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The brain of the tree pangolin (Manis tricuspis). VII. The amygdaloid body

Abstract Here, we describe the cytoarchitecture and chemoarchitecture of the amygdaloid body of the tree pangolin. Our definition of the amygdaloid body includes the pallial portions of the amygdala, and the centromedial group that is a derivative of the subpallium and part of the extended amygdala. The remainder of the extended amygdala is not described herein. Within the amygdaloid body of the tree pangolin, we identified the basolateral group (composed of the lateral, basal, and accessory basal amygdaloid nuclei), the superficial, or cortical nuclei (the anterior and posterior cortical nuclei, the periamygdaloid cortex, and nuclei of the olfactory tract), the centromedial group (the central amygdaloid nucleus and the medial nuclear cluster), and other amygdaloid nuclei (the anterior amygdaloid area, the amygdalohippocampal area, the intramedullary group, and intercalated islands). The location within and relative to each other within the amygdaloid body and the internal subdivisions of these groups were very similar to that reported in other mammalian species, with no clearly derived features specific to the tree pangolin. The only variation was the lack of an insular appearance of the intercalated islands, which in the tree pangolin were observed as a continuous band of neurons located dorsomedial to the basolateral group similar in appearance to and almost continuous with the intramedullary group. In carnivores, the closest relatives of the pangolins, and laboratory rats, a similar appearance of portions of the intercalated islands has been noted.


INTRODUCTION
Pangolins are generally nocturnal, solitary animals, but there are a range of behaviors that have been observed in captive specimens that could be classified as affective. These include territorial marking, defensive behaviors and postures, vocalizations, aggression, as well as reproductive and social behaviors such as courtship, chasing, mounting, copulation, and maternal interactions (Mohapatra & Panda, 2014;Zhang et al., 2020). These behaviors are observed frequently across mammalian species and indicate that affective states and behaviors processed and initiated by the pangolin brain, specifically the amygdaloid complex, are likely the result of similarly organized circuits, albeit these circuits being refined in such a way as to process affect in a manner relevant to pangolin life history and the survival of individual pangolins as members of their species (Rolls, 1999;Sah et al., 2003;Panskepp, 2011).
The amygdaloid complex, composed of the amygdaloid body in the rostral pole of the temporal lobe and the extended amygdala within the subpallium, is known to be involved in the processing of rage/anger, fear/anxiety, and lust/sexuality (Pankseep, 2011). Understanding the consistencies and variations in the organization of the amygdaloid complex of mammals may provide insights into affective behaviors and how they may be altered as species traverse independent evolutionary trajectories or when they inhabit and adapt to different environments (Rubenstein & Wrangham, 2014). The majority of our knowledge of the structure and function of the amygdaloid complex has emerged from studies of laboratory rodents, primates, and humans (e.g., Krettek & Price, 1978;Price et al., 1987;Pitkänen & Amaral, 1993a, b;Pitkänen et al., 1997;Kemppainen & Pitkänen, 2000), with few studies providing detailed analyses of rarely or less frequently studied mammals (e.g., Limacher-Burrell, et al., 2016;Limacher-Burrell et al., 2018;Pillay et al., 2021).
The amygdaloid body within the brain of the tree pangolin occupies the rostral pole of the temporal lobe, a location typical for mammals, has a volume that would be expected for a mammal with a brain mass of around 10 g (Imam et al., 2017), and exhibits a distinct nucleus of the lateral olfactory tract (Imam et al., 2018a); however, no comprehensive reports of the internal organization are available for either the amygdaloid body or the subpallial located extended amygdala in this species.
In the current study, we extend our observations on the anatomy of the central nervous system of the tree pangolin (Imam et al., 2017;Imam et al., 2018a, b;Imam et al., 2019a;Imam et al., 2019b;Imam et al., 2019c) by providing a detailed description of the nuclear and cortical organization of the amygdaloid body.
The amygdaloid complex of mammals contains both nuclear and cortical portions (within the amygdaloid body), as well as nuclei forming the extended amygdala (e.g., Alheid, 2003;de Olmos et al., 2004). Developmental gene expression studies have shown that the amygdaloid complex, the amygdaloid body and extended amygdala, derive from both pallial and subpallial domains, but in a complex manner (e.g., Puelles, 2017;Watson et al., 2017). In the current study, we describe the regions of the amygdaloid complex found within the rostral temporal pole (that we refer to as the amygdaloid body), including the nuclei forming the deep/basolateral group, the superficial/cortical-like/corticomedial nuclei, other amygdaloid nuclei (intramedullary group [IMG], amygdalohippocampal area [AHiA], intercalated cell masses [I], anterior amygdaloid area [AAA]), and the centromedial group. The regions assigned to the extended amygdala (apart from the centromedial group due to its topological continuity with pallial amygdala structures) such as the bed nuclei of the stria terminalis, shell of nucleus accumbens, and substantia innominata (Alheid, 2003) will be described in a subsequent article detailing the anatomy of the tree pangolin subpallial telencephalon.

