Ferret as a model system for studying the anatomy and function of the prefrontal cortex: A systematic review.

There is a lack of consensus on anatomical nomenclature, standards of documentation, and functional equivalence of the frontal cortex between species. There remains a major gap between human prefrontal function and interpretation of findings in the mouse brain that appears to lack several key prefrontal areas involved in cognition and psychiatric illnesses. The ferret is an emerging model organism that has gained traction as an intermediate model species for the study of top-down cognitive control and other higher-order brain functions. However, this research has yet to benefit from synthesis. Here, we provide a summary of all published research pertaining to the frontal and/or prefrontal cortex of the ferret across research scales. The targeted location within the ferret brain is summarized visually for each experiment, and the anatomical terminology used at time of publishing is compared to what would be the appropriate term to use presently. By doing so, we hope to improve clarity in the interpretation of both previous and future publications on the comparative study of frontal cortex.

The diencephalon of two carnivore species: The feliform banded mongoose and the caniform domestic ferret.

This study provides an analysis of the cytoarchitecture, myeloarchitecture, and chemoarchitecture of the diencephalon (dorsal thalamus, ventral thalamus, and epithalamus) of the banded mongoose (Mungos mungo) and domestic ferret (Mustela putorius furo). Using architectural and immunohistochemical stains, we observe that the nuclear organization of the diencephalon is very similar in the two species, and similar to that reported in other carnivores, such as the domestic cat and dog. The same complement of putatively homologous nuclei were identified in both species, with only one variance, that being the presence of the perireticular nucleus in the domestic ferret, that was not observed in the banded mongoose. The chemoarchitecture was also mostly consistent between species, although there were a number of minor variations across a range of nuclei in the density of structures expressing the calcium-binding proteins parvalbumin, calbindin, and calretinin. Thus, despite almost 53 million years since these two species of carnivores shared a common ancestor, strong phylogenetic constraints appear to limit the potential for adaptive evolutionary plasticity within the carnivore order. Apart from the presence of the perireticular nucleus, the most notable difference between the species studied was the physical inversion of the dorsal lateral geniculate nucleus, as well as the lateral posterior and pulvinar nuclei in the domestic ferret compared to the banded mongoose and other carnivores, although this inversion appears to be a feature of the Mustelidae family. While no functional sequelae are suggested, this inversion is likely to result from the altricial birth of Mustelidae species.

The hippocampal formation of two carnivore species: The feliform banded mongoose and the caniform domestic ferret.

Employing cyto-, myelo-, and chemoarchitectural staining techniques, we analyzed the structure of the hippocampal formation in the banded mongoose and domestic ferret, species belonging to the two carnivoran superfamilies, which have had independent evolutionary trajectories for the past 55 million years. Our observations indicate that, despite the time since sharing a last common ancestor, these species show extensive similarities. The four major portions of the hippocampal formation (cornu Ammonis, dentate gyrus, subicular complex, and entorhinal cortex) were readily observed, contained the same internal subdivisions, and maintained the topological relationships of these subdivisions that could be considered typically mammalian. In addition, adult hippocampal neurogenesis was observed in both species, occurring at a rate similar to that observed in other mammals. Despite the overall similarities, several differences to each other, and to other mammalian species, were observed. We could not find evidence for the presence of the CA2 and CA4 fields of the cornu Ammonis region. In the banded mongoose the dentate gyrus appears to be comprised of up to seven lamina, through the sublamination of the molecular and granule cell layers, which is not observed in the domestic ferret. In addition, numerous subtle variations in chemoarchitecture between the two species were observed. These differences may contribute to an overall variation in the functionality of the hippocampal formation between the species, and in comparison to other mammalian species. These similarities and variations are important to understanding to what extent phylogenetic affinities and constraints affect potential adaptive evolutionary plasticity of the hippocampal formation.

The amygdaloid body of two carnivore species: The feliform banded mongoose and the caniform domestic ferret.

