Nuclear organization of cholinergic,catecholaminergic, serotonergic and orexinergic systems in the brain of the Tasmanian devil (Sarcophilus harrisii)

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brains of three male Tasmanian devils (Sarcophilus harrisii), which had a mean brain mass of 11.6 g. We found that the nuclei generally observed for these systems in other mammalian brains were present in the brain of the Tasmanian devil. Despite this, specific differences in the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems appear to carry a phylogenetic signal. In the cholinergic system, only the dorsal hypothalamic cholinergic nucleus could be observed, while an extra dorsal subdivision of the laterodorsal tegmental nucleus and cholinergic neurons within the gelatinous layer of the caudal spinal trigeminal nucleus were observed. Within the catecholaminergic system the A4 nucleus of the locus coeruleus complex was absent, as was the caudal ventrolateral serotonergic group of the serotonergic system. The organization of the orexinergic system was similar to that seen in many mammals previously studied. Overall, while showing strong similarities to the organization of these systems in other mammals, the specific differences observed in the Tasmanian devil reveal either order specific, or class specific, features of these systems. Further studies will reveal the extent of change in the nuclear organization of these systems in marsupials and how these potential changes may affect functionality.     

Distribution of orexin-A immunoreactive neurons and their terminal networks in the brain of the rock hyrax, Procavia capensis

The present study describes the distribution of orexin-A immunoreactive neurons and terminal networks in relation to the previously described catecholaminergic, cholinergic and serotonergic systems within the brain of the rock hyrax, Procavia capensis. Adult female rock hyrax brains were sectioned and immunohistochemically stained with an antibody to orexin-A. The staining revealed that the neurons were mainly located within the hypothalamus as with other mammals. The orexinergic terminal network distribution also resembled the typical mammalian plan. High-density orexinergic terminal networks were located within regions of the diencephalon (e.g. paraventricular nuclei), midbrain (e.g. serotonergic nuclei) and pons (locus coeruleus), while medium density orexinergic terminal networks were evident in the telencephalic (e.g. basal forebrain), diencephalic (e.g. hypothalamus), midbrain (e.g. periaqueductal gray matter), pontine (e.g. serotonergic nuclei) and medullary regions (e.g. serotonergic and catecholaminergic nuclei). Although the distribution of the orexinergic terminal networks was typically mammalian, the rock hyrax did show one atypical feature, the presence of a high-density orexinergic terminal network within the anterodorsal nucleus of the dorsal thalamus (AD). The dense orexinergic innervation of the AD nucleus has only been reported previously in the Nile grass rat, Arvicanthis niloticus and Syrian hamster, Mesocricetus auratus, both diurnal mammals. It is possible that orexinergic innervation of the AD nucleus might be a unique feature associated with diurnal mammals. It was also noted that the dense orexinergic innervation of the AD nucleus coincided with previously identified cholinergic neurons and terminal networks in this particular nucleus of the rock hyrax brain. It is possible that this dense orexinergic innervation of the AD nucleus in the brain of the rock hyrax may act in concert with the cholinergic neurons and/or the cholinergic axonal terminals, which in turn may influence arousal states and motivational processing.     

Organization of cholinergic,putative catecholaminergic and serotonergic nuclei in the diencephalon,mibrain and pons of sub-adult male giraffes

The current study describes the nuclear organization and neuronal morphology of the cholinergic, putative catecholaminergic and serotonergic systems within the diencephalon, midbrain and pons of the giraffe using immunohistochemistry for choline acetyltransferase, tyrosine hydroxylase and serotonin. The giraffe has a unique phenotype (the long neck), a large brain (over 500 g) and is a non-domesticated animal, while previous studies examining the brains of other Artiodactyls have all been undertaken on domesticated animals. The aim of the present study was to investigate possible differences in the nuclear organization and neuronal morphology of the above-mentioned systems compared to that seen in other Artiodactyls and mammals. The nuclear organization of all three systems within the giraffe brain was similar to that of other Artiodactyls. Some features of interest were noted for the giraffe and in comparison to other mammals studied. The cholinergic neuronal somata of the laterodorsal tegmental nucleus were slightly larger than those of the pedunculopontine tegmental nucleus, a feature not described in other mammals. The putative catecholaminergic system of the giraffe appeared to lack an A15 dorsal nucleus, which is commonly seen in other mammals but absent in the Artiodactyls, had a large and expanded substantia nigra pars reticulata (A9 ventral), a small diffuse portion of the locus coerueleus (A6d), an expansive subcoeruleus (A7sc and A7d), and lacked the A4 nucleus of the locus coeruleus complex. The nuclear organization of the serotonergic system of the giraffe was identical to that seen in all other eutherian mammals studied to date. These observations in the giraffe demonstrate that despite significant changes in life history, phenotype, brain size and time of divergence, species within the same order show the same nuclear organization of the systems investigated.     