Sectioning and immunohistochemical staining
Prior to sectioning, each brain was allowed to equilibrate in 30% sucrose in 0.1 M PB at 4 • C. The brains were then frozen in crushed dry ice and sectioned into 50-μm-thick sections on a freezing microtome.
The whole brain of MT1 was sectioned in the coronal plane and a one in 10 series of sections taken and stained for Nissl, myelin, tyrosine hydroxylase (TH), orexin-A (OxA), serotonin (5HT), parvalbumin (PV), calbindin (CB), calretinin (CR), neurofilament H (NFH), and vesicular glutamate transporter 2 (vGlut2). The whole brain of MT3 was sectioned in the coronal plane and a one in five series of sections taken and stained for neuronal nuclear marker (NeuN), choline acetyltransferase (ChAT), PV, CB, and CR. The right half of the brain of MT5 was antibody characterization, specificity, and the protocol followed for all immunostains listed above, have been provided previously (Imam et al., 2018a; but see Table 1 for antibody details).

Analysis and iconography
A low power stereomicroscope was used to examine the sections and camera lucida drawings outlining architectural borders were made.
Architectonic borders of the amygdaloid nuclei and cortical regions were first defined using the standard Nissl and myelin stains. The parcellation of the nuclear and cortical regions of the amygdaloid body was then confirmed and refined using the immunohistochemical stains.
The drawings were then scanned and redrawn using the Canvas Draw across studies and species (e.g., Sorvari, Soininen et al., 1995;Pillay et al., 2021), where the terminology used in these studies was not

RESULTS
In the tree pangolin, the amygdaloid body, as noted previously (Imam et al., 2017), was observed in the rostral aspect of the temporal portion, or lobe, of the cerebral hemisphere (Figures 1 and 2). Within the amygdaloid body, we identified four major groups including the basolateral (or deep) group (or complex), the superficial (or cortical-like, or corticomedial) nuclei, the centromedial group, and other amygdaloid nuclei ( Figure 2). As mentioned, the current study only describes the nuclei and cortical areas of the amygdaloid complex found in the rostral temporal region, which we refer to as the amygdaloid body. We note here that the development of the amygdala is very complex and that our assignation of structures as belonging to the "amygdaloid body" may need to be altered, as portions (such as the centromedial group) may be assigned to the extended amygdala (Alheid, 2003;de Olmos et al., 2004).

