However, the labeled profiles were consisted of variably shaped elements in sizes much smaller than that of all neuronal cell types in the cerebellum. It should be noted that the very large somata of Purkinje cells were sometimes visible on the background, but they only displayed faint IR. There were lightly labeled small-sized cells in the molecular layer ML , which appeared to represent basket cells. A few small-sized cells in the GCL were encountered, also exhibiting light intensity in general Figure 8B.
Sortilin IR was observed in all deep cerebellar nuclei apparently in association with neuronal somal profiles. In the dentate nucleus, the labeled cells showed light to moderate intensity, and were round, multipolar or irregular in shape, with neuronal processes representing dendritic elements identifiable on and between the labeled somata Figures 8C,D. Figure 8. Panel A is low power view of the cerebellar cortex, with an impressive band-like labeling located in the middle of the cerebellar lobules.
The framed area in A is enlarged as B , which shows that at high magnification, this band reflects small granular but non-cellular elements distributed along the Purkinje cell layer PCL. The somata of Purkinje cells pointed by arrows exhibit faint labeling. Lightly stained small neurons pointed by arrowheads representing basket cells are present in the molecular layer ML , with a few also in the granule cell layer GCL.
Cells in the dentate nucleus DN are lightly stained with their proximal processes visible. The cerebellar white matter WM shows no labeling. In transverse section of the upper part of the pons E , the most impressive sortilin IR occurs in the locus coeruleus LC that is occupied by a group of densely labeled neurons, as enlarged in F.
In transverse sections of the pons at the level passing the upper part of the forth ventricle 4V , sortilin labeled cells were mostly impressive in the locus coeruleus LC located at the ventrolateral part of the ventricular floor Figure 8E. The cells exhibited strong reactivity, were mostly round or oval in shape, and had short processes connected to the somata Figure 8F , insert.
Lightly stained cells were present in the PAG, and the dorsal raphe nucleus DR at the midline of ventricular floor. Additionally labeled cells were sparsely seen in other areas of the dorsal areas of the pons corresponding the RF.
In transverse sections of the medulla oblongata MO at the levels of the inferior olivary IO complex, groups of labeled cells corresponding to the cranial nerve nuclei were visualized ventral and lateral to the 4V, while the main and accessory nuclei of the IO were clearly marked in the ventral part of the oblongata Figure 9A. Thus, the facial nucleus 7N , cochlear and vestibular nuclear complex 8N , nucleus of the solitary tract SoN , sensory trigeminal nuclei 5V , dorsal nucleus of vagus 10N and hypoglossal nucleus 12N were readily distinguishable in the immunolabeled sections at low magnification.
No labeling was seen in the cavity or hilus of the IO nuclei, suggestive of a lack of labeling to the axons leaving from the IO nuclei Figures 9E,F. The pyramidal tract Py occupying the most medioventral part of the oblongata was essentially avoid of immunolabeling. There existed a small group of labeled cells around the medioventral surface of the pyramid, representing the neurons of the arcuate nucleus AN Figure 9A.
Figure 9. At low magnification, labeled elements are mainly arranged as groups of cells located medial and lateral to the fourth ventricle 4V , and in the ventrolateral areas of the pons corresponding to the main and accessory nuclei dorsal and medial, IOd and IOm of the inferior olive IO.
Thus, labeled neurons are located in all cranial nerve nuclei at this level, including the cochlear and vestibular nuclei 8N , dorsal motor nucleus of vagus 10N , hypoglossal nucleus 12N , facial nucleus 7N , trigeminal sensory nucleus 5N , the solitary tract nucleus SoN and the reticular formation RF B—D. Neurons in the arcuate nucleus AN are also labeled, whereas the pyramid Py is unlabeled A.
Labeled neurons in the IO are mostly multipolar in shape by closer examination E,F. Scale bars are as indicated in the panels. On examination of sections from different anatomical locations, it was impressive that neurons exhibiting the strongest sortilin IR were often the largest in size relative to other labeled cells in the same region.
For examples, the large-sized pyramidal neurons in the neocortical layers III and V were heavily labeled, as were the large spiny neurons in both the neo- and paleo-striatum. This led us to speculate if there exists a correlation between somal size and sortilin expression among morphologically similar neurons or even different neurons.
Therefore, we carried out quantitative cellular analyses over the subnuclei of the amygdaloid complex, included the BLd, LA and BM subdivisions, as well as overall layers II—VI of the temporal neocortex from 3 brains case 3, 5, and 6. Figure Correlative analysis on somal area and immunoreactivity of sortilin-labeled neurons in the basolateral dorsal nucleus BLd , lateral nucleus LA and basomedial nucleus BM of the amygdaloid complex A—G , and the temporal neocortex H.
Panel A is a combined image consisted of microscopic fields from the above nuclei as indicated, obtained from the same Motic-scanned image. Panel B is a screen-print working document viewed on the OptiQuant interface, which is in gray-scale TIFF format with pseudocolor representation of optic density variability among labeled profiles.
