The quantitative anatomy of the hippocampus in homing pigeons and other pigeon breeds: implications for spatial cognition.

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Andrew N Iwaniuk, Kelsey J Racicot, Audrey EM Guyonnet, Ben Brinkman This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4459634/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Dec, 2024 Read the published version in Brain Structure and Function → Version 1 posted 11 You are reading this latest preprint version Abstract The artificial selection for specific behavioural and physical traits domesticated animals has resulted in a wide variety of breeds. One of the most widely recognized examples of behavioural selection is the homing pigeon ( Columba livia ), which has undergone intense selection for fast and efficient navigation, likely resulting in significant anatomical changes to the hippocampal formation. Previous neuroanatomical comparisons between homing and other pigeon breeds yielded mixed results, but only focused on volumes. We completed a more systematic test for differences in hippocampal formation anatomy between homing and other pigeon breeds by measuring volumes, neuron numbers and neuron densities in the hippocampal formation and septum across homing pigeons and seven other breeds. Overall, we found few differences in hippocampal formation volume across breeds, but large, significant differences in neuron numbers and densities. More specifically, homing pigeons have significantly more hippocampal neurons and at higher density than most other pigeon breeds, with nearly twice as many neurons as feral pigeons. These findings suggest that neuron numbers may be important component of homing behaviour in homing pigeons. Our data also provide the first evidence that neuronal density can be modified by artificial selection, which has significant implications for the study of domestication and interbreed variation in anatomy and behaviour. (4 to 6 words): hippocampus artificial selection septum pigeon neurons Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Domestic species are selected for a variety of behavioural and morphological traits by humans for aesthetics, meat quality, eggs, fur/plumage, companionship, competitions, and to perform specific tasks (Kruska 1988 ; Francis 2015 ). In some domesticated species, this has resulted in a plethora of breeds that can markedly in anatomy, physiology, and behaviour, often exceeding natural variation in the wild (Sanchez-Villagra 2022 ; Darwin 1888 ; Francis 2015 ; Hecht et al. 2023 ). Of the many species and breeds that humans have domesticated, the homing pigeon ( Columba livia ) is perhaps one of the most famous examples of artificial selection for a behavioural trait. Wild rock doves, the ancestor of domestic pigeons, use homing to navigate between nesting and foraging sites 20 km apart (Alleva et al. 1975 ; Baldaccini et al. 2000 ), but homing pigeons can navigate home from a novel location over 1,000 km away as a result of training and intense selection over decades by competitive pigeon racers (Hiatt and Esposito 2000 ; Kligerman 1978 ). In racing competitions, pigeons must typically navigate from novel locations, a process dependent on multisensory inputs to successfully orient homewards (Wallraff 2005 ; Walcott 1996 ; Walcott et al. 2018 ; Wiltschko and Wiltschko 2019 ) and visuospatial cognition (Bingman 2018 , 2024 ; Gagliardo et al. 2020 ) to locate their home loft. By selecting for fast and efficient homing over long distances, pigeon racers have likely selected for several physiological, anatomical, and behavioural traits, including improved spatial cognition (Bingman 2018 ; Herold et al. 2015 ). Spatial cognition in birds is mediated by the hippocampal formation (HF), a telencephalic region encompassing the hippocampus proper (Hp) and area parahippocampalis (APH) (Atoji 2006 ; Herold et al. 2015 ; Striedter 2016 ) that is analogous in function (and likely homologous) to the mammalian hippocampus and entorhinal cortex (Atoji et al. 2016 ; Striedter 2016 ; Colombo 2000 ). Accordingly, HF lesions impair homing pigeon performance on spatial tasks in the lab (Strasser and Bingman 1999 , 1997 ; White et al. 2002 ). When tested outside of the lab, HF lesioned homing pigeons are less likely to successfully return to the home loft, take longer to return, take longer paths with greater circuitousness, and have an impaired ability to recognize landmarks, including the loft itself (Bingman 2005, 1984, 1990; Gagliardo et al. 1999 ; Gagliardo et al. 2004 ; Gagliardo et al. 2020 ; Herold et al. 2015 ). Also in intact homing pigeons, the HF, particularly the APH, is active during homing based on early gene expression (Shimizu et al. 2004 ), providing further evidence that the HF plays an important role in homing behaviour. Given the importance of the HF in homing (Herold et al. 2015 ) and spatial cognition (Sherry 2006 ; Pravosudov and Roth II 2013 ), intense selection for fast and efficient navigation could have driven anatomical differences in the HF in homing pigeons compared to other pigeons. The homing pigeon Hp is larger than that of wild rock doves, but does not differ in size from several other domestic breeds that are not selected for homing (Rehkämper et al. 2008 ; Rehkamper et al. 1988 ). These mixed findings are, however, problematic for several reasons. First, Rehkämper et al. (1988, 2008 ) measured only the Hp whereas the APH is most active during homing (Shimizu et al. 2004 ), both Hp and APH are lesioned in homing pigeon experiments (Bingman 2005, 1984, 1990; Bingman et al. 1998 ; Gagliardo et al. 1999 ; Gagliardo et al. 2004 ; Strasser and Bingman 1997 ; Strasser et al. 1998 ; Strasser et al. 2004 ; White et al. 2002 ), and the total HF is what is measured in other species (Ward et al. 2012 ; Sherry 2006 ; Pravosudov and Roth Ii 2013 ). In addition, Rehkämper et al. (1988, 2008 ) used body mass as a scaling variable to test for differences in relative Hp volume, but larger and smaller body sizes are selected for independently of homing and other behaviours in pigeon breeds (Levi 1965 ). Instead, telencephalon volume is a more relevant scaling variable and consistent with other literature on the avian HF (Ward et al. 2012 ; Sherry 2006 ; Pravosudov and Roth Ii 2013 ). Last, a focus on volume neglects other aspects of hippocampal anatomy related to spatial cognition. For example, more HF neurons and higher rates of neurogenesis are associated with better performance in food caching tasks (Chancellor et al. 2011 ; Pravosudov and Roth II 2013 ; Gould et al. 2013 ). In fact, several lines of evidence indicate that neuron number and density may be more appropriate proxies of cognition than volume of a brain region (Pravosudov and Roth II 2013 ; Roth II et al. 2010; Kverkova et al. 2022 ; Sol et al. 2022 ). It is therefore crucial to go beyond simply measuring volume to compare the hippocampal anatomy of homing pigeons with other breeds. Here, we provide a robust test of whether the anatomy of the homing pigeon HF differs quantitatively from that of other pigeon breeds. We quantified HF volume and neuron numbers and density across homing, show, sport, and feral pigeon breeds, all of which differ in free flight experience and behavioural and physical traits under selection. In addition, to the HF, we quantified the same parameters, volume, neuron numbers, and neuron density, in the septum. The avian septum is connected directly with the HF (Atoji and Wild 2004 ), plays a role in some aspects of spatial and working memory (Coppola 2021; Peterson and Bingman 2011 ), but is not known to be involved in homing. Instead, the avian septum is typically associated with other behaviours, such as social communication, pair-bonding, sexual behaviour, and aggression (Goodson et al. 2004a ; Ramirez et al. 2009 ; Corrales Parada et al. 2021 ; Taziaux et al. 2006 ). Including the septum in our analyses therefore allows us to test if any interbreed differences in HF anatomy are specific to the HF or not as well as the potential effects of artificial selection on the septum. Based on what we know about the involvement of HF in homing (Bingman 2018 , 1990, 2024 ; Gagliardo et al. 2020 ; Herold et al. 2015 ; Shimizu et al. 2004 ) and the intense selection that homing pigeons have been under over centuries, we first predict that homing pigeons will have relatively larger HFs that contain more neurons than other breeds. Second, we predict that show breeds with have the smallest HFs with the fewest neurons because they are not flown outside of their lofts or cages and lack homing experience (Cnotka et al. 2008 ). Third, we predict that sporting breeds that regularly have free flight experience will have HF size and neuron numbers in between the homing and show pigeons. Last, we cannot predict differences in septum size or neuron numbers because of its diverse functions (Goodson et al. 2004a ; Ramirez et al. 2009 ; Corrales Parada et al. 2021 ; Taziaux et al. 2006 ), but if it is larger in homing pigeons, that could be taken as correlative evidence of its role in some aspect of spatial cognition (Coppola 2021; Peterson and Bingman 2011 ). MATERIALS AND METHODS Animals HF measurements were made from 51 male and female domestic pigeons, including feral pigeons (n = 8), homing pigeons (n = 10), sporting breeds (rollers, n = 6; highflyers, n = 7), and show breeds (capuchines, n = 4; Norwich croppers, n = 5; American show homers, n = 5; American show rollers, n = 6). Both males and females were sampled within each breed, but not evenly such that it was not possible to test for sex differences. Due to tissue damage, only a subset of these specimens (n = 35) could be used for our septum measurements. Thus, the analyses of septum are on fewer specimens and breeds (none of the capuchine pigeons were quantifiable). Homing pigeons (Fig. 1 A) were all trained and used in local races (based from Lethbridge, Alberta Canada) of over 300 km in distance. Feral pigeons, which are largely derived from homing pigeons (Giunchi et al. 2020 ; Stringham et al. 2012 ), were trapped using standard wire baited traps in Lethbridge and Delia, Alberta, Canada. For our sporting breeds, we sampled experienced rollers and highflyers from private breeders (Alberta and Saskatchewan, Canada). Rollers (Fig. 1 B) were selected for performing backwards somersaults in flight (Levi 1965 ; Mowrer 1940 ). Highflyers (Fig. 1 C), as the name implies, were selected for flying high above the loft, staying airborne for over eight hours (Hiatt and Esposito 2000 ). Both of these sporting breeds are flown regularly, but are not trained in homing flights. Show breeds, in contrast, are kept primarily in cages or lofts throughout their lives and are not flown outside. There are hundreds of show breeds that vary dramatically in size, plumage, and even behaviour (Hiatt and Esposito 2000 ; Levi 1965 ). We were able to obtain four show breeds from breeders: American show roller, American show homer, capuchine, and Norwich croppers. America show rollers (Fig. 1 D) are a relatively recent breed, that are derived from sporting rollers, but are selected for plumage so have largely lost their ability to roll (Levi 1965 ). Similarly, American show homers (Fig. 1 E) were derived from homing pigeons, but were selected for larger bodies, large heads, and plumage and have not been used for homing for over a century (Levi 1965 ). The capuchine is an old breed (300 + years old, (Levi 1965 )) selected for an upturned ruff of feathers (Fig. 1 F) and comes in a diverse range of colours. Last, the Norwich cropper, was selected for an exaggerated sexual display (Fig. 1 G) including elongated legs and more upright posture and was established over a century ago (Levi 1965 ). All pigeons were weighed with a spring balance, deeply anaesthetized with an intracoelomic injection of sodium pentobarbital (2 mL/kg body weight) and then perfused transcardially with 0.1 M phosphate buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde (PFA). The brains were removed and stored in 4% PFA at 4°C for 1–2 weeks. Brains were weighed, cryoprotected in 30% sucrose PBS solution, transferred into an antifreeze solution (Hoffman and Le 2004 ) and stored at -20°C until embedding and sectioning. Histology Brains were embedded in gelatin blocks and sectioned coronally on a freezing stage microtome at 40 µm thickness. All sections were collected in PBS + 1% sodium azide solution in multi-well plates and mounted in 1:4 series onto gelatinised slides. Once dry, sections were washed in chloroform and stained for Nissl substance using thionin acetate followed by a graded ethanol series. Sections were cleared in Hemo-De (Thermo Fisher Scientific, HD150A) and coverslipped with Permount (Thermo Fisher Scientific, #SP15500). Eighteen to 22 sections equally spaced throughout the rostrocaudal extent of HF were immunolabeled for the neuron specific antigen, NeuN (Mullen et al. 1992 ). Note that these same sections were also used for the septum. Sections were rinsed in PBS (pH 7.4), then incubated in 10% normal goat serum (Jackson ImmunoResearch, 005-000-121) in PBS + 0.025M Triton (PBST) for 1 hour. The sections were then incubated in a monoclonal mouse anti-NeuN primary antibody (clone A60, Sigma Aldrich, MAB377) at a 1:1000 dilution in PBST on a shaker plate for 24 hours at room temperature. The sections were rinsed again in PBS and then incubated for 4 hours in fluorescein (FITC) goat anti-mouse secondary antibody (Jackson ImmunoResearch, 111-095-144) at a 1:200 dilution in PBST on a shaker plate at room temperature. Finally, the sections were rinsed and mounted onto gelatinised slides. Stereology All volumetric measurements and neuron counts were made using unbiased stereology (West 2012 ). Volumetric measurements were made on a Zeiss Axio Imager M2 microscope using the Cavalieri Estimator Probe in StereoInvestigator™ with a 400 µm grid size for all regions of interest of the Nissl stained series. Regions of interest (telencephalon, HF, septum) were differentiated following the boundaries shown in stereotaxic atlases of the pigeon (Karten and Hodos 1967 ) and chick (Puelles et al. 2018 ). Quantification of total brain and telencephalon were taken using a 1x objective lens; all other regions were quantified using a 2.5x objective lens. Coefficients of error (Gunderson, m = 1) for all volumes were < 0.055. Neuron numbers were estimated using the optical fractionator method (West 2012 ) as implemented in StereoInvestigator™. For HF analysis, the following parameters were used across all mounted sections (15–20 sections per specimen): a grid spacing of 650 µm, a grid size of 40 µm, a dissector zone of 15 µm, and upper and lower guard zones of 5 µm. For the septum, five to ten sections spanning its rostrocaudal extent were sampled using the following parameters: a grid spacing of 350 µm, a grid size of 40 µm, a dissector zone of 15 µm, and upper and lower guard zones of 5 µm. Neuron counts were completed using a 40x immersion oil lens on a Zeiss Axio Imager M2 microscope. Coefficients of error (Gunderson, m = 1) for all cell counts were < 0.10 for all specimens. Means and standard deviations of data for all breeds are shown in Table 1 . Table 1 Average (± standard deviation) values for all of the neuroanatomical measurements made of the eight pigeon breeds examined. Sample sizes are provided for each breed in brackets below each value. Measurement Homing Feral Highflyer Roller Capuchine Show Roller Show Homer Norwich Cropper Telencephalon volume (mm 3 ) 992.09 ± 136.54 (n = 10) 977.14 ± 158.02 (n = 8) 923.53 ± 62.29 (n = 7) 907.18 ± 71.94 (n = 6) 960.91 ± 114.82 (n = 4) 880.49 ± 87.07 (n = 6) 928.31 ± 75.29 (n = 5) 1166.33 ± 69.53 (n = 5) Hippocampal formation volume (mm 3 ) 64.15 ± 8.47 (n = 10) 57.72 ± 5.79 (n = 8) 51.04 ± 7.33 (n = 7) 57.31 ± 3.43 (n = 6) 57.38 ± 4.78 (n = 4) 55.17 ± 7.54 (n = 6) 56.71 ± 1.94 (n = 5) 61.27 ± 6.22 (n = 5) Number of hippocampal neurons 2,426,353 ± 593,592 (n = 10) 1,072,185 ± 255,997 (n = 8) 1,276,222 ± 245,974 (n = 7) 1,348,679 ± 193,011 (n = 6) 1,328,841 ± 587,279 (n = 4) 1,851,884 ± 333,582 (n = 6) 1,853,808 ± 172,313 (n = 5) 2,428,458 ± 442,159 (n = 5) Hippocampal formation neuron density (#neurons/ mm 3 ) 37,601 ± 6,925 (n = 10) 18,708 ± 4,657 (n = 8) 25,288 ± 5,162 (n = 7) 23,538 ± 2,970 (n = 6) 22,720 ± 8,483 (n = 4) 33,787 ± 6,117 (n = 6) 32,745 ± 3,532 (n = 5) 39,980 ± 8,859 (n = 5) Septum volume (mm 3 ) 10.35 ± 1.04 (n = 8) 10.94 ± 1.68 (n = 5) 10.11 ± 1.63 (n = 4) 10.31 ± 1.14 (n = 4) - 13.12 ± 1.41 (n = 4) 12.81 ± 1.34 (n = 5) 14.70 ± 2.38 (n = 5) Number of septal neurons 337,330 ± 125,379 (n = 8) 222,269 ± 76,782 (n = 5) 409,978 ± 94,782 (n = 4) 225,684 ± 120,630 (n = 4) - 468,748 ± 97,394 (n = 4) 402,362 ± 70,850 (n = 5) 585,469 ± 72,107 (n = 5) Septum neuron density (#neurons/ mm 3 ) 32,549 ± 11,870 (n = 8) 21,908 ± 9,152 (n = 5) 42,036 ± 14,681 (n = 4) 21,851 ± 11,860 (n = 4) - 36,475 ± 10,842 (n = 4) 31,557 ± 70,850 (n = 5) 40,612 ± 7890 (n = 5) Statistical Analyses All statistical analyses were performed in Jamovi (The jamovi project 2024 ). Absolute values were analysed using one-way analyses of variance (ANOVAs) and Tukey’s honestly significant difference (HSD) post-hoc tests. Relative values were analysed using analyses of covariance (ANCOVAs) of log-transformed data and Tukey’s HSD post-hoc tests. Covariates were body mass, whole brain volume (minus region of interest), or telencephalon volume (minus region of interest). Interaction effects (breed x covariate) were not significant across all of our analyses, indicating no significant differences in slopes among breeds. Interaction effects were therefore removed from all final ANCOVA models. RESULTS Hippocampal formation (HF) Absolute HF volume differed significantly among breeds (F = 2.87, df = 7, 43, p = 0.015, Fig. 2 A). Homing pigeons had the largest HF volumes, whereas rollers had the smallest HF volumes. A Tukey’s HSD post-hoc test revealed that homers had significantly larger HF volumes than highflyers, but no other significant differences were detected among the other breeds. HF volume varied significantly with telencephalon volume (F = 12.88, df = 1, 42, p < 0.0001, Fig. 2 B) and there was a significant difference among breeds (F = 2.73, df = 7, 42, p = 0.02). As with the analysis of absolute volumes, the post-hoc test indicated that homing pigeons have relatively larger HF volumes than highflyers, but no other significant differences. In contrast to volumes, the absolute number of HF neurons varied far more across breeds (Fig. 2 C). Norwich croppers and homing pigeons had the most neurons (both means = 2.4 x 10 6 ), and feral pigeons had the fewest neurons, less than half of that of homing pigeons (1.1 x 10 6 ). Accordingly, our analyses found a significant difference across breeds in HF neuron number (F = 12.77, df = 7, 43, p < 0.001). Post-hoc tests revealed that homing pigeons and Norwich croppers had significantly more neurons than feral, highflyer, roller, and capuchine pigeons. Conversely, feral pigeons had significantly fewer neurons than homing, show roller, show homer, and Norwich cropper pigeons. When we compared the number of HF neurons to HF volume, we found a similar pattern (Fig. 2 D). The number of HF neurons increased with HF volume (F = 8.26, df = 1, 42, p = 0.006), but there was also a significant difference across breeds (F = 10.94, df = 7, 42, p < 0.001). Specifically, our post-hoc tests showed that homing pigeons and croppers had significantly more neurons, relative to HF volume, than feral, highflyer, roller, and capuchine pigeons whereas feral pigeons had significantly fewer neurons than homing, highflyer, show roller, show homer, and cropper pigeons. Last, HF neuronal density also differed greatly among breeds (Table 1 , Fig. 2 E). Norwich cropper and homing pigeons had the highest HF neuron densities (Table 1 ), and feral pigeons had the lowest neuron density, again less than half of that of homing pigeons (Table 1 , Fig. 2 E). These differences in HF neuron number and density between homing and feral pigeons are even apparent when looking at the sections; homing pigeons have far more neurons labeled (Fig. 2 F) than feral pigeons (Fig. 2 G) in sections taken from the same location within HF. Similar to our previous analysis of neuron numbers relative to HF volume, HF neuronal density differed significantly among breeds (F = 11.15, df = 7, 43, p < 0.001). Post-hoc tests revealed that homing and cropper pigeons had significantly higher neuron densities than feral, highflyer, roller, and capuchine pigeons. Conversely, feral pigeons had significantly lower neuron densities than homing, show roller, show homer, and cropper pigeons. Septum Norwich croppers had the largest septum volumes, whereas rollers, homers and highflyers shared the smallest septum volumes (Table 1 , Fig. 3 A). Absolute septum volume differed significantly among breeds (F = 6.64, df = 6, 28, p < 0.01). Norwich croppers had significantly larger septum volumes than feral, homer, highflyer, and roller pigeons in our post-hoc tests, but no other significant differences were found. Unlike HF volume, septum volume did not vary significantly with telencephalon volume (F = 0.78, df. = 1, 27, p = 0.39), but differed significantly among breeds (F = 5.34, df = 6, 27, p < 0.01). Post-hoc tests revealed homers and feral pigeons had relatively smaller septum volumes than croppers (Fig. 3 B). On average, Norwich cropper pigeons had the most septal neurons, and feral pigeons had the fewest septal neurons (Table 1 ) and the absolute number of septum neurons differed significantly among breeds (F = 8.23, df = 6, 28, p < 0.01); Norwich croppers had significantly more septal neurons than feral and roller pigeons and show rollers had more neurons than feral and roller pigeons (Fig. 3 C). The number of neurons did not vary significantly with septum volume (F = 0.06, F = 1, 27, p = 0.81), but differed significantly among breeds (F = 5.15, df = 6, 27, p < 0.01, Fig. 3 D). Post-hoc tests revealed that feral and roller pigeons had relatively fewer neurons than croppers and show rollers. Finally, we also compared neuron density by dividing neuron number by septum volume. Highflyer and Norwich cropper pigeons had the highest septum neuron densities, and feral and roller pigeons had the lowest septum neuron densities (Table 1 , Fig. 3 E). Overall, breeds differed significantly in septal neuron density (F = 2.79, df = 6, 28, p = 0.03), but the post-hoc tests yielded no significant differences. Although the neuronal density appeared to differ among some breed pairs, such as the homing pigeon and cropper sections shown in Fig. 3 (F and G, respectively), based on the boxplot (Fig. 3 E) there is considerable overlap among breeds in neuronal density. DISCUSSION Similar to previous studies (Ebinger and Lohmer 1984 ; Rehkämper et al. 2008 ; Rehkamper et al. 1988 ) we found that homing pigeons do not differ greatly in relative HF volume from other pigeon breeds, including feral pigeons. However, the data partially supported our prediction that homing pigeons would have more HF neurons and higher neuronal densities than other breeds. Furthermore, while there were some among-breed differences in septum neuroanatomy, homing pigeons largely did not differ from other breeds. As we discuss below, the relationship between differences in HF or septal quantitative anatomy and behaviour requires testing, but our data suggest that neuron numbers could be important for the remarkable navigational abilities of homing pigeons. Differences in septum anatomy across breeds As mentioned previously, the septum plays a significant role in modulating agonistic and courtship behaviour in birds (Goodson et al. 2004a ; Ramirez et al. 2009 ; Corrales Parada et al. 2021 ; Taziaux et al. 2006 ), but it is also connected directly with the HF (Atoji and Wild 2004 ) and involved in spatial