Nuclear organization of some immunohistochemically identifiable neural systems in two species of the Euarchontoglires: A Lagomorph,Lepus capensis,and a Scandentia,Tupaia belangeri

The present study describes the organization of the nuclei of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of two members of Euarchontoglires, Lepus capensis and Tupaia belangeri. The aim of the present study was to investigate the nuclear complement of these neural systems in comparison to previous studies on Euarchontoglires and generally with other mammalian species. Brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The majority of nuclei revealed in the current study were similar between the species investigated and to mammals generally, but certain differences in the nuclear complement highlight potential phylogenetic interrelationships within the Euarchontoglires and across mammals. In the northern tree shrew the nucleus of the trapezoid body contained neurons immunoreactive to the choline acetyltransferase antibody with some of these neurons extending into the lamellae within the superior olivary nuclear complex (SON). The cholinergic nature of the neurons of this nucleus, and the extension of cholinergic neurons into the SON, has not been noted in any mammal studied to date. In addition, cholinergic neurons forming the medullary tegmental field were also present in the northern tree shrew. Regarding the catecholaminergic system, the cape hare presented with the rodent specific rostral dorsal midline medullary nucleus (C3), and the northern tree shrew lacked both the ventral and dorsal divisions of the anterior hypothalamic group (A15v and A15d). Both species were lacking the primate/megachiropteran specific compact portion of the locus coeruleus complex (A6c). The nuclei of the serotonergic and orexinergic systems of both species were similar to those seen across most Eutherian mammals. Our results lend support to the monophyly of the Glires, and more broadly suggest that the megachiropterans are more closely related to the primates than are any other members of Euarchontoglires studied to date.     

Nuclear organization of cholinergic, putative catecholaminergic, serotonergic and orexinergic systems in the brain of the African pygmy mouse (Mus minutoides): organizational complexity is preserved in small brains

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brain of the African pygmy mouse (Mus minutoides). The African pygmy mice studied had a brain mass of around 275 mg, making these the smallest rodent brains to date in which these neural systems have been investigated. In contrast to the assumption that in this small brain there would be fewer subdivisions of these neural systems, we found that all nuclei generally observed for these systems in other rodent brains were also present in the brain of the African pygmy mouse. As with other rodents previously studied in the subfamily Murinae, we observed the presence of cortical cholinergic neurons and a compactly organized locus coeruleus. These two features of these systems have not been observed in the non-Murinae rodents studied to date. Thus, the African pygmy mouse displays what might be considered a typical Murinae brain organization, and despite its small size, the brain does not appear to be any less complexly organized than other rodent brains, even those that are over 100 times larger such as the Cape porcupine brain. The results are consistent with the notion that changes in brain size do not affect the evolution of nuclear organization of complex neural systems. Thus, species belonging to the same order generally have the same number and complement of the subdivisions, or nuclei, of specific neural systems despite differences in brain size, phenotype or time since evolutionary divergence.     