Deep or basolateral group/complex
The basolateral group was composed of three nuclei, the lateral, basal, and accessory basal amygdaloid nuclei (Figures 2 and 3).  (NeuN, a, c, e, g, j, k, l), parvalbumin (f), calbindin (b, h), and calretinin (d, i). The associated diagrams are from the same series of sections from which the photomicrographs were taken, but represent the combined architectural analysis from all sections, as compared to the single representation shown in the photomicrographs. In all photomicrographs and diagrams, dorsal is to the top and medial to the left. Photomicrograph and diagram (a) represent the most rostral portion of the amygdaloid body, while photomicrograph and diagram (l) represent the most caudal portion. Each photomicrograph and diagram are approximately 250 μm apart. The various nuclear/cortical groups of the amygdaloid body are represented with differing colors, including the deep or basolateral group in yellow, the superficial or cortical-like or corticomedial group in purple, the centromedial group in green, and the remaining amygdaloid nuclei in red. See list for abbreviations F I G U R E 2 Continued and 3). The largest and most distinct of these parts was the Bmc, which was located dorsally within the nucleus and was composed of relatively larger neurons than the Bi and Bpc and the dorsally adjacent LA (Figures 3(a) and 3(f)); with the relatively smaller neurons occupying the Bpc distinguishing this part from the slightly larger neurons of the Bi (Figure 3(a)). In addition, fascicles of myelinated fibers were observed to course through the Bmc, with a lower density of these fascicles in the Bi and their near absence from the Bpc  (Figures 3(a) and 4). The ABi had slightly smaller neurons than those of the ABmc, with those of the ABpc being smaller than those of the ABi, while those in the ABsh were smaller than the ABmc neurons and exhibited a slightly higher packing density (Figures 3(a) and 4(a)). Across the four parts, we observed a consistent low density of myelinated fibers (Figures 3(b) and 4(b)).

Superficial or cortical-like or corticomedial nuclei
The nuclei or cortical areas that are assigned to the superficial nuclei usually comprise the nuclei of the lateral (NLOT) and accessory (NAOT) olfactory tracts, the anterior and posterior cortical nuclei, and the periamygdaloid cortical areas. Both the NLOT (Figures 2(a)-2(e) and 5) and NAOT have been described previously (Imam et al., 2018a) and are thus not considered further herein.

3.2.1
The anterior and posterior cortical nuclei A low to moderate density of myelinated fibers was observed in both nuclei ( Figure 5(b)). Within the CoA, scattered multipolar neurons immunopositive for parvalbumin was observed, but a low to moderate density of these neurons was observed in the CoP (Figure 5(c)).
While there was clear parvalbumin-immunopositive dendritic networks in both nuclei, the density of these networks was higher in the CoP than the CoA (Figure 5(c)). Calbindin-immunopositive neurons and neuropil staining was noted in both nuclei, although these immunopositive neurons exhibited slightly smaller soma and were in a higher density in the CoA compared with the CoP ( Figure 5(d)).

The periamygdaloid cortex
The periamygdaloid cortex, located in the ventral most aspect of the amygdaloid body, was composed of medial (PACm) and lateral (PACl) periamygdaloid cortical areas (Figure 2). Both areas were composed of a three-layered cortex, including a distinct neuron-sparse molecular layer (layer 1, bordered externally by the lateral olfactory tract), a neuron dense layer 2, and a less neuron dense layer 3 (Figures 6(a), 6(e), and 6(j)). Layer 1 in both the PACm and PACl contained a low density of scattered neurons, a low density of parvalbumin-immunopositive apical dendrites, a low density calbindin-and calretinin-immunopositive  (Figure 6). Layer 2 in both areas contained the highest density of neurons, although the packing density in the PACm appears slightly higher than in the PACl (Figures 6(a), 6(e), and 6(j)).
In both areas, a low density of parvalbumin-immunopositive neurons were noted, and the density of parvalbumin-immunopositive structures in layer 2 appears higher in the PACl when compared with the PACm (Figures 6(b), 6(f), and 6(k)). In layer 2 of the PACm, very few calbindin-immunopositive neurons were observed, with these being more numerous in PACl, with some of these neurons being relatively large multipolar neurons (Figures 6(c), 6(g), and 6(l)). In contrast, the density of the calbindin-immunopositive terminal network in the PACm appeared higher in the PACm compared with the PACl (Figures 6(c), 6(g), and 6(l)). A few scattered, small calretinin-immunopositive neurons were observed in layer 2 of the PACm, but these were not readily These similarities and differences allowed the delineation of these two cortical areas, from each other and the adjacent cortical territories.  Figure 7(a)). In both the CeC and CeL, a slightly higher density of myelinated fibers was observed compared with the CeM, while the CeI exhibited the lowest density of myelinated fibers (Figure 7(b)).
A low density of calbindin-immunpositive neurons were observed in the CeC, CeL, and CeM, but in the CeI, only a few scattered calbindin-immunpositive neurons were observed (Figure 7(c)). In both the CeL and CeM, a moderately intense neuropil immunostaining for calbindin was present, the intensity of which was lower in the CeC and lowest in the CeI (Figure 7(c)). The highest density of neurofilament H-immunopositive structures were observed in the CeC and CeL, with a lower density being observed in the CeM and the lowest density in the CeI (Figure 7(d)). The remaining stains used in the current F I G U R E 6 Lower (a-d) and higher (e-n) magnification photomicrographs of the periamygdaloid cortex (PAC) within the amygdaloid body of the tree pangolin stained for neuronal nuclear marker (NeuN, a, e, j), parvalbumin (PV, b, f, k), calbindin (CB, c, g, l), calretinin (CR, d, h, m), and neurofilament H (NFH, i, n). Both the medial (PACm, e-i) and lateral (PACl, j-n) periamygdaloid cortical areas exhibit a trilaminate organization (layers 1, 2, 3). In all images, dorsal is to the top and medial to the left. Scale bar in (d) = 1 mm and applies to a-d. Scale bar in (n) = 100 μm and applies to (e)-(n). See list for abbreviations study showed no differentiation between the divisions, but using the variances noted we could postulate the presence of and delineate these four divisions.