and working memory (Coppola 2021; Peterson and Bingman 2011 ). Despite the role of the septum in some aspects of memory, homing pigeons have smaller septal volumes and fewer neurons than most show breeds (Fig. 3 ). From this, we conclude that if the septum is contributing to homing behaviour, it is not dependent on more neurons or a larger volume. Unexpectedly, croppers had larger septal volumes and more septal neurons than several other breeds whereas feral pigeons have smaller septal volumes and fewer neurons (Fig. 3 . A-D). As discussed previously, croppers were selected for exaggerated courtship displays (Fig. 1 E) (Levi 1965 ). Given the role of the septum in regulating sexual and courtship behaviour (Goodson et al. 2004a ; Taziaux et al. 2006 ; Goodson et al. 2004b ), it is possible that selection for these exaggerated displays resulted in neuroanatomical changes in the septum. How a larger septum or more septal neurons might be associated with courtship display is unclear though as much of the interspecific variation in social behaviour associated with the septum is typically due to changes in receptor and nonpeptide expression (Goodson et al. 2006 ; Goodson et al. 2009 ). With respect to the feral pigeons, we speculate that a smaller septum with fewer neurons may be an effect of chronic stress. Feral pigeons are exposed to many stressors not experienced by domesticated pigeons (e.g., predation, variable food availability, lethal control efforts (Murton et al. 1972 )) and chronic stress can have detrimental effects on neurogenesis in birds (Smulders 2017 ; Brenowitz and Larson 2015 ). Further, this stress hypothesis could also explain the relatively neuron poor HF of feral pigeons (see below). Differences in hippocampal formation anatomy across breeds Previous studies of hippocampus size across pigeon breeds focused on Hp and not the entire HF and lacked data on neuron numbers and densities (Ebinger and Lohmer 1984 ; Rehkämper et al. 2008 ; Rehkamper et al. 1988 ), making it difficult to ascertain to what extent HF quantitative anatomy differs between homing and other pigeons. Although homing pigeons tended to have relatively and absolutely large HF volumes (Fig. 2 A,B), few significant differences were detected, which is consistent with (Rehkämper et al. 2008 ). More importantly, we show that there are significant differences in neuron numbers and densities such that homing pigeons tend to have more neurons than feral, sporting, and some show breeds (Fig. 2 C-G). There are at least a couple of reasons why homing pigeon HF size and neuron numbers differ from some, but not all, other domestic breeds. First, domestic pigeon breeds are frequently interbred (Hiatt and Esposito 2000 ; Levi 1965 ; Pacheco et al. 2020 ). For example, show homers are derived from and often backcrossed with homing pigeons and American show rollers are a recently developed breed that often involves crossing with other breeds, including homing pigeons. Pigeon fanciers, apart from some racing pigeon lines, also do not publish breeding records so purebred registries are lacking. As a result, pigeon breeds are not as genetically distinct (Pacheco et al. 2020 ) as purebred dog breeds (Parker et al. 2017 ) or other domesticated animal breeds (Rasali et al. 2011 ). If some neuroanatomical traits have a strong genetic basis (e.g., large HF), then mixing of different pigeon breeds could lead to similarities in HF size across breeds. Second, it is possible that changes in neuronal density have accompanied selection for traits other than homing. For example, selection for the exaggerated sexual displays of croppers could be associated with changes in androgen levels and/or steroid hormone receptors, resulting in higher survivorship of new neurons (Balthazart and Ball 2016 ; Chen et al. 2013 ). Neuroanatomical changes in the HF of some breeds could therefore be a by-product of selection for traits other than homing. Interestingly, the biggest difference in neuron numbers and densities was between homing and feral pigeons (Fig. 2 C-G). Feral pigeons are largely derived from homing pigeons (Stringham et al. 2012 ; Giunchi et al. 2020 ), but experience markedly different selection pressures. Feral pigeons have similar mortality rates to wild pigeons (both rock doves and Columba palumbus ) and must search for food and water, recognize and avoid predators, and cope with environmental variability (Murton et al. 1972 ), all in stark contrast to the captive environments of homing pigeons and other domesticated pigeons. The stressors that face feral pigeons could mean that they experience chronic stress, which significantly impairs neurogenesis and new neuron survivorship in HF (Gualtieri et al. 2019 ; Robertson et al. 2017 ; Smulders 2017 ). This would then lead to fewer neurons and lower neuronal density, as observed in our data (Fig. 2 G). Whether there are behavioural consequences of having fewer HF neurons remains to be tested. Feral pigeons do not home as effectively as homing pigeons, but this is thought to be a motivational issue as they tend to stop more frequently and associate with other pigeons (Edrich and Keeton 1977 ; Chelazzi and Pineschi 1974 ). That said, if these differences in HF anatomy arise from stress, then the homing-feral pigeon difference should be lessened in feral pigeons raised in captivity. In stark contrast to the feral pigeons, homing pigeons tended to have more and denser neurons than the other breeds sampled (Fig. 2 C-E). It is tempting to conclude that having more neurons and higher neuronal density in the HF provides some benefit to spatial memory and cognition in pigeons. Data from chickadees ( Poecile spp.) indicates that populations and individuals with more neurons tend to perform better on spatial memory tasks, providing a link between performance and neuron numbers (Pravosudov and Roth II 2013 ). However, there are several unknowns with respect to homing that prevent us from making a direct link between neuron numbers and cognition from our data. First, data are lacking on homing performance across pigeon breeds. Homing experiments with feral pigeons and wild rock doves (Baldaccini et al. 2001 ; Edrich and Keeton 1977 ; Chelazzi and Pineschi 1974 ) suggest that they lack the motivation to fly long distances to return home. Data on homing rate (i.e., percentage of released birds that return to the loft) shown in (Shao et al. 2019 ) indicate that some non-homing breeds are largely incapable of returning from distances greater than 10km, but detailed tracking data as used in modern homing experiments (e.g., (Gagliardo et al. 2020 ; Gagliardo et al. 2018 )) were lacking. These non-homing breeds could have the same issue with motivation as feral pigeons (Chelazzi and Pineschi 1974 ; Edrich and Keeton 1977 ), not be physically capable, or lack the cognitive ability to home like homing pigeons. Without behavioural testing across breeds, we simply do not know. Second, we do not know what role training has on HF anatomy in pigeons. Restricting homing pigeons to a loft and not providing them with homing experience results in a smaller Hp (Cnotka et al. 2008 ), which likely arises from fewer neurons but this has yet to be tested. That said, flight experience appears to be insufficient to drive changes in neuron numbers because highflyer and roller pigeons all fly regularly outdoors, yet all have fewer HF neurons and lower HF neuron density than homing pigeons. Last, much of the research on pigeon homing over the past several decades has focused primarily on sensory processing (Wiltschko and Wiltschko 2019 ; Wallraff 2005 ; Gagliardo 2013 ) with the cognitive components of homing not researched as extensively (Bingman 2018 ). HF activity certainly increases during homing, especially within the APH (Shimizu et al. 2004 ), and hippocampal lesions impair homing (Bingman 2005, 1984, 1990; Gagliardo et al. 1999 ; Gagliardo et al. 2004 ; Gagliardo et al. 2020 ; Herold et al. 2015 ), but how the HF contributes to the homing pigeon’s cognitive map has remained elusive (Bingman 2018 ; Ben-Tov and Gutfreund 2022 ). Nevertheless, based on data from food caching species (Pravosudov and Roth II 2013 ; Croston et al. 2015 ) and contrasts between caching and non-caching songbirds (Payne et al. 2021 ; Sherry 2006 ), one would predict that homing pigeons would have some cognitive advantages over the other breeds examined. Although place cells have proved challenging to locate in pigeons, more neurons could allow new memories to form without interfering with old memories (Payne et al. 2021 ) or increase the speed of spatial memory processing. Of course, these are speculative and require testing, but it is unlikely that the addition of many mature neurons in the HF is unrelated to some difference in function. The relationship between neuron numbers and spatial cognition in pigeons may be unclear, but our data demonstrates clearly that breeds vary in neuronal density. Almost all of the literature on the effects of domestication on the brain and variation among domesticated breeds has focused on volumes of brain regions (Kruska 2005 ; Hecht et al. 2023 ; Rehkämper et al. 2008 ; Rehkamper et al. 1988 ; Rehkamper et al. 2003 ; Ebinger 1972 , 1975 , 1995 ; Ebinger and Lohmer 1984 , 1987 ; Ebinger and Rohrs 1995 ; Brusini et al. 2018 ), with little to no data on neuron numbers (Racicot et al. 2021 ). Our data is the first to show that artificial selection can drive significant changes in neuronal density, in stark contrast to previous claims (Jardim-Messeder et al. 2017 ), and without a major increase in brain region volume. The fact that brain region volumes and neuronal density can vary significantly among breeds within a species has significant implications for studying the neurobehavioural effects of domestication and artificial selection. Importantly, inter-breed and wild-domesticate comparisons need to move beyond volumetrics and examine neuronal density, neuron size and morphology, and connectivity to better understand the effects of different selection regimes on brain anatomy. Declarations Acknowledgements We wish to thank the Lethbridge Homing Pigeon Racing Club, Facilities staff at the University of Lethbridge, various pigeon breeders in Alberta and Saskatchewan, and the Brinkman farm for providing pigeons for use in this study, and Dr. Maurice Needham for assisting with microscopy. Funding Support for this study was provided by scholarships from the University of Lethbridge to AG and JKR and grants from the Natural Sciences and Engineering Research Council (NSERC), Canada Research Chairs Program, and Canada Foundation for Innovation to ANI. Data availability All of the data reported herein is provided in supplementary material. Declaration Conflict of Interest : All the authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. Research involving animals/ethical approval: All of the procedures outlined herein adhered to the Canada Council for Animal Care Guidelines and were approved by the University of Lethbridge Animal Welfare Committee (Protocol #2011). References Alleva E, Baldaccini NE, Foa A, Visalberghi E (1975) Homing behaviour of the rock pigeon. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4459634","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309995177,"identity":"2a6f1ffb-f2ac-4d3a-b576-b36307792409","order_by":0,"name":"Andrew N Iwaniuk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYHACAxDBAyIOMBywYeBjYGAjSUsaUD0zcVqg4MBhwlr4Zzdv+/CxjUHG4PjZg4d5zpxPbGPvP/aAocYOpxaJO8eKZ85sY+AxOJOXcJjnxu3ENp7D7AYMx5JxW3Mjx5iZdxsDj9mBHIPDPB+AWiSS2SQYG5hx6pAHafkL0nL+DUjLucQ2+ccgLfU4tRiAtDCCtNwA2XLjANAWZpCWwzi1GN5IK2bs/SfBY3/jjcHBOWeSjdt4ks0kEo4dx6lF7kbyZoYfZ2zsJftzjD+8OWYn289+8JnEh5pq3N6HAAk0fgIhDaNgFIyCUTAK8AIAKBVT5DMIX2sAAAAASUVORK5CYII=","orcid":"","institution":"University of Lethbridge","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"N","lastName":"Iwaniuk","suffix":""},{"id":309995178,"identity":"f59bf028-51f2-40b7-80fc-7536b48323b7","order_by":1,"name":"Kelsey J Racicot","email":"","orcid":"","institution":"University of Lethbridge","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kelsey","middleName":"J","lastName":"Racicot","suffix":""},{"id":309995179,"identity":"dc81591e-c3c2-4eef-88ed-bf46926e96c2","order_by":2,"name":"Audrey EM Guyonnet","email":"","orcid":"","institution":"University of Lethbridge","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Audrey","middleName":"EM","lastName":"Guyonnet","suffix":""},{"id":309995180,"identity":"4d3bb0eb-8e74-49eb-b81b-621aadf9100d","order_by":3,"name":"Ben Brinkman","email":"","orcid":"","institution":"University of Lethbridge","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ben","middleName":"","lastName":"Brinkman","suffix":""}],"badges":[],"createdAt":"2024-05-22 09:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4459634/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4459634/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00429-024-02882-5","type":"published","date":"2024-12-17T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57699585,"identity":"2e52d692-fcb9-4f49-ab4d-b0d9a6016e97","added_by":"auto","created_at":"2024-06-04 13:35:11","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":430802,"visible":true,"origin":"","legend":"\u003cp\u003ePhotos of the domesticated pigeon (\u003cem\u003eColumba livia\u003c/em\u003e) breeds sampled. A. homing pigeon. B. highflyer, bred for flying at high altitudes for long periods of time. C. roller, bred for performing aerial, backwards somersaults. D. American show roller, a show breed derived from performing rollers, but no longer rolls and is not flown outdoors. E. show homer, a large breed derived from homing pigeons that is selected for large body size and plumage. F. capuchine, an old show breed selected for an upturned ruff of neck feathers. G. Norwich cropper, a breed selected for exaggerated sexual displays and an upright posture.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4459634/v1/1dccff4a14ec9675445312c4.jpeg"},{"id":57699587,"identity":"16316b8d-89fe-4ada-96b9-926979f31f35","added_by":"auto","created_at":"2024-06-04 13:35:12","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":379660,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative data on hippocampal formation (HF) volume, neuron numbers, and neuron density. A. Boxplots (min-max, mean shown by horizontal line) of absolute hippocampal formation volume (mm\u003csup\u003e3\u003c/sup\u003e) across all eight breeds sampled. B. log-transformed HF volume plotted against log-transformed telencephalon (minus HF) volume. Each of the breeds is shown in a different symbol. The dashed line represents the least-squares linear regression line calculated across all breeds. C. Boxplots of the total number of neurons in the HF across all eight breeds sampled. D. log-transformed number of hippocampal formation neurons plotted against log-transformed hippocampal formation volume. The dashed line represents the least-squares linear regression line calculated across all breeds. The symbols are the same as those used in Fig. 2B. E. Boxplots of hippocampal formation neuron density, expressed as total number of neurons divided by hippocampal formation volume. F. A photomicrograph of NeuN labeling through the hippocampal formation of a homing pigeon (scale bar = 50 mm). G. A photomicrograph of NeuN labeling through the hippocampal formation of a feral pigeon (scale bar = 50 mm).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4459634/v1/230b417526adabd6a9c020f6.jpeg"},{"id":57699588,"identity":"c1e85aee-f878-401d-9bf2-d5ec5ba51d47","added_by":"auto","created_at":"2024-06-04 13:35:12","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":772646,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative data on septum volume, neuron numbers, and neuron density. A. Boxplots (min-max, mean shown by horizontal line) of absolute septum volume (mm\u003csup\u003e3\u003c/sup\u003e) across seven breeds sampled. B. log-transformed septum volume plotted against log-transformed telencephalon (minus septum) volume. Each of the breeds is shown in a different symbol, as shown in the legend. The dashed line represents the least-squares linear regression line calculated across all breeds. C. Boxplots of the total number of neurons in the septum across seven breeds sampled. D. log-transformed number of septum neurons plotted against log-transformed septum volume. The dashed line represents the least-squares linear regression line calculated across all breeds. The symbols are the same as those used in Fig. 3B. E. Boxplots of septum neuron density, expressed as number of neurons divided by septum volume. F. A photomicrograph of NeuN labeling through the hippocampal formation of a homing pigeon (scale bar = 50 mm). G. A photomicrograph of NeuN labeling through the hippocampal formation of a Norwich cropper pigeon (scale bar = 50 mm).\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4459634/v1/5dcdd5207e9731b8330fb3b2.jpeg"},{"id":72201687,"identity":"7b6e9a22-7141-4bc9-b662-bb3e148be60d","added_by":"auto","created_at":"2024-12-23 16:09:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2148728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4459634/v1/e2f931de-6b41-4bfe-9389-fb6378d82d41.pdf"},{"id":57700281,"identity":"d0b4e89f-9f10-4a54-afaa-aa59348724c0","added_by":"auto","created_at":"2024-06-04 13:43:11","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18580,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.Data.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4459634/v1/5d611ec9fe174b2d04cec42b.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The quantitative anatomy of the hippocampus in homing pigeons and other pigeon breeds: implications for spatial cognition.","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDomestic species are selected for a variety of behavioural and morphological traits by humans for aesthetics, meat quality, eggs, fur/plumage, companionship, competitions, and to perform specific tasks (Kruska \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Francis \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In some domesticated species, this has resulted in a plethora of breeds that can markedly in anatomy, physiology, and behaviour, often exceeding natural variation in the wild (Sanchez-Villagra \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Darwin \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1888\u003c/span\u003e; Francis \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Hecht et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Of the many species and breeds that humans have domesticated, the homing pigeon (\u003cem\u003eColumba livia\u003c/em\u003e) is perhaps one of the most famous examples of artificial selection for a behavioural trait. Wild rock doves, the ancestor of domestic pigeons, use homing to navigate between nesting and foraging sites 20 km apart (Alleva et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Baldaccini et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), but homing pigeons can navigate home from a novel location over 1,000 km away as a result of training and intense selection over decades by competitive pigeon racers (Hiatt and Esposito \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Kligerman \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). In racing competitions, pigeons must typically navigate from novel locations, a process dependent on multisensory inputs to successfully orient homewards (Wallraff \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Walcott \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Walcott et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wiltschko and Wiltschko \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and visuospatial cognition (Bingman \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Gagliardo et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) to locate their home loft. By selecting for fast and efficient homing over long distances, pigeon racers have likely selected for several physiological, anatomical, and behavioural traits, including improved spatial cognition (Bingman \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Herold et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpatial cognition in birds is mediated by the hippocampal formation (HF), a telencephalic region encompassing the hippocampus proper (Hp) and area parahippocampalis (APH) (Atoji \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Herold et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Striedter \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) that is analogous in function (and likely homologous) to the mammalian hippocampus and entorhinal cortex (Atoji et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Striedter \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Colombo \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Accordingly, HF lesions impair homing pigeon performance on spatial tasks in the lab (Strasser and Bingman \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; White et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). When tested outside of the lab, HF lesioned homing pigeons are less likely to successfully return to the home loft, take longer to return, take longer paths with greater circuitousness, and have an impaired ability to recognize landmarks, including the loft itself (Bingman 2005, 1984, 1990; Gagliardo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Gagliardo et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Gagliardo et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Herold et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Also in intact homing pigeons, the HF, particularly the APH, is active during homing based on early gene expression (Shimizu et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), providing further evidence that the HF plays an important role in homing behaviour.\u003c/p\u003e \u003cp\u003eGiven the importance of the HF in homing (Herold et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and spatial cognition (Sherry \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pravosudov and Roth II \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), intense selection for fast and efficient navigation could have driven anatomical differences in the HF in homing pigeons compared to other pigeons. The homing pigeon Hp is larger than that of wild rock doves, but does not differ in size from several other domestic breeds that are not selected for homing (Rehk\u0026auml;mper et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rehkamper et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). These mixed findings are, however, problematic for several reasons. First, Rehk\u0026auml;mper et al. (1988, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) measured only the Hp whereas the APH is most active during homing (Shimizu et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), both Hp and APH are lesioned in homing pigeon experiments (Bingman 2005, 1984, 1990; Bingman et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Gagliardo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Gagliardo et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Strasser and Bingman \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Strasser et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Strasser et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; White et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and the total HF is what is measured in other species (Ward et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sherry \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pravosudov and Roth Ii \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In addition, Rehk\u0026auml;mper et al. (1988, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) used body mass as a scaling variable to test for differences in relative Hp volume, but larger and smaller body sizes are selected for independently of homing and other behaviours in pigeon breeds (Levi \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). Instead, telencephalon volume is a more relevant scaling variable and consistent with other literature on the avian HF (Ward et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sherry \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pravosudov and Roth Ii \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Last, a focus on volume neglects other aspects of hippocampal anatomy related to spatial cognition. For example, more HF neurons and higher rates of neurogenesis are associated with better performance in food caching tasks (Chancellor et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pravosudov and Roth II \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gould et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In fact, several lines of evidence indicate that neuron number and density may be more appropriate proxies of cognition than volume of a brain region (Pravosudov and Roth II \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Roth II et al. 2010; Kverkova et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sol et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is therefore crucial to go beyond simply measuring volume to compare the hippocampal anatomy of homing pigeons with other breeds.