Nuclear organization of cholinergic,putative catecholaminergic and serotonergic nuclei in the brain of the eastern rock elephant shrew,Elephantulus myurus

The organization of the nuclear subdivisions of the cholinergic, putative catecholaminergic and serotonergic systems of the brain of the elephant shrew (Elephantulus myurus) were determined following immunohistochemistry for choline acetyltransferase, tyrosine hydroxylase and serotonin, respectively. This was done in order to determine if differences in the nuclear organization of these systems in comparison to other mammals were evident and how any noted differences may relate to specialized behaviours of the elephant shrew. The elephant shrew belongs to the order Macroscelidea, and forms part of the Afrotherian mammalian cohort. In general, the organization of the nuclei of these systems resembled that described in other mammalian species. The cholinergic system showed many features in common with that seen in the rock hyrax, rodents and primates; however, specific differences include: (1) cholinergic neurons were observed in the superior and inferior colliculi, as well as the cochlear nuclei; (2) cholinergic neurons were not observed in the anterior nuclei of the dorsal thalamus as seen in the rock hyrax; and (3) cholinergic parvocellular nerve cells forming subdivisions of the laterodorsal and pedunculopontine tegmental nuclei were not observed at the midbrain/pons interface as seen in the rock hyrax. The organization of the putative catecholaminergic system was very similar to that seen in the rock hyrax and rodents except for the lack of the rodent specific C3 nucleus, the dorsal division of the anterior hypothalamic group (A15d) and the compact division of the locus coeruleus (A6c). The nuclear organization of the serotonergic system was identical to that seen in all eutherian mammals studied to date. The additional cholinergic neurons found in the cochlear nucleus and colliculi may relate to a specific acoustic signalling system observed in elephant shrews expressed when the animals are under stress or detect a predator. These neurons may then function to increase attention to this type of acoustic signal termed foot drumming.     

Quantitative Analysis of the Timing of Development of the Cerebellum and Precerebellar Nuclei in Monotremes,Metatherians, Rodents,and Humans

We have used a quantitative statistical approach to compare the pace of development in the cerebellum and precerebellar systems relative to body size in monotremes and metatherians with that in eutherians (rodents and humans). Embryos, fetuses, and early postnatal mammals were scored on whether key structural events had been reached in the development of the cerebellum itself (CC—corpus cerebelli; 10 milestones), or the pontine and inferior olivary precerebellar nuclear groups (PC; 4 milestones). We found that many early cerebellar and precerebellar milestones (e.g., formation of Purkinje cell layer and deep cerebellar nuclei) were reached at a smaller absolute body length in both metatherians and eutherians together, compared to monotremes. Some later milestones (e.g., formation of the external granular layer and primary fissuration) were reached at a smaller body length in metatherians than eutherians. When the analysis was performed with proportional body length expressed as a natural log-transformed ratio of length at birth, milestones were reached at a much smaller proportional body length in rodents and humans than in the metatherians or monotremes. The findings are consistent with the slower pace of metabolic activity and embryonic development in monotremes. They also indicate slightly advanced maturation of some early features of the cerebellum in some metatherians (i.e., early cerebellar development in dasyurids relative to body size), but do not support the notion of an accelerated development of the cerebellum to cope with the demands of early birth. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1998–2013, 2020. © 2019 American Association for Anatomy     

Morphological and Histochemical Description of Quail Feather Development

The current study investigated the macroscopic and microscopic differences between pennaceous and plumulaceous feathers. The morphology of the barbules distinguished pennaceous and plumulaceous feathers, particularly the shape of barbules during their development. In pennaceous feathers, the initial barbules were large and elongated or pyriform in shape, while plumulaceous feathers had small, thin, elongated initial barbules. The spinous barbules were characteristic of pennaceous feathers. The histochemical reactivity of both feather types for Mallory trichrome, orange G, and acridine orange, safranin O, PAS, and methylene blue was determined. Keratin was detected by Mallory trichrome, orange G, and acridine orange. In conclusion, the histochemical properties of pennaceous and plumulaceous feathers of quail, particularly the distribution and nature of keratin during development, should be considered in future studies. The unique morphological features of pennaceous and plumulaceous feathers could be used as a guide for phylogenetic identification. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1865–1883, 2020. © 2019 American Association for Anatomy     