The current study provides an analysis of the cytoarchitecture, myeloarchitecture, and chemoarchitecture of the amygdaloid body of the banded mongoose (Mungos mungo) and domestic ferret (Mustela putorius furo). Using architectural and immunohistochemical stains, we observe that the organization of the nuclear and cortical portions of the amygdaloid complex is very similar in both species. The one major difference is the presence of a cortex-amygdala transition zone observed in the domestic ferret that is absent in the banded mongoose. In addition, the chemoarchitecture is, for the most part, quite similar in the two species, but several variances, such as differing densities of neurons expressing the calcium-binding proteins in specific nuclei are noted. Despite this, certain aspects of the chemoarchitecture, such as the cholinergic innervation of the magnocellular division of the basal nuclear cluster and the presence of doublecortin expressing neurons in the shell division of the accessory basal nuclear cluster, appear to be consistent features of the Eutherian mammal amygdala. The domestic ferret presented with an overall lower myelin density throughout the amygdaloid body than the banded mongoose, a feature that may reflect artificial selection in the process of domestication for increased juvenile-like behavior in the adult domestic ferret, such as a muted fear response. The shared, but temporally distant, ancestry of the banded mongoose and domestic ferret allows us to generate observations relevant to understanding the relative influence that phylogenetic constraints, adaptive evolutionary plasticity, and the domestication process may play in the organization and chemoarchitecture of the amygdaloid body.

Seasonal weight changes in laboratory ferrets.

Ferrets (Mustela putorius furo) are a valuable animal model used in biomedical research. Like many animals, ferrets undergo significant variation in body weight seasonally, affected by photoperiod, and these variations complicate the use weight as an indicator of health status. To overcome this requires a better understanding of these seasonal weight changes. We provide a normative weight data set for the female ferret accounting for seasonal changes, and also investigate the effect of fluid regulation on weight change. Female ferrets (n = 39) underwent behavioural testing from May 2017 to August 2019 and were weighed daily, while housed in an animal care facility with controlled light exposure. In the winter (October to March), animals experienced 10 hours of light and 14 hours of dark, while in summer (March to October), this contingency was reversed. Individual animals varied in their body weight from approximately 700 to 1200 g. However, weights fluctuated with light cycle, with animals losing weight in summer, and gaining weight in winter such that they fluctuated between approximately 80% and 120% of their long-term average. Ferrets were weighed as part of their health assessment while experiencing water regulation for behavioural training. Water regulation superimposed additional weight changes on these seasonal fluctuations, with weight loss during the 5-day water regulation period being greater in summer than winter. Analysing the data with a Generalised Linear Model confirmed that the percentage decrease in weight per week was relatively constant throughout the summer months, while the percentage increase in body weight per week in winter decreased through the season. Finally, we noted that the timing of oestrus was reliably triggered by the increase in day length in spring. These data establish a normative benchmark for seasonal weight variation in female ferrets that can be incorporated into the health assessment of an animal's condition.

Ferret brain possesses young interneuron collections equivalent to human postnatal migratory streams.

The human early postnatal brain contains late migratory streams of immature interneurons that are directed to cortex and other focal brain regions. However, such migration is not observed in rodent brain, and whether other small animal models capture this aspect of human brain development is unclear. Here, we investigated whether the gyrencephalic ferret cortex possesses human-equivalent postnatal streams of doublecortin positive (DCX+) young neurons. We mapped DCX+ cells in the brains of ferrets at P20 (analogous to human term gestation), P40, P65, and P90. In addition to the rostral migratory stream, we identified three populations of young neurons with migratory morphology at P20 oriented toward: (a) prefrontal cortex, (b) dorsal posterior sigmoid gyrus, and (c) occipital lobe. These three neuronal collections were all present at P20 and became extinguished by P90 (equivalent to human postnatal age 2 years). DCX+ cells in such collections all expressed GAD67, identifying them as interneurons, and they variously expressed the subtype markers SP8 and secretagogin (SCGN). SCGN+ interneurons appeared in thick sections to be oriented from white matter toward multiple cortical regions, and persistent SCGN-expressing cells were observed in cortex. These findings indicate that ferret is a suitable animal model to study the human-relevant process of late postnatal cortical interneuron integration into multiple regions of cortex.