3.3.2
The STIA The small STIA was located on the most dorsal aspect of the medial cortical region of the amygdaloid body of the tree pangolin (Figures 2(g)-2(j)). This division evinced a weakly defined bilaminar appearance, with a superficial lamina composed of a lower density of larger neurons, while a higher density of smaller neurons comprised the deeper lamina (Figure 8(a)). No parvalbumin-or neurofilament H-immunoreactive structures were observed in the STIA (Figures 8(d) and 8(g)), with calbindin-immunopositive neurons being found primarily in the deeper lamina (Figure 8(e)) and calretinin-immunopositive neurons being found throughout both laminae (Figure 8(f)).

3.3.3
The medial amygdaloid nucleus  (Figures 8(b) and 8(c)). No distinct differences in the chemoarchitecture of layer 3 were evident that contributed to the delineation of these two subdivisions (Figures 8(d)-8(g)). Thus, while we identify two subdivisions of the medial amygdaloid nucleus the differences between these subdivisions are subtle.

The AAA
The AAA was located on the medial aspect of the anterior portion of the amygdaloid body, lying between the ventral pallidum (VP) and the nucleus of the lateral olfactory tract (NLOT) (Figures 2(a)-2(d) and 9).
This moderately neuron-dense area (Figure 9(a)) was readily distinguished from the VP medially and NLOT laterally by the absence of distinct immunostaining for choline acetyltransferase (Figure 9(b)), a F I G U R E 8 Low and higher magnification photomicrographs of the medial amygdaloid nucleus within the amygdaloid body of the tree pangolin stained for neuronal nuclear marker (NeuN, a,  significantly lower density of parvalbumin-immunopositive structures than the VP, and the presence of parvalbumin-immunopositive neurons in the NLOT (Figure 9(c)). In addition, a moderate density of calbindinimmunopositive neurons were observed in the AAA, which exhibited a much lower density in the VP and were almost absent in the NLOT (Figure 9(d)). A moderate density of calretinin-immunopositive neurons were observed in the AAA, with these neurons being absent in the NLOT and of a lower density in the VP (Figure 9(e)). A high density of neurofilament H-immunopositive soma and dendrites characterized the VP, while in the AAA, the immunopositive soma were absent and a much lower density of dendrites were observed, both of which are absent in the NLOT (Figure 9(f)). This combination of features allowed the delineation of the AAA from surrounding structures.

The IMG and I
The IMG in the brain of the tree pangolin was observed to lie between the lateral amygdaloid nucleus and the putamen (  (Figures 11(c) and 11(d)), as well as a less intense calretinin-immunopositive neuropil staining that the dorsally located basal amygdaloid nucleus (Figure 11(d)).