The Claustrum in the Squirrel Monkey

The claustrum (CLA) is a subcortical structure that is reciprocally and topographically connected with the cerebral cortex. The complexity of the cerebral cortex varies dramatically across mammals, raising the question of whether there might also be differences in CLA organization, circuitry, and function. Species variations in the shape of the CLA are well documented. Studies in multiple species have identified subsets of neurochemically distinct interneurons; some data suggest species variations in the nature, distribution, and numbers of different neurochemically identified neuronal types. We have studied the CLA in a smooth-brained primate, the squirrel monkey, using Nissl-stained sections and immunohistochemistry. We found that the shape of the CLA is different from that in other primates. We found several different neurochemically defined populations of neurons equally distributed throughout the CLA. Immunoreactivity to GAD_65/67 and GABA_A receptors suggest that GABAergic interneurons provide widespread inhibitory input to CLA neurons. Immunoreactivity to glutamate transporters suggests widespread and overlapping excitatory input from cortical and possibly subcortical sources. Comparison of CLA organization in different species suggests that there may be major species differences both in the organization and in the functions of the CLA. Anat Rec, 303:1439–1454, 2020. © 2019 American Association for Anatomy     

Organization of cholinergic,catecholaminergic, serotonergic and orexinergic nuclei in three strepsirrhine primates: Galago demidoff,Perodicticus potto and Lemur catta

The nuclear organization of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of three species of strepsirrhine primates is presented. We aimed to investigate the nuclear complement of these neural systems in comparison to those of simian primates, megachiropterans and other mammalian species. The brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The nuclei identified were identical among the strepsirrhine species investigated and identical to previous reports in simian primates. Moreover, a general similarity to other mammals was found, but specific differences in the nuclear complement highlighted potential phylogenetic interrelationships. The central feature of interest was the structure of the locus coeruleus complex in the primates, where a central compactly packed core (A6c) of tyrosine hydroxylase immunopositive neurons was surrounded by a shell of less densely packed (A6d) tyrosine hydroxylase immunopositive neurons. This combination of compact and diffuse divisions of the locus coeruleus complex is only found in primates and megachiropterans of all the mammalian species studied to date. This neural character, along with variances in a range of other neural characters, supports the phylogenetic grouping of primates with megachiropterans as a sister group.     

Organization and number of orexinergic neurons in the hypothalamus of two species of Cetartiodactyla: a comparison of giraffe (Giraffa camelopardalis) and harbour porpoise (Phocoena phocoena)

The present study describes the organization of the orexinergic (hypocretinergic) neurons in the hypothalamus of the giraffe and harbour porpoise--two members of the mammalian Order Cetartiodactyla which is comprised of the even-toed ungulates and the cetaceans as they share a monophyletic ancestry. Diencephalons from two sub-adult male giraffes and two adult male harbour porpoises were coronally sectioned and immunohistochemically stained for orexin-A. The staining revealed that the orexinergic neurons could be readily divided into two distinct neuronal types based on somal volume, area and length, these being the parvocellular and magnocellular orexin-A immunopositive (OxA+) groups. The magnocellular group could be further subdivided, on topological grounds, into three distinct clusters--a main cluster in the perifornical and lateral hypothalamus, a cluster associated with the zona incerta and a cluster associated with the optic tract. The parvocellular neurons were found in the medial hypothalamus, but could not be subdivided, rather they form a topologically amorphous cluster. The parvocellular cluster appears to be unique to the Cetartiodactyla as these neurons have not been described in other mammals to date, while the magnocellular nuclei appear to be homologous to similar nuclei described in other mammals. The overall size of both the parvocellular and magnocellular neurons (based on somal volume, area and length) were larger in the giraffe than the harbour porpoise, but the harbour porpoise had a higher number of both parvocellular and magnocellular orexinergic neurons than the giraffe despite both having a similar brain mass. The higher number of both parvocellular and magnocellular orexinergic neurons in the harbour porpoise may relate to the unusual sleep mechanisms in the cetaceans.     