Cortical and thalamic connectivity of occipital visual cortical areas 17, 18, 19, and 21 of the domestic ferret (Mustela putorius furo).

The present study describes the ipsilateral and contralateral corticocortical and corticothalamic connectivity of the occipital visual areas 17, 18, 19, and 21 in the ferret using standard anatomical tract-tracing methods. In line with previous studies of mammalian visual cortex connectivity, substantially more anterograde and retrograde label was present in the hemisphere ipsilateral to the injection site compared to the contralateral hemisphere. Ipsilateral reciprocal connectivity was the strongest within the occipital visual areas, while weaker connectivity strength was observed in the temporal, suprasylvian, and parietal visual areas. Callosal connectivity tended to be strongest in the homotopic cortical areas, and revealed a similar areal distribution to that observed in the ipsilateral hemisphere, although often less widespread across cortical areas. Ipsilateral reciprocal connectivity was observed throughout the visual nuclei of the dorsal thalamus, with no contralateral connections to the visual thalamus being observed. The current study, along with previous studies of connectivity in the cat, identified the posteromedial lateral suprasylvian visual area (PMLS) as a distinct network hub external to the occipital visual areas in carnivores, implicating PMLS as a potential gateway to the parietal cortex for dorsal stream processing. These data will also contribute to a macro connectome database of the ferret brain, providing essential data for connectomics analyses and cross-species analyses of connectomes and brain connectivity matrices, as well as providing data relevant to additional studies of cortical connectivity across mammals and the evolution of cortical connectivity variation.

Cortical and thalamic connectivity of temporal visual cortical areas 20a and 20b of the domestic ferret (Mustela putorius furo).

The present study describes the ipsilateral and contralateral corticocortical and corticothalamic connectivity of the temporal visual areas 20a and 20b in the ferret using standard anatomical tract-tracing methods. The two temporal visual areas are strongly interconnected, but area 20a is primarily connected to the occipital visual areas, whereas area 20b maintains more widespread connections with the occipital, parietal and suprasylvian visual areas and the secondary auditory cortex. The callosal connectivity, although homotopic, consists mainly of very weak anterograde labeling which was more widespread in area 20a than area 20b. Although areas 20a and 20b are well connected with the visual dorsal thalamus, the injection into area 20a resulted in more anterograde label, whereas more retrograde label was observed in the visual thalamus following the injection into area 20b. Most interestingly, comparisons to previous connectional studies of cat areas 20a and 20b reveal a common pattern of connectivity of the temporal visual cortex in carnivores, where the posterior parietal cortex and the central temporal region (PMLS) provide network points required for dorsal and ventral stream interaction enroute to integration in the prefrontal cortex. This pattern of network connectivity is not dissimilar to that observed in primates, which highlights the ferret as a useful animal model to understand visual sensory integration between the dorsal and ventral streams. The data generated will also contribute to a connectomics database, to facilitate cross species analysis of brain connectomes and wiring principles of the brain.

Cortical and thalamic connectivity of posterior parietal visual cortical areas PPc and PPr of the domestic ferret (Mustela putorius furo).

The present study describes the ipsilateral and contralateral cortico-cortical and cortico-thalamic connectivity of the parietal visual areas, posterior parietal caudal cortical area (PPc) and posterior parietal rostral cortical area (PPr), in the ferret using standard anatomical tract-tracing methods. The two divisions of posterior parietal cortex of the ferret are strongly interconnected, however area PPc shows stronger connectivity with the occipital and suprasylvian visual cortex, while area PPr shows stronger connectivity with the somatomotor cortex, reflecting the functional specificity of these two areas. This pattern of connectivity is mirrored in the contralateral callosal connections. In addition, PPc and PPr are connected with the visual and somatomotor nuclei of the dorsal thalamus. Numerous connectional similarities exist between the posterior parietal cortex of the ferret (PPc and PPr) and the cat (area 7 and 5), indicative of the homology of these areas within the Carnivora. These findings highlight the existence of a frontoparietal network as a shared feature of the organization of parietal cortex across Euarchontoglires and Laurasiatherians, with the degree of expression varying in relation to the expansion and areal complexity of the posterior parietal cortex. This observation indicates that the ferret is a potentially valuable experimental model animal for understanding the evolution and function of the posterior parietal cortex and the frontoparietal network across mammals. The data generated will also contribute to a connectomics database, to further cross-species analyses of connectomes and illuminate wiring principles of cortical connectivity across mammals.