DISCUSSION
The amygdaloid complex, being responsible for the generation of affective states/emotions in relation to stimuli relevant to the survival of individuals as members of a particular species (e.g., Rolls, 1999;Panskepp, 2011), is an important structure within the brain of all vertebrates. Despite the species-specificity of the stimuli that generate affective states, cross-species comparisons of the nuclear organization of the amygdaloid body of mammals has revealed only minor variations (e.g., Fox, 1940;Krettek & Price, 1978;Price et al., 1987;Pitkänen & Amaral, 1993a, b;Pitkänen et al., 1997;Kemppainen & Pitkänen, 2000;Ashwell, 2010;Limacher-Burrell et al., 2016, 2018Pillay et al., 2021).
The tree pangolin is not an exception to this conserved structural organization, and in the current study, we could readily determine the organization of the tree pangolin amygdaloid body by relating our observations to studies in other mammals. In this sense, while the stimuli that evoke affective state generation by the amygdaloid body the tree pangolin may differ to other species, it appears that the broad neuronal circuits that process these stimuli, and initiate affective states and behaviors, are conserved across mammalian brains.

Pangolin and carnivore amygdalae-similarities and differences
As the Philodota (or Manidae) and Carnivora are considered sisterorders within the Laurasiatherian mammal radiation (Arnason et al., 2008;Yu et al., 2011;Foley et al., 2016), we compared the results of our analysis to similar studies undertaken in carnivores to determine whether we could reveal any derived traits of the tree pangolin amygdaloid body in comparison with their closest relatives. Architectonic analyses of the amygdaloid body among the carnivores are available for the domestic dog (Kosmal & Nitecka, 1977), domestic cat (Fox, 1940), domestic ferret, and banded mongoose (Pillay et al., 2021). The general pattern of main nuclear subdivisions and compartmentalization of these subdivisions is shared across the species for which data are available. There does not appear to be any specific variances in the general organization of the tree pangolin amygdala that is not readily observed in the carnivoran amygdala. This indicates the likelihood that the neural processing and generation of affect-laden stimuli and affect-driven behavior is similar across these related species. The sole difference of note when comparing the tree pangolin amygdala to that of carnivores is the dorsal extension of the CoA and the deep involution of the NLOT within the tree pangolin amygdaloid body that is not seen in the carnivores that have been studied (Kosmal & Nitecka, 1977;Fox, 1940;Pillay et al., 2021). This potentially indicates a shift toward more of the amygdaloid body being devoted to the processing of affect related to olfactory information than might be observed in carnivores, although F I G U R E 1 0 Lower (a-d) and higher (e-j) magnification photomicrographs of the amygdaloid intramedullary gray matter (IMG) and intercalated island, or band, of the amygdala (I) within the amygdaloid body of the tree pangolin stained for neuronal nuclear marker (NeuN, a, e, h), calbindin (b, f, i), calretinin (c), neurofilament H (d), and tyrosine hydroxylase (g, j). Note how both the IMG and I form distinct bands of small neurons (e, h) between the lateral amygdaloid nucleus and putamen (IMG) and basal amygdaloid nucleus and central amygdaloid nucleus (I). Note the specific absence of calretinin immunostaining in these nuclei (c), and the marked neuropil staining for neurofilament H in both nuclei (d). Some of these neurons are immunopositive for calbindin (f, i), and in both the IMG and I there is a moderately dense tyrosine hydroxylase immunopositive terminal network (g, j). In all images, dorsal is to the top and medial to the left. Scale bar in (d) = 1 mm and applies to (a)-(d). Scale bar in (j) = 50 μm and applies to (e)-(j). See list for abbreviations F I G U R E 1 1 Lower (a-d) and higher (insets of a-d) magnification photomicrographs of the amygdalohippocampal area (AHiA) of the amygdaloid body of the tree pangolin stained for neuronal nuclear marker (NeuN, a), parvalbumin (b), calbindin (c), and calretinin (d). The insets are high magnification images of immunostained neurons in the CeI (NeuN, inset a), CeM (calbindin, inset c), and CeL (neurofilament H, inset d). In all images, dorsal is to the top and medial to the left. Scale bar in (d) = 1 mm and applies to (a)-(d). Scale bar in inset (d) = 25 μm and applies to all insets. See list for abbreviations. this would require further investigation to determine the validity of this possibility.