Klebsiella pneumoniae Redistributes Katanin Severing Proteins and Alters Astral Microtubules during Mitosis

Klebsiella pneumoniae has become a growing concern within hospitals due to multidrug resistant strains and increasing mortality rates. Recently, we showed that at the subcellular level, K. pneumoniae compromises the integrity of the epithelia by disassembling the microtubule networks of cells through the actions of katanin microtubule severing proteins. In this study, we report on the observation that mitotic cells are targeted by K. pneumoniae and that during infections, the katanin proteins are excluded from the microtubule organizing centers of dividing cells, resulting in the alteration of the microtubule cytoskeleton. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1859–1864, 2020. © 2019 American Association for Anatomy     

Cellular location and major terminal networks of the orexinergic system in the brain of two megachiropterans

The present study describes the distribution of orexin-A immunoreactive neurons and their terminal networks in the brains of two species of megachiropterans. In general the organization of the orexinergic system in the mammalian brain is conserved across species, but as one of two groups of mammals that fly and have a high metabolic rate, it was of interest to determine whether there were any specific differences in the organization of this system in the megachiropterans. Orexinergic neurons were limited in distribution to the hypothalamus, and formed three distinct clusters, or nuclei, a main cluster with a perifornical location, a zona incerta cluster in the dorsolateral hypothalamus and an optic tract cluster in the ventrolateral hypothalamus. The nuclear parcellation of the orexinergic system in the megachiropterans is similar to that seen in many mammals, but differs from the microchiropterans where the optic tract cluster is absent. The terminal networks of the orexinergic neurons in the megachiropterans was similar to that seen in a range of mammalian species, with significant terminal networks being found in the hypothalamus, cholinergic pedunculopontine and laterodorsal tegemental nuclei, the noradrenergic locus coeruleus complex, all serotonergic nuclei, the paraventricular nuclei of the epithalamus and adjacent to the habenular nuclei. While the megachiropteran orexinergic system is typically mammalian in form, it does differ from that reported for microchiropterans, and thus provides an additional neural character arguing for independent evolution of these two chiropteran suborders.     

Orexinergic bouton density is lower in the cerebral cortex of cetaceans compared to artiodactyls

The species of the cetacean and artiodactyl suborders, which constitute the order Cetartiodactyla, exhibit very different sleep phenomenology, with artiodactyls showing typical bihemispheric slow wave and REM sleep, while cetaceans show unihemispheric slow wave sleep and appear to lack REM sleep. The aim of this study was to determine whether cetaceans and artiodactyls have differently organized orexinergic arousal systems by examining the density of orexinergic innervation to the cerebral cortex, as this projection will be involved in various aspects of cortical arousal. This study provides a comparison of orexinergic bouton density in the cerebral cortex of twelve Cetartiodactyla species (ten artiodactyls and two cetaceans) by means of immunohistochemical staining and stereological analysis. It was found that the morphology of the axonal projections of the orexinergic system to the cerebral cortex was similar across all species, as the presence, size and proportion of large and small orexinergic boutons were similar. Despite this, orexinergic bouton density was lower in the cerebral cortex of the cetaceans studied compared to the artiodactyls studied, even when corrected for brain mass, neuron density, glial density and glial:neuron ratio. Results from correlational and principal component analyses indicate that glial density is a major determinant of the observed differences between artiodactyl and cetacean cortical orexinergic bouton density.     

Mysticetes to MiniConference to Manuscripts: Introduction to Thematic Issue on Mysticete Anatomy

This issue of the Anatomical Record is focused on the theme of Mysticete Anatomy. There are six included articles that explore the anatomy of the nasal region (Marquez et al., 2018; Maust-Mohl et al., 2018), larynx (Damien et al., 2018), lungs (Fetherston et al., 2018), sublingual fascia (Werth et al., 2018), and brain (Raghanti et al., 2018). These papers document anatomical features exhibited by mysticetes (baleen whales) and their related cousins (including other whales, and the semiaquatic moose and hippopotamus). This theme stems from a 2-day MiniConference on Mysticete Anatomy, hosted at the Icahn School of Medicine at Mount Sinai in New York City on May 2016. Anatomy is explored in the contexts of function and evolution of aquatic adaptations. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc. Anat Rec, 302:663–666, 2019. © 2019 Wiley Periodicals, Inc.     