Characterization of the Inner and Outer Fiber Layers in the Developing Cerebral Cortex of Gyrencephalic Ferrets.

Changes in the cerebral cortex of mammals during evolution have been of great interest. Ferrets, monkeys, and humans have more developed cerebral cortices compared with mice. Although the features of progenitors in the developing cortices of these animals have been intensively investigated, those of the fiber layers are still largely elusive. By taking the advantage of our in utero electroporation technique for ferrets, here we systematically investigated the cellular origins and projection patterns of axonal fibers in the developing ferret cortex. We found that ferrets have 2 fiber layers in the developing cerebral cortex, as is the case in monkeys and humans. Axonal fibers in the inner fiber layer projected contralaterally and subcortically, whereas those in the outer fiber layer sent axons to neighboring cortical areas. Furthermore, we performed similar experiments using mice and found unexpected similarities between ferrets and mice. Our results shed light on the cellular origins, the projection patterns, the developmental processes, and the evolution of fiber layers in mammalian brains.

The Effect of Onset Age of Visual Deprivation on Visual Cortex Surface Area Across-Species.

Blindness early in life induces permanent alterations in brain anatomy, including reduced surface area of primary visual cortex (V1). Bilateral enucleation early in development causes greater reductions in primary visual cortex surface area than at later times. However, the time at which cortical surface area expansion is no longer sensitive to enucleation is not clearly established, despite being an important milestone for cortical development. Using histological and MRI techniques, we investigated how reductions in the surface area of V1 depends on the timing of blindness onset in rats, ferrets and humans. To compare data across species, we translated ages of all species to a common neuro-developmental event-time (ET) scale. Consistently, blindness during early cortical expansion induced large (~40%) reductions in V1 surface area, in rats and ferrets, while blindness occurring later had diminishing effects. Longitudinal measurements on ferrets confirmed that early enucleation disrupted cortical expansion, rather than inducing enhanced pruning. We modeled the ET associated with the conclusion of the effect of blindness on surface area at maturity (ETc), relative to the normal conclusion of visual cortex surface area expansion, (ETdev). A final analysis combining our data with extant published data confirmed that ETc occurred well before ETdev.

Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size.

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability) 1 , studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans2-4, but have little effect on the brains of mice5-9; this probably reflects evolutionarily divergent functions of ASPM10,11. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity12-14 than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25-40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients3,4, suggesting that loss of 'cortical units' has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates12-16. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.

Characterization of White Matter Tracts by Diffusion MR Tractography in Cat and Ferret that Have Similar Gyral Patterns.

The developmental relationships between gyral structures and white matter tracts have long been debated, but it is still difficult to discern whether they influence each other's development or are causally related. To explore this topic, this study used cats and ferrets as models for species that share similar gyral folding patterns and imaged with diffusion magnetic resonance imaging to compare white matter innervations in homologous gyri and other brain regions. Adult cat and ferret brains were analyzed via diffusion spectrum imaging tractography and homologous regions of interest were compared. Although similar genetic lineage and gyral structures would suggest analogous white matter tracts, tractography reveals significantly differing white matter connectivity in both the visual and auditory cortices. Similarities in connectivity were concentrated primarily in the highly conserved cerebellar region. These results correlate well with existing histological and functional studies of both species. Our results indicate that, while the 2 species may share similar gyral structures, they utilize different white matter connectivity; suggesting that while species may share similar gyral structures, they can develop different underlying white matter connectivity.