\u003c/p\u003e \u003cp\u003eHere, we provide a robust test of whether the anatomy of the homing pigeon HF differs quantitatively from that of other pigeon breeds. We quantified HF volume and neuron numbers and density across homing, show, sport, and feral pigeon breeds, all of which differ in free flight experience and behavioural and physical traits under selection. In addition, to the HF, we quantified the same parameters, volume, neuron numbers, and neuron density, in the septum. The avian septum is connected directly with the HF (Atoji and Wild \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), plays a role in some aspects of spatial and working memory (Coppola 2021; Peterson and Bingman \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), but is not known to be involved in homing. Instead, the avian septum is typically associated with other behaviours, such as social communication, pair-bonding, sexual behaviour, and aggression (Goodson et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e; Ramirez et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Corrales Parada et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Taziaux et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Including the septum in our analyses therefore allows us to test if any interbreed differences in HF anatomy are specific to the HF or not as well as the potential effects of artificial selection on the septum.\u003c/p\u003e \u003cp\u003eBased on what we know about the involvement of HF in homing (Bingman \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, 1990, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Gagliardo et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Herold et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shimizu et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and the intense selection that homing pigeons have been under over centuries, we first predict that homing pigeons will have relatively larger HFs that contain more neurons than other breeds. Second, we predict that show breeds with have the smallest HFs with the fewest neurons because they are not flown outside of their lofts or cages and lack homing experience (Cnotka et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Third, we predict that sporting breeds that regularly have free flight experience will have HF size and neuron numbers in between the homing and show pigeons. Last, we cannot predict differences in septum size or neuron numbers because of its diverse functions (Goodson et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e; Ramirez et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Corrales Parada et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Taziaux et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), but if it is larger in homing pigeons, that could be taken as correlative evidence of its role in some aspect of spatial cognition (Coppola 2021; Peterson and Bingman \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eHF measurements were made from 51 male and female domestic pigeons, including feral pigeons (n\u0026thinsp;=\u0026thinsp;8), homing pigeons (n\u0026thinsp;=\u0026thinsp;10), sporting breeds (rollers, n\u0026thinsp;=\u0026thinsp;6; highflyers, n\u0026thinsp;=\u0026thinsp;7), and show breeds (capuchines, n\u0026thinsp;=\u0026thinsp;4; Norwich croppers, n\u0026thinsp;=\u0026thinsp;5; American show homers, n\u0026thinsp;=\u0026thinsp;5; American show rollers, n\u0026thinsp;=\u0026thinsp;6). Both males and females were sampled within each breed, but not evenly such that it was not possible to test for sex differences. Due to tissue damage, only a subset of these specimens (n\u0026thinsp;=\u0026thinsp;35) could be used for our septum measurements. Thus, the analyses of septum are on fewer specimens and breeds (none of the capuchine pigeons were quantifiable).\u003c/p\u003e \u003cp\u003eHoming pigeons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) were all trained and used in local races (based from Lethbridge, Alberta Canada) of over 300 km in distance. Feral pigeons, which are largely derived from homing pigeons (Giunchi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Stringham et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), were trapped using standard wire baited traps in Lethbridge and Delia, Alberta, Canada. For our sporting breeds, we sampled experienced rollers and highflyers from private breeders (Alberta and Saskatchewan, Canada). Rollers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) were selected for performing backwards somersaults in flight (Levi \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Mowrer \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1940\u003c/span\u003e). Highflyers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), as the name implies, were selected for flying high above the loft, staying airborne for over eight hours (Hiatt and Esposito \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Both of these sporting breeds are flown regularly, but are not trained in homing flights. Show breeds, in contrast, are kept primarily in cages or lofts throughout their lives and are not flown outside. There are hundreds of show breeds that vary dramatically in size, plumage, and even behaviour (Hiatt and Esposito \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Levi \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). We were able to obtain four show breeds from breeders: American show roller, American show homer, capuchine, and Norwich croppers. America show rollers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) are a relatively recent breed, that are derived from sporting rollers, but are selected for plumage so have largely lost their ability to roll (Levi \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). Similarly, American show homers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) were derived from homing pigeons, but were selected for larger bodies, large heads, and plumage and have not been used for homing for over a century (Levi \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). The capuchine is an old breed (300\u0026thinsp;+\u0026thinsp;years old, (Levi \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1965\u003c/span\u003e)) selected for an upturned ruff of feathers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) and comes in a diverse range of colours. Last, the Norwich cropper, was selected for an exaggerated sexual display (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) including elongated legs and more upright posture and was established over a century ago (Levi \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1965\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll pigeons were weighed with a spring balance, deeply anaesthetized with an intracoelomic injection of sodium pentobarbital (2 mL/kg body weight) and then perfused transcardially with 0.1 M phosphate buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde (PFA). The brains were removed and stored in 4% PFA at 4\u0026deg;C for 1\u0026ndash;2 weeks. Brains were weighed, cryoprotected in 30% sucrose PBS solution, transferred into an antifreeze solution (Hoffman and Le \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and stored at -20\u0026deg;C until embedding and sectioning.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eBrains were embedded in gelatin blocks and sectioned coronally on a freezing stage microtome at 40 \u0026micro;m thickness. All sections were collected in PBS\u0026thinsp;+\u0026thinsp;1% sodium azide solution in multi-well plates and mounted in 1:4 series onto gelatinised slides. Once dry, sections were washed in chloroform and stained for Nissl substance using thionin acetate followed by a graded ethanol series. Sections were cleared in Hemo-De (Thermo Fisher Scientific, HD150A) and coverslipped with Permount (Thermo Fisher Scientific, #SP15500).\u003c/p\u003e \u003cp\u003eEighteen to 22 sections equally spaced throughout the rostrocaudal extent of HF were immunolabeled for the neuron specific antigen, NeuN (Mullen et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Note that these same sections were also used for the septum. Sections were rinsed in PBS (pH 7.4), then incubated in 10% normal goat serum (Jackson ImmunoResearch, 005-000-121) in PBS\u0026thinsp;+\u0026thinsp;0.025M Triton (PBST) for 1 hour. The sections were then incubated in a monoclonal mouse anti-NeuN primary antibody (clone A60, Sigma Aldrich, MAB377) at a 1:1000 dilution in PBST on a shaker plate for 24 hours at room temperature. The sections were rinsed again in PBS and then incubated for 4 hours in fluorescein (FITC) goat anti-mouse secondary antibody (Jackson ImmunoResearch, 111-095-144) at a 1:200 dilution in PBST on a shaker plate at room temperature. Finally, the sections were rinsed and mounted onto gelatinised slides.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStereology\u003c/h2\u003e \u003cp\u003eAll volumetric measurements and neuron counts were made using unbiased stereology (West \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Volumetric measurements were made on a Zeiss Axio Imager M2 microscope using the Cavalieri Estimator Probe in StereoInvestigator\u0026trade; with a 400 \u0026micro;m grid size for all regions of interest of the Nissl stained series. Regions of interest (telencephalon, HF, septum) were differentiated following the boundaries shown in stereotaxic atlases of the pigeon (Karten and Hodos \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1967\u003c/span\u003e) and chick (Puelles et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Quantification of total brain and telencephalon were taken using a 1x objective lens; all other regions were quantified using a 2.5x objective lens. Coefficients of error (Gunderson, m\u0026thinsp;=\u0026thinsp;1) for all volumes were \u0026lt;\u0026thinsp;0.055.