to the mammalian NDBb. This latter finding nurtures the hypothesis that aging may not affect the brain of birds as intensely as it does mammals. Older pigeons, like mammals, do display reductions in adult neurogenesis [14] and decreased HF

[17] and septal activation during spatial learning. But in contrast to mammals, birds, or at least homing pigeons, do not display atrophy of the telencephalon [40], HF [13], septum and NDB [41], reduced NDB activation during spatial learning, nor any apparent loss of cholinergic neurons in the NDB. One potential explanation for the suspected partial resistance to the effects of aging on the avian septo-hippocampal system is that, compared to similarly sized mammals, birds have

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higher plasma levels of antioxidants. Antioxidants are capable of providing various tissues, including the brain, with protection against cellular damage caused by reactive oxygen species

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(ROS) accumulation (i.e., byproducts of oxygen, which can cause damage to DNA, lipids and proteins through oxidation; see [42, 43]). In summary, the accumulating data on neurocognitive

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aging in birds are revealing shared characteristics with mammals, similarities that are important

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for furthering the development of general theories of aging processes. But of perhaps more interest are the differences between birds and mammals, and more broadly variation across all vertebrate

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classes, which could inspire further research into the origins of such differences and how understanding those differences could be exploited to develop translational interventions to

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promote brain health during aging.

In conclusion, the current paper highlights the importance of the avian septum for understanding declining spatial cognition in birds and serves as a reminder that the hippocampus does not function in isolation. The benefits of a comparative approach toward understanding

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neurocognitive aging should be apparent, and we should note that not only pigeons could be of interest as complementary models for neurocognitive aging research. Future research in birds should aim to more robustly characterize the full extent of age-related changes to the cholinergic and other neurotransmitter systems that regulate septo-hippocampal function. For example, GABAergic projections from the septum work in concert with cholinergic projections to create

hippocampal theta oscillations (whereas cholinergic signals might initiate theta oscillations, the inhibitory effect of GABAergic signaling may control theta oscillation frequency [44]). Obvious directions for future research thus include an assessment of age-related changes to avian septohippocampal cholinergic-GABAergic interactions, HF oscillatory activity, especially in the theta range [45], and the properties of HF cholinergic and GABAergic signals.

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Author Contributions Vincent J. Coppola: conceptualization; methodology; validation; formal analysis; investigation; resources; writing – original draft; visualization; project administration.

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Daniele Nardi: conceptualization; writing – review and editing; supervision

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Verner P. Bingman: conceptualization; methodology; validation; resources; writing – review and editing; supervision; project administration

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Acknowledgements

The authors would like to thank Drs. Patricia Sharp, Richard Anderson, and Brooks Vostal

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for their contributions towards the planning of this study, Dr. Uwe Mayer for his technical (immunohistochemistry) support, Jari Willing for his assistance in photographing tissue, Joan

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Phillip for her careful review of earlier versions of the manuscript, and the BGSU Department of Psychology for its financial support. The research was conducted while VPB was supported by

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NSF grant IOS-1457304.

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Figure Captions

Figure 1. (A) Schematic of a birds-eye view of the testing environment. The 8 feeders are shown arranged in a circle. The area labeled (x) represents a curtained off viewing area for the experimenter. Various landmarks are present to create a spatially heterogeneous environment; specifically, a 83 ×165 × 122 cm foam trapezoid (a), door (b), wash area (c), 57 cm tall stool (d),

91 × 91 × 74 cm table (e), overturned five-gallon bucket (f), and a 61 cm tall artificial tree (g). In addition, 4 posters (P1-4) were placed on 3 of the 4 walls. Posters consisted of a colored background with different colored shapes on them (e.g., P1 had a green background with 2 orange rectangles side-by-side). (B) Schematic of the feeder design (F = feeder; R = ramp). All

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measurements are in cm.

Figure 2. (A) Schematic of coronal sections of the pigeon brain shown at 5 anterior-posterior levels

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(A9.50, A9.00, A8.50, A8.00, and A7.50) corresponding to the pigeon brain atlas (Karten & Hodos, 1967). The three regions of interest are indicated: lateral septum (SL), medial septum (SM),

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and nucleus of the diagonal band (NDB). The black squares (not to scale) within each region of

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interest represent the approximate positioning of the sampling boxes used to count c-Fos and

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ChAT labels. (B) An example of c-Fos (left) and ChAT (right) labeling in the septum (A7.50).

Figure 3. Mean (±SEM) number of c-Fos labels in the total septum (A), left septum (B), right

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septum (C), left SL (D), right SL (E), left SM (F), and right SM (G) separated by age group (young: gray bars; old: white bars). Individual data points are overlaid. (*) = p < .05.

Figure 4. Mean (±SEM) number of ChAT labels in the total septum (A), left septum (B), right

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septum (C), left SL (D), right SL (E), left SM (F), and right SM (G) separated by age group (young: gray bars; old: white bars). Individual data points are overlaid.

Figure 5. Mean (±SEM) number of c-Fos labels in the total NDB (A), left NDB (B), and right NDB (C), as well as the number of ChAT labels in the total NDB (D), right NDB (E), and left

NDB (F) separated by age group (young: gray bars; old: white bars). Individual data points are

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Correlations between septal c-Fos measures and TOT-TEST on the last trial of testing. Right Septum

Left Septum

Left SL

Right SL

Left SM

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TOT-TEST (Young)

r = .09 p = 0.84

r = .31 p = 0.50

r = -.04 p = 0.94

r = .16 p = 0.73

r = .33 p = 0.47

r = -.11 p = 0.81

r = .22 p = 0.63

TOT-TEST (Old)

r = -.34 p = 0.57

r = -.59 p = 0.29

r = -.27 p = 0.65

r = -.55 p = 0.34

r = -.53 p = 0.36

r = -.21 p = 0.74

r = -.45 p = 0.45

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Total Septum