The hippocampal formation in migratory birds seems to be essential for navigation based on recognition of landmarks around breeding and wintering sites and resting and feeding stopover sites during migration (Bingman & MacDougall-Shackleton, 2017). In previous studies, we investigated hippocampal morphological changes associated with long-distance migration (Carvalho-Paulo et al., 2017; de Morais Magalhães et al., 2017; Diniz et al., 2016; Henrique et al., 2020; Mendes De Lima et al., 2019) and demonstrated that the migratory route affects astrocyte morphology and number, as well as neurogenesis and hippocampal volume, suggesting that contrasting factors along the migratory journey affect these elements differentially. A single study using a stereological approach has provided an estimation of the total number of NeuN-immunolabeled neurons and a reconstruction of the three-dimensional microglial morphology in the hippocampal formation (HF) of the spotted sandpiper (A. macularius; Diniz et al., 2016). However, no information is available related to the plasticity of inhibitory neurons in the HF of this species in the wintering period.

Previous work in birds has shown that calcium-binding protein (parvalbumin; PV) is a neuroplastic marker that increases as a result of multisensory stimulation in regions responsible for learning and memory, including the hippocampus (Barkan et al., 2017) and the songbird vocal-control nuclei (Brenowitz & Larson, 2015; Zengin-Toktas & Woolley, 2017). Seasonal changes in parvalbuminergic neurons and their perineuronal nets have been described in vocal-control nuclei, with differential expression in different songbird species, and these changes have been proposed as a key cellular mechanism mediating species variation (Cornez et al., 2017; Cornez, Collignon, Müller, Ball, et al., 2020; Cornez, Collignon, Müller, Cornil, et al., 2020). The numbers of this neuronal subpopulation are modified in the face of social (Rytova et al., 2019), multisensory, and cognitive stimuli both in mammals (Arida et al., 2007; Caroni, 2015a, 2015b; Donato et al., 2015; Gainey & Feldman, 2017; Murueta-Goyena et al., 2018; Placencia et al., 2019) and in birds (Chaudhury et al., 2006; Cornez et al., 2017; Wada et al., 2004). Although detailed information is available regarding the participation of parvalbuminergic neurons in hippocampus-dependent tasks in mammals (Capogna et al., 2020), no information is available about a role for these neurons in long-distance migratory behavior in shorebirds. In the present report, employing a parvalbuminergic neuronal subpopulation marker, we searched for progressive changes in brain plasticity during the wintering period in two distinct neuroanatomical and functional regions of A. macularius. We focused our attention on the wintering period of A. macularius in the mangroves of the Amazon River estuary in the state of Pará, Brazil. Spotted sandpipers migrate singly or in small groups to their wintering grounds, which extend from the extreme southern United States to southern South America (Billerman et al., 2020). Previous data based on field observations, museum collections, ringing information, and censuses have revealed a large distribution of A. macularius in the Brazilian territory, including greater numbers along the coasts of the states of Amapá, Pará, Maranhão, and Rio Grande do Norte, where higher density is found in September and between November and April (Serrano, 2010).

To identify a differential neuroplastic response, we also estimated the number of parvalbuminergic neurons in the magnocellular nucleus of the tectal isthmus (IMC), which receives organized projections from the overlying layers of the tectum opticum and projects its axons highly divergently back to the tectum (Faunes et al., 2013). Visuotopic maps representing the visual field and allowing for spatial location of moving objects warranting attention throughout the migratory process must remain unchanged after their construction, which is largely genetically defined (Huberman et al., 2008; Karten, 2015). Therefore, we predicted that by comparing these two regions in the same period, we would find significant differences in estimates of parvalbuminergic neuron numbers in the hippocampus but not in the IMC.

Methods

Birds were captured in four different windows (newcomers: August–October; Migratory Rest 1: November–January; Migratory Rest 2: February–April; and premigration: May–July) of the wintering period in the island of Otelina (0° 45' 42.57" S 46° 55' 51.86" W) in the tropical coastal zone of northern Brazil. However, in this study, we examined only birds collected in Migratory Rest 1 and premigration periods. These captures were performed under the approval of the university institutional ethics committee CEUA/UFPA No. 1840281116 and license No. 44551-5 from Instituto Brasileiro do Meio Ambiente–IBAMA, which regulates Brazilian wildlife use for research. At dusk, mist nets (12 m × 2.5 m) were opened for capture. The nets were inspected every 10 minutes for the removal of the captured birds. Migratory Rest 1 and Premigration individuals were captured on December 12 and May 17, respectively.