\u003c/p\u003e \u003cp\u003eNeuron numbers were estimated using the optical fractionator method (West \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) as implemented in StereoInvestigator\u0026trade;. For HF analysis, the following parameters were used across all mounted sections (15\u0026ndash;20 sections per specimen): a grid spacing of 650 \u0026micro;m, a grid size of 40 \u0026micro;m, a dissector zone of 15 \u0026micro;m, and upper and lower guard zones of 5 \u0026micro;m. For the septum, five to ten sections spanning its rostrocaudal extent were sampled using the following parameters: a grid spacing of 350 \u0026micro;m, a grid size of 40 \u0026micro;m, a dissector zone of 15 \u0026micro;m, and upper and lower guard zones of 5 \u0026micro;m. Neuron counts were completed using a 40x immersion oil lens on a Zeiss Axio Imager M2 microscope. Coefficients of error (Gunderson, m\u0026thinsp;=\u0026thinsp;1) for all cell counts were \u0026lt;\u0026thinsp;0.10 for all specimens. Means and standard deviations of data for all breeds are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAverage (\u0026plusmn;\u0026thinsp;standard deviation) values for all of the neuroanatomical measurements made of the eight pigeon breeds examined. Sample sizes are provided for each breed in brackets below each value.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeasurement\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHoming\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFeral\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHighflyer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRoller\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCapuchine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eShow Roller\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShow Homer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNorwich Cropper\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTelencephalon volume (mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e992.09\u0026thinsp;\u0026plusmn;\u0026thinsp;136.54\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e977.14\u0026thinsp;\u0026plusmn;\u0026thinsp;158.02\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e923.53\u0026thinsp;\u0026plusmn;\u0026thinsp;62.29\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e907.18\u0026thinsp;\u0026plusmn;\u0026thinsp;71.94\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e960.91\u0026thinsp;\u0026plusmn;\u0026thinsp;114.82\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e880.49\u0026thinsp;\u0026plusmn;\u0026thinsp;87.07\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e928.31\u0026thinsp;\u0026plusmn;\u0026thinsp;75.29\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1166.33\u0026thinsp;\u0026plusmn;\u0026thinsp;69.53\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHippocampal formation volume (mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.15\u0026thinsp;\u0026plusmn;\u0026thinsp;8.47\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.72\u0026thinsp;\u0026plusmn;\u0026thinsp;5.79\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.04\u0026thinsp;\u0026plusmn;\u0026thinsp;7.33\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57.31\u0026thinsp;\u0026plusmn;\u0026thinsp;3.43\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e57.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.78\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e55.17\u0026thinsp;\u0026plusmn;\u0026thinsp;7.54\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e56.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e61.27\u0026thinsp;\u0026plusmn;\u0026thinsp;6.22\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of hippocampal neurons\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,426,353\u0026thinsp;\u0026plusmn;\u0026thinsp;593,592\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,072,185\u0026thinsp;\u0026plusmn;\u0026thinsp;255,997\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,276,222\u0026thinsp;\u0026plusmn;\u0026thinsp;245,974\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,348,679\u0026thinsp;\u0026plusmn;\u0026thinsp;193,011\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1,328,841\u0026thinsp;\u0026plusmn;\u0026thinsp;587,279\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1,851,884\u0026thinsp;\u0026plusmn;\u0026thinsp;333,582\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1,853,808\u0026thinsp;\u0026plusmn;\u0026thinsp;172,313\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2,428,458\u0026thinsp;\u0026plusmn;\u0026thinsp;442,159\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHippocampal formation neuron density\u003c/p\u003e \u003cp\u003e(#neurons/ mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37,601\u0026thinsp;\u0026plusmn;\u0026thinsp;6,925\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18,708\u0026thinsp;\u0026plusmn;\u0026thinsp;4,657\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25,288\u0026thinsp;\u0026plusmn;\u0026thinsp;5,162\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23,538\u0026thinsp;\u0026plusmn;\u0026thinsp;2,970\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22,720\u0026thinsp;\u0026plusmn;\u0026thinsp;8,483\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33,787\u0026thinsp;\u0026plusmn;\u0026thinsp;6,117\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32,745\u0026thinsp;\u0026plusmn;\u0026thinsp;3,532\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e39,980\u0026thinsp;\u0026plusmn;\u0026thinsp;8,859\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeptum volume (mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of septal neurons\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e337,330\u0026thinsp;\u0026plusmn;\u0026thinsp;125,379\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e222,269\u0026thinsp;\u0026plusmn;\u0026thinsp;76,782\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e409,978\u0026thinsp;\u0026plusmn;\u0026thinsp;94,782\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e225,684\u0026thinsp;\u0026plusmn;\u0026thinsp;120,630\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e468,748\u0026thinsp;\u0026plusmn;\u0026thinsp;97,394\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e402,362\u0026thinsp;\u0026plusmn;\u0026thinsp;70,850\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e585,469\u0026thinsp;\u0026plusmn;\u0026thinsp;72,107\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeptum neuron density\u003c/p\u003e \u003cp\u003e(#neurons/ mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32,549\u0026thinsp;\u0026plusmn;\u0026thinsp;11,870\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21,908\u0026thinsp;\u0026plusmn;\u0026thinsp;9,152\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42,036\u0026thinsp;\u0026plusmn;\u0026thinsp;14,681\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21,851\u0026thinsp;\u0026plusmn;\u0026thinsp;11,860\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36,475\u0026thinsp;\u0026plusmn;\u0026thinsp;10,842\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e31,557\u0026thinsp;\u0026plusmn;\u0026thinsp;70,850\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e40,612\u0026thinsp;\u0026plusmn;\u0026thinsp;7890\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed in Jamovi (The jamovi project \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Absolute values were analysed using one-way analyses of variance (ANOVAs) and Tukey\u0026rsquo;s honestly significant difference (HSD) post-hoc tests. Relative values were analysed using analyses of covariance (ANCOVAs) of log-transformed data and Tukey\u0026rsquo;s HSD post-hoc tests. Covariates were body mass, whole brain volume (minus region of interest), or telencephalon volume (minus region of interest). Interaction effects (breed x covariate) were not significant across all of our analyses, indicating no significant differences in slopes among breeds. Interaction effects were therefore removed from all final ANCOVA models.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHippocampal formation (HF)\u003c/h2\u003e \u003cp\u003eAbsolute HF volume differed significantly among breeds (F\u0026thinsp;=\u0026thinsp;2.87, df\u0026thinsp;=\u0026thinsp;7, 43, p\u0026thinsp;=\u0026thinsp;0.015, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Homing pigeons had the largest HF volumes, whereas rollers had the smallest HF volumes. A Tukey\u0026rsquo;s HSD post-hoc test revealed that homers had significantly larger HF volumes than highflyers, but no other significant differences were detected among the other breeds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHF volume varied significantly with telencephalon volume (F\u0026thinsp;=\u0026thinsp;12.88, df\u0026thinsp;=\u0026thinsp;1, 42, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and there was a significant difference among breeds (F\u0026thinsp;=\u0026thinsp;2.73, df\u0026thinsp;=\u0026thinsp;7, 42, p\u0026thinsp;=\u0026thinsp;0.02). As with the analysis of absolute volumes, the post-hoc test indicated that homing pigeons have relatively larger HF volumes than highflyers, but no other significant differences.\u003c/p\u003e \u003cp\u003eIn contrast to volumes, the absolute number of HF neurons varied far more across breeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Norwich croppers and homing pigeons had the most neurons (both means\u0026thinsp;=\u0026thinsp;2.4 x 10\u003csup\u003e6\u003c/sup\u003e), and feral pigeons had the fewest neurons, less than half of that of homing pigeons (1.1 x 10\u003csup\u003e6\u003c/sup\u003e). Accordingly, our analyses found a significant difference across breeds in HF neuron number (F\u0026thinsp;=\u0026thinsp;12.77, df\u0026thinsp;=\u0026thinsp;7, 43, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Post-hoc tests revealed that homing pigeons and Norwich croppers had significantly more neurons than feral, highflyer, roller, and capuchine pigeons. Conversely, feral pigeons had significantly fewer neurons than homing, show roller, show homer, and Norwich cropper pigeons.\u003c/p\u003e \u003cp\u003eWhen we compared the number of HF neurons to HF volume, we found a similar pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The number of HF neurons increased with HF volume (F\u0026thinsp;=\u0026thinsp;8.26, df\u0026thinsp;=\u0026thinsp;1, 42, p\u0026thinsp;=\u0026thinsp;0.006), but there was also a significant difference across breeds (F\u0026thinsp;=\u0026thinsp;10.94, df\u0026thinsp;=\u0026thinsp;7, 42, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Specifically, our post-hoc tests showed that homing pigeons and croppers had significantly more neurons, relative to HF volume, than feral, highflyer, roller, and capuchine pigeons whereas feral pigeons had significantly fewer neurons than homing, highflyer, show roller, show homer, and cropper pigeons.