Biphasic aspect of sexually dimorphic ontogenetic trajectory of gyrification in the ferret cerebral cortex.

The present study characterized quantitatively sexual dimorphic development of gyrification by MRI-based morphometry. High spatial-resolution 3D MR images (using RARE sequence with short TR and minimum TE setting) were acquired from fixed brain of male and female ferrets at postnatal days (PDs) 4-90 using 7-tesla preclinical MRI system. The gyrification index was evaluated either throughout the cerebral cortex (global GI) or in representative primary sulci (sulcal GI). The global GI increased linearly from PD 4, and reached a peak at PD 42, marking 1.486±0.018 in males and 1.460±0.010 in females, respectively. Sexual difference was obtained by greater global GI in males than in females on PD 21 and thereafter. Rostrocaudal GI distribution revealed an overall male-over-female sulcal infolding throughout the cortex on PD 21. Then, an adult pattern of sexually dimorphic cortical convolution was achieved so that gyrification in the temporo-parieto-occipital region was more progressive in males than in females on PD 42, and slightly extended posteriorly in males until PD 90. In the sulcal GI, sulcus-specific male-over-female GI was revealed in the rhinal fissure, and presylvian sulcus on PD 42, and additionally in the coronal, splenial, lateral, and caudal suprasylvian sulci on PD 90. The current results suggest that age-related sexual dimorphism of the gyrification was biphasic in the ferret cortex. A male-over-female gyrification was allometric by PD 21, and was thereafter specific to primary sulci located on phylogenetically newer multimodal cortical regions.

Reproductive Medicine in Ferrets.

In the United States, desexing is performed routinely in ferrets at the age of 6 weeks, therefore reproductive tract diseases are not so common. However, in Europe most ferrets are desexed when they are several months old, or they are kept as intact animals. For this reason, diseases of the reproductive organs and a prolonged estrus are far more frequent in Europe than in the United States. This article summarizes and reviews the anatomy, reproductive physiology, management of reproduction (including surgical and hormonal contraception) and reproductive tract diseases in male and female ferrets.

Population based MRI and DTI templates of the adult ferret brain and tools for voxelwise analysis.

Non-invasive imaging has the potential to play a crucial role in the characterization and translation of experimental animal models to investigate human brain development and disorders, especially when employed to study animal models that more accurately represent features of human neuroanatomy. The purpose of this study was to build and make available MRI and DTI templates and analysis tools for the ferret brain as the ferret is a well-suited species for pre-clinical MRI studies with folded cortical surface, relatively high white matter volume and body dimensions that allow imaging with pre-clinical MRI scanners. Four ferret brain templates were built in this study - in-vivo MRI and DTI and ex-vivo MRI and DTI - using brain images across many ferrets and region of interest (ROI) masks corresponding to established ferret neuroanatomy were generated by semi-automatic and manual segmentation. The templates and ROI masks were used to create a web-based ferret brain viewing software for browsing the MRI and DTI volumes with annotations based on the ROI masks. A second objective of this study was to provide a careful description of the imaging methods used for acquisition, processing, registration and template building and to demonstrate several voxelwise analysis methods including Jacobian analysis of morphometry differences between the female and male brain and bias-free identification of DTI abnormalities in an injured ferret brain. The templates, tools and methodological optimization presented in this study are intended to advance non-invasive imaging approaches for human-similar animal species that will enable the use of pre-clinical MRI studies for understanding and treating brain disorders.

Anatomy and Disorders of the Oral Cavity of Ferrets and Other Exotic Companion Carnivores.

Exotic companion carnivores such as ferrets, skunks, fennec foxes, coatimundis, raccoons, and kinkajous presented in clinical practice share similar dental anatomy, function, and diseases. The domestic ferret serves as the representative species for this group with its anatomy, diseases, and conditions described in detail. Dog and cat guidelines for veterinary and home care seem to be relevant and applicable, including dental endodontic procedures. Annual or biannual dental examinations and prophylaxis are recommended. The most common dental and oral problems are tooth wear, plaque and calculus, teeth fractures, gingivitis and periodontitis, tooth loss, abscesses, oral ulceration, tonsillitis, and neoplasia.