Perfusion and immunohistochemistry

All perfusions were performed during the field work immediately after capture under deep isoflurane anesthesia. All efforts were made to minimize the number of animals used and their stress and discomfort. The animals were perfused transcardially with heparinized 0.9% saline solution (1 ml/L) for 10 minutes, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.2–7.4, for 10 minutes. The beginning of saline flow was initiated after the jugular vein was cut with an incision in the right side of the neck, allowing the brain to be better permeated by saline and 4% paraformaldehyde. The brains were dissected and postfixed in 4% paraformaldehyde overnight at 4 °C, stored in 0.05 M phosphate-buffered saline, and sliced using a Vibratome (Leica VT1000S) into sections 100 μm thick to yield four anatomical serial sections.

The selected sections were washed with 0.1 M (pH 7.2–7.4) PB prior to incubation for 60 minutes in 0.2 M boric acid (pH 9.0) at 70°C. Nonspecific binding sites were blocked by incubation of sections in 10% casein for 60 minutes. Incubation of the sections in the primary antibody against PV (1:200 dilution, Millipore, mab code 1572) for immunostaining occurred for 3 days at 4°C with gentle agitation. Endogenous peroxidase was inactivated by incubation of sections in 0.3% hydrogen peroxide in PB saline for 15 minutes. After washes, the sections were incubated for 60 minutes in the avidin–biotin–peroxidase complex (Vector ABC Elite kit, Vector Laboratories, Burlingame, CA, USA). Peroxidase enzyme activity was detected using 3'3-diaminobenzidine (Sigma) and β-d-glucose/glucose oxidase reaction (Shu et al., 1988). After the reaction product was formed, a dark blue–gray precipitate was generated at the antigen–antibody interaction sites, and the reaction was stopped in 0.1 M PB. The sections were mounted on gelatinized glass slides and dehydrated in an ethanol series (70%, 80%, 90%, 100%, and 100%) followed by a xylene series.

Stereological cell counts

For cell counting, we used an optical microscope (Eclipse 80i, Nikon) with a motorized stage and analog-to-digital converters (MAC6000 system, Ludl Electronic Products, Hawthorne, NY, USA). Three-dimensional digital conversions of the x, y, and z axes of each digitized point were stored in a computer that controlled the movements of the motorized stage with the aid of specialized software (Stereo Investigator, Microbrightfield, Williston, VT, USA). To avoid ambiguity in identifying objects of interest and ensure greater accuracy in counting, the 4.0× objective was replaced by another Plan Fluor 100× objective (N.A. = 1.3; df = 0.2 μm; Nikon, Japan) for counting.

To estimate cell numbers in the region of the IMC and HF, we used an optical fractionator, which allows estimation of the total cells in the region of interest. For this purpose, the number of neurons within the counting blocks arranged in the area of interest was multiplied by the probability values of the sample fractions using the following equation:

$$ \mathrm{N}=\Sigma \mathrm{Q}\times 1/\mathrm{ssf}\times 1/\mathrm{asf}\times 1/\mathrm{tsf}, $$
(1)

where N is the estimate of the total number of cells, ΣQ is the sum of counted neurons, ssf is the sample fraction of the number of sections, asf is the sample fraction of the section area, and tsf is the sample fraction of the section thickness (West, 2002).

Table 1 shows stereological parameters and volume estimates of the IMC of five birds captured in two different time windows of the wintering period (Migratory Rest 1 and premigration). Table 2 shows stereological parameters and the total number of PV neurons in the HF of five individuals of A. macularius captured in the Migratory Rest 1 and premigration time windows of the wintering period.

Table 1 Stereological parameters for counting parvalbumin neurons and volume estimates of the magnocellular isthmus nuclei of 5 individuals of Actitis macularius captured in two different time windows (Migratory Rest 1 and Pre-migration)
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Table 2 Stereological parameters for counting parvalbumin neurons and volume estimates of the hippocampal formation of Actitis macularius in Pre-migration and Migratory Rest 1 time windows
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Figure 1 illustrates a random and systematic sampling approach for stereological counts and volume estimate. We used the optical fractionator to estimate the total number of cells of the areas of interest (HF and IMC). This method relies on thick sections for estimating the total number of cells from the number sampled with a systematic randomly sampled set of unbiased digitally defined counting boxes, covering the entire area of interest. A uniform distance between unbiased selected counting boxes, located over preselected grids covering the whole area of interest, is part of the fractionator method. To measure the HF and IMC volumes, we also used StereoInvestigator software (Microbrightfield, Inc.), which relies on a standard stereological method to estimate volumes based on the Cavalieri procedure, and the values for statistical analyses were extracted from a PV-immunolabeled anatomical series of sections. The volumes were measured beginning from the first tissue section of the IMC and HF through the last section of these areas, as previously suggested (Roth & Pravosudov, 2009). Figure 2 shows the IMC and the objects of interest for cell counting (immunolabeled PV neurons). Figure 3 shows the area (HF) and the objects of interest (PV-immunolabeled neurons) from A. macularius individuals captured in the Migratory Rest 1 and premigratory time windows of the wintering period.