Extreme Anatomy: The Lottery Winners,Specialists, and Extreme Adaptations That Are No More

This special issue of The Anatomical Record explores extravagant adaptions that vertebrates have evolved from their base groups to survive in the most challenging environments. It stems from a symposium entitled “Extreme Anatomy: Living beyond the edge,” which was held April 23, 2017, at the annual meeting of the American Association for Anatomy, in Chicago, IL. In Part 1 of this issue, we examined extreme morphologies that allow exploration of new niches. In this issue, we return to the evolution of terrestriality by digging deeply into the fossil history of the piscine antecedent of tetrapods. These were truly “lottery winners” among vertebrates. This issue also bears on extreme specialists that once thrived but are now long extinct and some extant species that thrive in the hottest terrestrial niches. Herein, several contributions discuss developmental strategies that facilitate later demanding locomotor regimens and feeding strategies for accessing nutrients from less than ideal food sources. From mole-rats to short-faced breeds of domestic dogs, we encounter another host of the most unusual of earth's creatures, who have much to teach us about our world. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:214–217, 2020. © 2019 American Association for Anatomy     

Extreme Anatomy: Gear for the Pioneer

This special issue of The Anatomical Record explores extravagant adaptions that vertebrates have evolved from their base groups to survive in the most challenging environments. The special issue stems from a symposium entitled “Extreme Anatomy: Living beyond the edge,” which was held April 23, 2017, at the annual meeting of the American Association of Anatomists, (now called the American Association for Anatomy), in Chicago, IL. In part 1 of this issue, we encounter fossorial mammals and cave-dwelling fish and salamanders that have reduced visual systems accompanied by a variety of mechanosensory adaptations. In rivers and seas, teeth may not suffice in the pursuit of prey: aquatic vertebrates are adorned with armor or weaponry or elaborate keratinous sieves. As vertebrates exploit a great diversity of niches, selection has favored a dizzying array of specialized sensory and locomotor adaptions for deep diving, rapid flight, and navigation through dark and complex settings. Each special adaptation, some seemingly quite “extreme” deviations from an original Bauplan, becomes a tool for a pioneer-like diversification of vertebrates. Anat Rec, 2019. © 2019 American Association for Anatomy     

Comparing the Superficial Vasculature of the Central Nervous System in Six Laboratory Animals: A Hypothesis About the Role of the “Circle of Willis”

We provide images of the entire central nervous system vasculature, and compare the anatomical findings in six different laboratory animals. A detailed understanding of the specific anatomy for each is important in the design of experimental modeling and for understanding the specific function of each target organ . Six different types of animals, the Korean wild mouse, C57BL/6J mouse, F344 rat, mongolian gerbil, Syrian hamsters, and guinea pigs, were included. To stain the blood vessels in each of the animals, Alcian blue reagent was used to perfuse each species. The bifurcation and anastomotic patterns of the anterior cerebral arteries differed in each species. The vascular supply to the olfactory nerve was visualized as a single artery supplying both olfactory nerves, and arteries supplying the lateral portion of the olfactory nerves originating from the olfactory bulb area. The posterior communicating arteries of the six animals demonstrated unique morphologies. The shape of the hypophyseal portal system varied by species. Most animals used in this study had a hexagonal Circle of Willis, except for the Korean wild mouse. Using this approach, we successfully mapped the brain vascular system in six different species of animals. This information and the images created can guide other researchers as they design research studies and create experimental models for new surgical procedures and approaches. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc. Anat Rec, 302:2049–2061, 2019. © 2019 American Association for Anatomy     

The Role of miR-326 in Adipogenic Differentiation of Human Adipose-Derived Stem Cells by Targeting C/EBPα in vitro

The CCAAT-enhancer-binding protein α (C/EBPα) plays an important role in adipogenic differentiation of adipose-derived stem cells (ASC). Recent studies have shown that microRNAs (miRNAs) participate in the regulation of self-renewal, proliferation, and multi-directional differentiation of ASCs. In the present study, we analyzed the targeting miRNAs on C/EBPα and found that miR-326 played an essential role in it. The results of qPCR confirmed that the expression of miR-326 was reduced in adipogenic differentiation. In addition, the dual-luciferase reporter assay system verified binding between miR-326 and the 3′ untranslated region of C/EBPα. Furthermore, transfection of miR-326 into human adipose-derived stem cells caused a significant reduction in C/EBPα. Our results highlight the importance of miR-326 in adipogenic differentiation and provide a reliable basis for clinical treatment of adipose-related diseases. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:2054–2060, 2020. © 2019 American Association for Anatomy