\u003c/p\u003e \u003cp\u003eLast, HF neuronal density also differed greatly among breeds (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Norwich cropper and homing pigeons had the highest HF neuron densities (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and feral pigeons had the lowest neuron density, again less than half of that of homing pigeons (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These differences in HF neuron number and density between homing and feral pigeons are even apparent when looking at the sections; homing pigeons have far more neurons labeled (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) than feral pigeons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) in sections taken from the same location within HF. Similar to our previous analysis of neuron numbers relative to HF volume, HF neuronal density differed significantly among breeds (F\u0026thinsp;=\u0026thinsp;11.15, df\u0026thinsp;=\u0026thinsp;7, 43, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Post-hoc tests revealed that homing and cropper pigeons had significantly higher neuron densities than feral, highflyer, roller, and capuchine pigeons. Conversely, feral pigeons had significantly lower neuron densities than homing, show roller, show homer, and cropper pigeons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSeptum\u003c/h2\u003e \u003cp\u003eNorwich croppers had the largest septum volumes, whereas rollers, homers and highflyers shared the smallest septum volumes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Absolute septum volume differed significantly among breeds (F\u0026thinsp;=\u0026thinsp;6.64, df\u0026thinsp;=\u0026thinsp;6, 28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Norwich croppers had significantly larger septum volumes than feral, homer, highflyer, and roller pigeons in our post-hoc tests, but no other significant differences were found. Unlike HF volume, septum volume did not vary significantly with telencephalon volume (F\u0026thinsp;=\u0026thinsp;0.78, df. = 1, 27, p\u0026thinsp;=\u0026thinsp;0.39), but differed significantly among breeds (F\u0026thinsp;=\u0026thinsp;5.34, df\u0026thinsp;=\u0026thinsp;6, 27, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Post-hoc tests revealed homers and feral pigeons had relatively smaller septum volumes than croppers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn average, Norwich cropper pigeons had the most septal neurons, and feral pigeons had the fewest septal neurons (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the absolute number of septum neurons differed significantly among breeds (F\u0026thinsp;=\u0026thinsp;8.23, df\u0026thinsp;=\u0026thinsp;6, 28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); Norwich croppers had significantly more septal neurons than feral and roller pigeons and show rollers had more neurons than feral and roller pigeons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe number of neurons did not vary significantly with septum volume (F\u0026thinsp;=\u0026thinsp;0.06, F\u0026thinsp;=\u0026thinsp;1, 27, p\u0026thinsp;=\u0026thinsp;0.81), but differed significantly among breeds (F\u0026thinsp;=\u0026thinsp;5.15, df\u0026thinsp;=\u0026thinsp;6, 27, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Post-hoc tests revealed that feral and roller pigeons had relatively fewer neurons than croppers and show rollers.\u003c/p\u003e \u003cp\u003eFinally, we also compared neuron density by dividing neuron number by septum volume. Highflyer and Norwich cropper pigeons had the highest septum neuron densities, and feral and roller pigeons had the lowest septum neuron densities (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Overall, breeds differed significantly in septal neuron density (F\u0026thinsp;=\u0026thinsp;2.79, df\u0026thinsp;=\u0026thinsp;6, 28, p\u0026thinsp;=\u0026thinsp;0.03), but the post-hoc tests yielded no significant differences. Although the neuronal density appeared to differ among some breed pairs, such as the homing pigeon and cropper sections shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (F and G, respectively), based on the boxplot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) there is considerable overlap among breeds in neuronal density.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSimilar to previous studies (Ebinger and Lohmer \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Rehk\u0026auml;mper et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rehkamper et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) we found that homing pigeons do not differ greatly in relative HF volume from other pigeon breeds, including feral pigeons. However, the data partially supported our prediction that homing pigeons would have more HF neurons and higher neuronal densities than other breeds. Furthermore, while there were some among-breed differences in septum neuroanatomy, homing pigeons largely did not differ from other breeds. As we discuss below, the relationship between differences in HF or septal quantitative anatomy and behaviour requires testing, but our data suggest that neuron numbers could be important for the remarkable navigational abilities of homing pigeons.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDifferences in septum anatomy across breeds\u003c/h2\u003e \u003cp\u003eAs mentioned previously, the septum plays a significant role in modulating agonistic and courtship behaviour in birds (Goodson et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e; Ramirez et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Corrales Parada et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Taziaux et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), but it is also connected directly with the HF (Atoji and Wild \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and involved in spatial and working memory (Coppola 2021; Peterson and Bingman \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Despite the role of the septum in some aspects of memory, homing pigeons have smaller septal volumes and fewer neurons than most show breeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). From this, we conclude that if the septum is contributing to homing behaviour, it is not dependent on more neurons or a larger volume.\u003c/p\u003e \u003cp\u003eUnexpectedly, croppers had larger septal volumes and more septal neurons than several other breeds whereas feral pigeons have smaller septal volumes and fewer neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. A-D). As discussed previously, croppers were selected for exaggerated courtship displays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) (Levi \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). Given the role of the septum in regulating sexual and courtship behaviour (Goodson et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e; Taziaux et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Goodson et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2004b\u003c/span\u003e), it is possible that selection for these exaggerated displays resulted in neuroanatomical changes in the septum. How a larger septum or more septal neurons might be associated with courtship display is unclear though as much of the interspecific variation in social behaviour associated with the septum is typically due to changes in receptor and nonpeptide expression (Goodson et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Goodson et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). With respect to the feral pigeons, we speculate that a smaller septum with fewer neurons may be an effect of chronic stress. Feral pigeons are exposed to many stressors not experienced by domesticated pigeons (e.g., predation, variable food availability, lethal control efforts (Murton et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1972\u003c/span\u003e)) and chronic stress can have detrimental effects on neurogenesis in birds (Smulders \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Brenowitz and Larson \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Further, this stress hypothesis could also explain the relatively neuron poor HF of feral pigeons (see below).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDifferences in hippocampal formation anatomy across breeds\u003c/h2\u003e \u003cp\u003ePrevious studies of hippocampus size across pigeon breeds focused on Hp and not the entire HF and lacked data on neuron numbers and densities (Ebinger and Lohmer \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Rehk\u0026auml;mper et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rehkamper et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1988\u003c/span\u003e), making it difficult to ascertain to what extent HF quantitative anatomy differs between homing and other pigeons. Although homing pigeons tended to have relatively and absolutely large HF volumes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B), few significant differences were detected, which is consistent with (Rehk\u0026auml;mper et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). More importantly, we show that there are significant differences in neuron numbers and densities such that homing pigeons tend to have more neurons than feral, sporting, and some show breeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-G). There are at least a couple of reasons why homing pigeon HF size and neuron numbers differ from some, but not all, other domestic breeds. First, domestic pigeon breeds are frequently interbred (Hiatt and Esposito \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Levi \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Pacheco et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, show homers are derived from and often backcrossed with homing pigeons and American show rollers are a recently developed breed that often involves crossing with other breeds, including homing pigeons. Pigeon fanciers, apart from some racing pigeon lines, also do not publish breeding records so purebred registries are lacking. As a result, pigeon breeds are not as genetically distinct (Pacheco et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) as purebred dog breeds (Parker et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) or other domesticated animal breeds (Rasali et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). If some neuroanatomical traits have a strong genetic basis (e.g., large HF), then mixing of different pigeon breeds could lead to similarities in HF size across breeds. Second, it is possible that changes in neuronal density have accompanied selection for traits other than homing. For example, selection for the exaggerated sexual displays of croppers could be associated with changes in androgen levels and/or steroid hormone receptors, resulting in higher survivorship of new neurons (Balthazart and Ball \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Neuroanatomical changes in the HF of some breeds could therefore be a by-product of selection for traits other than homing.\u003c/p\u003e \u003cp\u003eInterestingly, the biggest difference in neuron numbers and densities was between homing and feral pigeons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-G). Feral pigeons are largely derived from homing pigeons (Stringham et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Giunchi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), but experience markedly different selection pressures. Feral pigeons have similar mortality rates to wild pigeons (both rock doves and \u003cem\u003eColumba palumbus\u003c/em\u003e) and must search for food and water, recognize and avoid predators, and cope with environmental variability (Murton et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1972\u003c/span\u003e), all in stark contrast to the captive environments of homing pigeons and other domesticated pigeons. The stressors that face feral pigeons could mean that they experience chronic stress, which significantly impairs neurogenesis and new neuron survivorship in HF (Gualtieri et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Robertson et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Smulders \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This would then lead to fewer neurons and lower neuronal density, as observed in our data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Whether there are behavioural consequences of having fewer HF neurons remains to be tested. Feral pigeons do not home as effectively as homing pigeons, but this is thought to be a motivational issue as they tend to stop more frequently and associate with other pigeons (Edrich and Keeton \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Chelazzi and Pineschi \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). That said, if these differences in HF anatomy arise from stress, then the homing-feral pigeon difference should be lessened in feral pigeons raised in captivity.