Fig. 1
figure 1

Optical fractionator approach for counting cells and Cavalieri volume estimate. a–c Magnocellular isthmus nucleus (IMC). d–f Hippocampal formation (HF). Counting boxes indicating the rejection (red lines) and acceptance (green lines) regions (b and e). Pink-shaded areas indicate the area of interest (a) = IMC; (d) HF. (Color figure online)

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Fig. 2
figure 2

Photomicrographs of PV-immunolabeled sections to illustrate IMC from Migratory Rest 1 (pink) and premigration (blue) individuals. Dotted squares indicate PV-immunolabeled neurons inside the area of interest. (Color figure online)

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Fig. 3
figure 3

PV-immunolabeled sections from A. macularius captured before departure (premigration) and during Migratory Rest 1 to illustrate area and object of interests. Scale bars: a–c = 250 μm; d = 25 μm. (Color figure online)

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Statistical analysis

For comparisons between the experimental groups, we used parametric analysis, adopting two-tailed t tests for independent samples with a confidence interval of 95% or 99% (p < .05 or p < .01, respectively). Outliers were removed based on standard deviation.

The main variability in the present analysis was biological, with the ratio CE2/CV2 < 0.5, where CE is the estimated coefficient of error (coefficient of Sheaffer) and CV is the coefficient of variation. The ratio between CE2 and observed variance of the group, CV2, was less than 0.5; for details, see Slomianka and West (2005). To minimize possible sources of variation, all data were collected and analyzed with the same unbiased methodology, and all samples were obtained using the same tissue processing protocols. In this way, we expected to reduce nonbiological sources of error to acceptable levels.

Results

The number of PV-immunolabeled interneurons in the HF were greater in the premigration period than in the Migratory Rest 1 period (see Fig. 4; t = 5.77, p = .0004). In contrast, the number of PV-immunolabeled interneurons in the IMC counts did not differ between the two groups (92,305 ± 5,812 vs. 95,694 ± 4,028, mean ± SE; t = 1.072, p = .3151). The CBV2 (coefficient of biological variation) was the main factor responsible for more than 90% of the total variation in HF cell counts, suggesting little contribution of methodological error (see Table 3). The CBV2 (coefficient of biological variation) was the main factor responsible for almost all variation in IMC cell counts, suggesting a negligible contribution of methodological errors (see Table 4).

Fig. 4
figure 4

Graphic representation of the mean values of stereologically counted PV-immunolabeled neuron number (a) and volumes (b) of the HF of five individuals of A. macularius, captured during the premigration and Migratory Rest 1 windows of the wintering period. As compared with Migratory Rest 1, the HF of individuals captured during the premigration window showed a significant increase in PV-immunolabeled neurons

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Table 3 Stereological detailed results of parvalbumin counting neurons of the hippocampal formation of five individuals of Actitis macularius in Pre-migration (PM) and Migratory Rest 1 (MR1)
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Table 4 Estimate of the total number of parvalbumin labeled neurons of the magnocellular isthmus nuclei of five individuals of Actitis macularius captured in the two distinct time windows of the wintering period
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Discussion

Based on previous evidence that the HF is involved in learning and memory (Barkan et al., 2017) and social interaction (Rytova et al., 2019) and that IMC is essential for controlling head, neck, and eye movements (Faunes et al., 2013), we tested the hypothesis that PV neurons would increase in the HF and remain unchanged in the IMC during the wintering period of A. macularius. We found no significant difference in the total number of IMC PV neurons between the Migratory Rest 1 and premigration periods, but the total number of PV neurons was significantly greater in the HF in the premigration group compared with the HF in the Migratory Rest 1 group. We infer from these results that the greater number of parvalbuminergic neurons in the HF reflects adaptive changes in the hippocampal circuits involved in the learning and memory and social interaction necessary for migration back to the breeding grounds in the northern hemisphere.