Broad similarities of amygdaloid body organization across mammals
The similarity of the organization of the amygdaloid body in pangolins and carnivores extends more broadly across mammals. In the mammal species that have been studied, the main nuclear groupings (such as lateral, basal, basal accessory, cortical, centromedial and other nuclei) appear to be present, as well as the internal divisions of these groups (e.g., sheep- Richard, 1967; laboratory rat- Krettek & Price, 1978, Pitkänen et al., 1997, Kemppainen & Pitkänen, 2000 Despite these broad similarities, there was one specific feature of the tree pangolin amygdaloid body, which appears to be shared by the carnivores, and partially by laboratory rats (Marowsky et al., 2005;Pinard et al., 2012), that was noted to be somewhat different to that observed in other mammal species. In the tree pangolin, the IMG and the intercalated islands (I) evince a very similar architecture and immunostaining pattern, as well as these neuronal clusters being located very close to each other. In addition, in the tree pangolin, the intercalated islands do not appear to be distinct islands, rather they have an appearance that does not differ from the IMG. In the domestic cat (Fox, 1940), domestic dog (Kosmal & Nitecka, 1977), domestic ferret, and banded mongoose (Pillay et al., 2021), and partially in laboratory rats (Marowsky et al., 2005;Pinard et al., 2012), a somewhat similar nonisolated architectural pattern of portions of the intercalated islands is depicted. In these species, the intercalated islands are located dorsomedial to the basolateral group. In contrast, in other studies of the laboratory rat (Krettek & Price, 1978;Pitkänen et al., 1997;Kemppainen & Pitkänen, 2000), crab-eating macaque (Pitkänen & Amaral, 1993a, b), human (Sorvari et al., 1995), rock hyrax (Limacher-Burrell et al., 2016), and African elephant (Limacher-Burrell et al., 2018), the intercalated islands are distinct islands, rather than a "band" of neurons, and are often located between the basal and accessory basal nuclei. These intercalated neurons play a role in the control of inhibition in the central amygdaloid nucleus (Sah et al., 2003), and thus may contribute to the modulation of fear conditioning. The observed differences in the architectural organization of the intercalated amygdaloid neurons between species may play a role in the differential expression and conditioning of fear responses in different species, but this possibility requires further study.

4.3
Are there any potentially adaptive aspects regarding affect in pangolins?
The range of affective behaviors observed in captive pangolins, as outlined by Mohapatra & Panda (2014) and Zhang et al. (2020), has been observed in many other mammalian species. In this sense, given the conserved organization of the amygdaloid body in the tree pangolin in comparison with other mammals, as determined with the methods employed in the current study, it would be difficult at this stage to posit any species-specific specializations of the neural processing and subsequent behavioral expression of affect in the tree pangolin. Directed studies of other aspects of amygdaloid organization in the tree pangolin may reveal clues to lineage or species-specific neural processing of affect, such as has been observed regarding the expression of aggression (Jacobs et al., 2006) or domestication (Saetre et al., 2004) in the domestic dog.
Despite these potential avenues, perhaps the most direct way to further develop our understanding of affect in the tree pangolin would be through the identification of the specific sensory stimuli that initiate affective behaviors. The investigation of such stimuli, involved in reproduction, fear, aggression, and other affective behaviors, would augment our understanding of these unusual mammalian species and may lead to better husbandry practices as well as improving, or developing, the possibility of captive breeding colonies to assist in the reintroduction of pangolin species where wild populations have been eliminated due to poaching and human encroachment on natural habitats.

ACKNOWLEDGMENTS
We thank Dr. Elizabeth Ebewele of the Department of Forestry, Federal Ministry of Environment, Nigeria, for her assistance in helping to facilitate the issuance of the CITES permit.

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS
P. R. M. conceptualized the study. A. I., A. B., M. S. A., and P. R. M. obtained the brains. A. I., A. B., and P. R. M. performed the staining and analysis. A. I. and P. R. M. wrote the manuscript, and the remaining authors contributed to the editing and improvement of the early drafts of the manuscript. All authors had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

DATA AVAILABILITY STATEMENT
Data have not been shared due to this study being based on histological sections.

TRANSPARENT PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/cne.25345