Differential cortical laminar structure revealed by NeuN immunostaining and myeloarchitecture between sulcal and gyral regions independent of sexual dimorphisms in the ferret cerebrum.

The purpose of this study was to quantitatively clarify differences in laminar structure and myeloarchitecture of sulcal and gyral regions of the cerebral cortex of ferrets. Histological sections of cerebrum from male and female ferrets at postnatal day 90 were made at the coronal plane, and were immunostained with anti-NeuN or anti-myelin basic protein (MBP). Thickness was estimated in the entire depth or three strata, that is, layer I, outer (layers II-III) and inner (layers IV-VI) strata of the neocortex in representative five sulcal and seven gyral regions. As with the entire cortical depth, outer and inner strata were significantly thinner in the sulcal bottoms than in the gyral crowns, whereas layer I had about twofold greater thickness in the sulcal bottoms. However, thicknesses of the entire cortical depth and each cortical stratum were not statistically different among five sulcal regions or seven gyral regions examined. By MBP immunostaining, myelin fibers ran tangentially through the superficial regions of layer I in gyral crowns. Those fibers were relatively denser in gyri of frontal and temporal regions, and relatively sparse in gyri of parietal and occipital regions, although their density in any gyri was not different between sexes. These results show a differential laminar structure and myeloarchitecture between the sulcal and gyral regions of the ferret cerebral cortex present in both sexes. Myelination of layer I tangential fibers varied among primary gyri and was weaker in phylogenetically higher-order cortical gyri. Anat Rec, 299:1003-1011, 2016. © 2016 Wiley Periodicals, Inc.

Spatial organization of astrocytes in ferret visual cortex.

Astrocytes form an intricate partnership with neural circuits to influence numerous cellular and synaptic processes. One prominent organizational feature of astrocytes is the "tiling" of the brain with non-overlapping territories. There are some documented species and brain region-specific astrocyte specializations, but the extent of astrocyte diversity and circuit specificity are still unknown. We quantitatively defined the rules that govern the spatial arrangement of astrocyte somata and territory overlap in ferret visual cortex using a combination of in vivo two-photon imaging, morphological reconstruction, immunostaining, and model simulations. We found that ferret astrocytes share, on average, half of their territory with other astrocytes. However, a specific class of astrocytes, abundant in thalamo-recipient cortical layers ("kissing" astrocytes), overlap markedly less. Together, these results demonstrate novel features of astrocyte organization indicating that different classes of astrocytes are arranged in a circuit-specific manner and that tiling does not apply universally across brain regions and species. J. Comp. Neurol. 524:3561-3576, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Male prevalent enhancement of leftward asymmetric development of the cerebellar cortex in ferrets (Mustela putorius).

The present study was conducted in MRI-based volumetry to characterize the sexual dimorphism of the cerebellum in young adult ferrets. High spatial resolution 3D anatomical MRI at 7-tesla were acquired ex vivo from fixed cerebella of 90-day-old male and female ferrets. The 3D morphology and topology of cerebellar structures were reproduced well by volume-rendered images obtained from MRI. Volume of the whole cerebellum was significantly larger in males than in females. The cerebellar cortex was further divided into five transverse domains: the anterior zone (AZ; lobules I-V), central zone anterior (lobule VI), central zone posterior (CZp; lobule VII), posterior zone (PZ; lobules VIII-IXa) and nodular zone (NZ; lobules IXb -X). Significantly greater volumes in males than in females were detected bilaterally in the AZ, CZp, and NZ, and leftward in PZ. Notably, the significant volume asymmetry was detected leftward in the CZp of males. By asymmetry quotient analysis, the counterclockwise torque asymmetry of the cerebellum was revealed, and it was more striking in males than in females. The present results suggest that sexual dimorphism of the ferret cerebellum is characterized by enhancing the leftward laterality in the CZp in males, forming the distinctive counterclockwise torque asymmetry.