Social interaction, PV neurons, spatial learning and memory, and migration

Social relationships between birds of the same group may facilitate collective behavior in following similar interaction rules to form flocks and organize flights during migration. Indeed, within flocks, paired birds interact with fewer neighbors than unpaired birds and flap their wings more slowly, which may result in energy savings. In contrast, flocks with more paired birds may have reduced efficiency in information transfer through the flock (Ling et al., 2019). Because social relationships and learning and memory are influenced by PV neurons in the hippocampus, it is tempting to speculate that a significant increase in PV-immunolabeled neurons in the HF of the premigration group may be important in establishing social relationships among group members. It is important to highlight that A. macularius migrates singly or in small groups (Reed et al., 2013), and in the latter case, social interaction may increase efficiency of information transfer through the flock.

PV neurons are important for learning and memory, which includes landscape recognition along the journey and small-scale local visuospatial stimuli to recognize stopovers associated with the migratory process (Bingman & MacDougall-Shackleton, 2017). The greater numbers of these neurons in the HF in the premigration group suggest that PV neurons may be at least part of the adaptive response of the local hippocampal circuitry associated with these tasks. In agreement, previous reports have demonstrated that PV number is modified in the response to somatomotor, visuospatial, and cognitive stimuli, both in mammals (Arida et al., 2007; Caroni, 2015a, 2015b; Donato et al., 2015; Gainey & Feldman, 2017; Murueta-Goyena et al., 2018; Placencia et al., 2019) and in birds (Chaudhury et al., 2006; Cornez et al., 2017; Wada et al., 2004). As a neuroplastic marker in learning and memory, PV increases in birds in the hippocampus (Barkan et al., 2017) and songbird vocal-control nuclei (Brenowitz & Larson, 2015; Zengin-Toktas & Woolley, 2017) following multisensory stimulation. A reasonable, if speculative, hypothesis is that during the winter period, the physiological changes required for migration, including in glia and hippocampal neurons, must be achieved before the boreal summer (da Costa et al., 2020). These changes may support the spatial learning demands required to search for food stored as fat needed for the migratory flight, as well as to increase social interaction by improving the efficiency of information transfer between individuals flying in groups towards the northern hemisphere.

Limited plasticity, visual maps, and head, neck, and eye-movement control

In contrast to the HF, PV neurons in the IMC remained unchanged. Indeed, the number of PV neurons in this region, which receives organized projections from the overlying layers of the tectum opticum (Faunes et al., 2013) remained similar between the premigration and Migratory Rest 1 groups. Because these neurons are topographically organized, they need to be preserved to perform their functions. Thus, nonsignificant neuronal plasticity in this region is expected during the migratory process. Visuotopic maps representing the visual field in the tectum and their projections that command IMC neurons, allowing for spatial location of moving objects that warrant attention throughout the migratory process, therefore must remain unchanged. After their construction, which is largely genetically defined (Huberman et al., 2008; Karten, 2015), their circuits must be maintained with little plasticity.

Methodological limitations

We want to highlight that an essential problem with using two time points for the HF data is that we cannot differentiate whether the change we detected represents an increase in PV+ interneurons during premigration or a decrease during Migratory Rest 1. This potential conflation is an important limitation to be considered in future studies. Another issue that remains to be investigated is whether the change in PV neuron counts results from an increase in the number of interneurons (neurogenesis), more interneurons expressing PV, or both. Because we did not use other markers to identify and count new interneurons or measure the expression of PV, it is difficult to answer this question, which is another limitation of the present report.

To quantify the number of PV neurons in the HF and IMC nucleus, we applied the optical fractionator, an accurate method of quantification combining properties of an optical dissector and fractionator that has been used in a variety of studies to determine cell numbers in multiple brain regions. Although the optical fractionator is unaffected by histological changes or shrinkage, edge effects remain an important issue when performing comparative analysis between experimental groups (Glaser & Glaser, 2000; West, 2002; West et al., 1991). It is not possible to correct for edge effects in the Z plane, but a guard zone was equally applied to all counting sites to minimize this effect.

Conclusions

In the present report, we focused our attention on the wintering period of the long-distance migratory spotted sandpiper (A. macularius) and studied the progressive changes in brain plasticity in two central nervous system regions, employing a parvalbuminergic neuronal subpopulation marker. In this subpopulation of inhibitory neurons, hippocampal numbers are modified in the face of social, multisensory, and cognitive stimuli, both in mammals and in birds. We found that the numbers of PV cells in the HF was greater in birds captured during the premigration window compared with those captured during the Migratory Rest 1 period. We suggest that the greater number of parvalbuminergic neurons in the premigratory period reflects adaptive changes in hippocampal circuits needed for the long-distance journey back to the reproductive niches in the northern hemisphere.