\u003c/p\u003e \u003cp\u003eIn stark contrast to the feral pigeons, homing pigeons tended to have more and denser neurons than the other breeds sampled (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). It is tempting to conclude that having more neurons and higher neuronal density in the HF provides some benefit to spatial memory and cognition in pigeons. Data from chickadees (\u003cem\u003ePoecile\u003c/em\u003e spp.) indicates that populations and individuals with more neurons tend to perform better on spatial memory tasks, providing a link between performance and neuron numbers (Pravosudov and Roth II \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, there are several unknowns with respect to homing that prevent us from making a direct link between neuron numbers and cognition from our data. First, data are lacking on homing performance across pigeon breeds. Homing experiments with feral pigeons and wild rock doves (Baldaccini et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Edrich and Keeton \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Chelazzi and Pineschi \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1974\u003c/span\u003e) suggest that they lack the motivation to fly long distances to return home. Data on homing rate (i.e., percentage of released birds that return to the loft) shown in (Shao et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) indicate that some non-homing breeds are largely incapable of returning from distances greater than 10km, but detailed tracking data as used in modern homing experiments (e.g., (Gagliardo et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gagliardo et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)) were lacking. These non-homing breeds could have the same issue with motivation as feral pigeons (Chelazzi and Pineschi \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Edrich and Keeton \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1977\u003c/span\u003e), not be physically capable, or lack the cognitive ability to home like homing pigeons. Without behavioural testing across breeds, we simply do not know. Second, we do not know what role training has on HF anatomy in pigeons. Restricting homing pigeons to a loft and not providing them with homing experience results in a smaller Hp (Cnotka et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), which likely arises from fewer neurons but this has yet to be tested. That said, flight experience appears to be insufficient to drive changes in neuron numbers because highflyer and roller pigeons all fly regularly outdoors, yet all have fewer HF neurons and lower HF neuron density than homing pigeons. Last, much of the research on pigeon homing over the past several decades has focused primarily on sensory processing (Wiltschko and Wiltschko \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wallraff \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gagliardo \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with the cognitive components of homing not researched as extensively (Bingman \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). HF activity certainly increases during homing, especially within the APH (Shimizu et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and hippocampal lesions impair homing (Bingman 2005, 1984, 1990; Gagliardo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Gagliardo et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Gagliardo et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Herold et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), but how the HF contributes to the homing pigeon\u0026rsquo;s cognitive map has remained elusive (Bingman \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ben-Tov and Gutfreund \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nevertheless, based on data from food caching species (Pravosudov and Roth II \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Croston et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and contrasts between caching and non-caching songbirds (Payne et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sherry \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), one would predict that homing pigeons would have some cognitive advantages over the other breeds examined. Although place cells have proved challenging to locate in pigeons, more neurons could allow new memories to form without interfering with old memories (Payne et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) or increase the speed of spatial memory processing. Of course, these are speculative and require testing, but it is unlikely that the addition of many mature neurons in the HF is unrelated to some difference in function.\u003c/p\u003e \u003cp\u003eThe relationship between neuron numbers and spatial cognition in pigeons may be unclear, but our data demonstrates clearly that breeds vary in neuronal density. Almost all of the literature on the effects of domestication on the brain and variation among domesticated breeds has focused on volumes of brain regions (Kruska \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hecht et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rehk\u0026auml;mper et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rehkamper et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Rehkamper et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Ebinger \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1972\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1975\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Ebinger and Lohmer \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Ebinger and Rohrs \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Brusini et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), with little to no data on neuron numbers (Racicot et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our data is the first to show that artificial selection can drive significant changes in neuronal density, in stark contrast to previous claims (Jardim-Messeder et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and without a major increase in brain region volume. The fact that brain region volumes and neuronal density can vary significantly among breeds within a species has significant implications for studying the neurobehavioural effects of domestication and artificial selection. Importantly, inter-breed and wild-domesticate comparisons need to move beyond volumetrics and examine neuronal density, neuron size and morphology, and connectivity to better understand the effects of different selection regimes on brain anatomy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe wish to thank the Lethbridge Homing Pigeon Racing Club, Facilities staff at the University of Lethbridge, various pigeon breeders in Alberta and Saskatchewan, and the Brinkman farm for providing pigeons for use in this study, and Dr. Maurice Needham for assisting with microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupport for this study was provided by scholarships from the University of Lethbridge to AG and JKR and grants from the Natural Sciences and Engineering Research Council (NSERC), Canada Research Chairs Program, and Canada Foundation for Innovation to ANI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the data reported herein is provided in supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e: All the authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch involving animals/ethical approval:\u003c/strong\u003e All of the procedures outlined herein adhered to the Canada Council for Animal Care Guidelines and were approved by the University of Lethbridge Animal Welfare Committee (Protocol #2011).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlleva E, Baldaccini NE, Foa A, Visalberghi E (1975) Homing behaviour of the rock pigeon. 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J R Soc Interface 16(158):20190295. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1098/rsif.2019.0295\u003c/span\u003e\u003cspan address=\"10.1098/rsif.2019.0295\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"brain-structure-and-function","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bsaf","sideBox":"Learn more about [Brain Structure and Function](https://www.springer.com/journal/429)","snPcode":"429","submissionUrl":"https://submission.nature.com/new-submission/429/3","title":"Brain Structure and Function","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"(4 to 6 words): hippocampus, artificial selection, septum, pigeon, neurons","lastPublishedDoi":"10.21203/rs.3.rs-4459634/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4459634/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe artificial selection for specific behavioural and physical traits domesticated animals has resulted in a wide variety of breeds. One of the most widely recognized examples of behavioural selection is the homing pigeon (\u003cem\u003eColumba livia\u003c/em\u003e), which has undergone intense selection for fast and efficient navigation, likely resulting in significant anatomical changes to the hippocampal formation. Previous neuroanatomical comparisons between homing and other pigeon breeds yielded mixed results, but only focused on volumes. We completed a more systematic test for differences in hippocampal formation anatomy between homing and other pigeon breeds by measuring volumes, neuron numbers and neuron densities in the hippocampal formation and septum across homing pigeons and seven other breeds. Overall, we found few differences in hippocampal formation volume across breeds, but large, significant differences in neuron numbers and densities. More specifically, homing pigeons have significantly more hippocampal neurons and at higher density than most other pigeon breeds, with nearly twice as many neurons as feral pigeons. These findings suggest that neuron numbers may be important component of homing behaviour in homing pigeons. Our data also provide the first evidence that neuronal density can be modified by artificial selection, which has significant implications for the study of domestication and interbreed variation in anatomy and behaviour.\u003c/p\u003e","manuscriptTitle":"The quantitative anatomy of the hippocampus in homing pigeons and other pigeon breeds: implications for spatial cognition.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-04 13:35:07","doi":"10.21203/rs.3.rs-4459634/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-18T13:40:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-03T20:35:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-27T14:10:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320248967871562167980338990091973688878","date":"2024-06-12T10:24:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80424434892969182845065253428215061325","date":"2024-06-12T07:32:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281939165594202092443465982049786081690","date":"2024-06-12T06:11:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275022129107269464287661978901807269488","date":"2024-06-12T05:18:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-12T04:51:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-26T12:51:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-22T15:07:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Brain Structure and Function","date":"2024-05-22T09:06:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"brain-structure-and-function","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bsaf","sideBox":"Learn more about [Brain Structure and Function](https://www.springer.com/journal/429)","snPcode":"429","submissionUrl":"https://submission.nature.com/new-submission/429/3","title":"Brain Structure and Function","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4eedb0d4-073a-406d-ba9d-314e85e4b976","owner":[],"postedDate":"June 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:00:58+00:00","versionOfRecord":{"articleIdentity":"rs-4459634","link":"https://doi.org/10.1007/s00429-024-02882-5","journal":{"identity":"brain-structure-and-function","isVorOnly":false,"title":"Brain Structure and Function"},"publishedOn":"2024-12-17 15:57:22","publishedOnDateReadable":"December 17th, 2024"},"versionCreatedAt":"2024-06-04 13:35:07","video":"","vorDoi":"10.1007/s00429-024-02882-5","vorDoiUrl":"https://doi.org/10.1007/s00429-024-02882-5","workflowStages":[]},"version":"v1","identity":"rs-4459634","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4459634","identity":"rs-4459634","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}