Specific optic densities are calculated by using the optic density measured over a section processed in parallel but omitting the primary antibody incubation as a cut-off threshold. Panel D plots the specific optic densities of corresponding groups of individual neurons. The medians are significantly different between individual paring groups.
The numbers n of neurons measured are also labeled in the graph panels. A total of sortilin labeled neurons in the BLd nucleus, neurons in the LA and neurons in the BM were measured for brain case 3. The mean somal area was significantly larger for the neurons in the BLd Measurements from the brains cases 5 and 6 revealed similar differences as the above in regard to the somal size and labeling intensity of neurons between the amygdaloid subnuclei graphs not shown , with a positive correlation between the two indices while the measurements from individual neurons were plotted together Figures 10F,G.
We carried out the same type of cellular quantification as above over cortical layers II—VI of the temporal neocortex also using sections passing the amygdaloid complex from brain case 3, 5 and 6.
Shown as an example case 3 , there was also a positive correlation between the somal size and intensity of sortilin immunolabeling among the populations of neocortical neurons Figure 10H. We carried out multiple sets of double immunofluorescent characterization to determine the cellular phenotypes of sortilin-labeled cells in selected brain structures, including the cerebral neocortex, hippocampal formation, cerebellar cortex and several subcortical locations.
As assessed in sections of temporal neocortex and hippocampal formation, sortilin IR appeared to mostly colocalize with NeuN, the common mature neuronal marker Figures 11A—H. For each of the above regions, About NeuN immunofluorescent cells per case used cases 3, 4, 5, and 6 were analyzed, with those displayed distinct sortilin colabeling counted.
Among the total amount of NeuN-labeled neurons, Confocal double immunofluorescent characterization of sortilin labeling with neuronal markers. Antibody markers and imaged areas are as indicated, with cell nuclei displayed by bisbenzimide Bis stain in blue.
Panels R1—3 plot quantification of the colocalization rates. We also examined sortilin colocalization with markers of GABAergic inhibitory interneurons with double immunofluorescence.
In double labeling for sortilin with Sp8, a putative common marker of cortical GABAergic interneurons, we did not find an impressive colocalization of the two markers data not shown. By overall scanning of the merged images, sortilin appeared to visualize largely a separate population of cells different from that of PV and CB labeled ones in the same microscopic field Figures 11I—P.
The somata and proximal dendrites of sortilin immunoreactive neurons were surrounded by PV immunoreactive terminals Figures 11I—L , characteristic of GABAergic perineuronal nets around excitatory cortical pyramidal neurons Enwright et al. Among the total CB-labeled interneurons based on — neurons counted per region per brain , In contrast to the neuronal markers, sortilin IR in the temporal neocortex was not found to colocalize with immunofluorescence of GFAP, the commonly used astrocytic marker Figures 12A—D.
There was also no any immunofluorescent colocalization of sortilin with Iba1, a microglial marker Figures 12E—H. Sortilin with PV and CB not shown double immunofluorescence was also carried in cerebellar cortical sections. Sortilin IR was faint or not readily detectable in the somata and dendrites of Purkinje cells Figure 12I , While PV immunofluorescence was distinct in somata, dendrites as well as axonal processes of Purkinje cells Figures 12J,K.
Consistent with the peroxidase-DAB labeling, small dot-like sortilin immunoreactive elements were seen around the Purkinje cell layer between the somata and proximal dendrites of the Purkinje cells Figure 12L. In addition to the above, we carried out confocal double immunofluorescence in several subcortical structures including the BNM, SNc, LC and brainstem. Sortilin and TH colocalization was invariably seen among neurons in the SNc Figures 12M—P as well as the LC not shown , indicating sortilin expression in dopaminergic neurons and noradrenergic neurons, respectively.
A complete colocalization of sortilin in ChAT positive multipolar cells was detected in sections covering the basal forebrain region over the area of BNM Figures 12Q—T. Confocal double immunofluorescent characterization of sortilin labeling relative to glial and additional neuronal markers.
Antibody markers and imaged areas are as indicated, with cell nuclei displayed by bisbenzimide Bis. Panel A—D show that sortilin labeling does not colocalize with immunofluorescence of glial fibrillary acidic protein GFAP , an astrocytic marker, around the border of layer VI and the white matter WM of the temporal cortex Ctx.
There is also no colocalization between sortilin and Iba1, a marker of microglia E—H. Sortilin labeled dot-like elements white arrows are present around the Purkinje cell somata. Panels M—P demonstrate a complete colocalization of sortilin in dopaminergic neurons in the pars compacta of the substantia nigra SNc as visualized by the tyrosine hydroxylase TH antibody.
The present study shows a widespread sortilin IR in the human brain across major anatomical locations, with considerable laminar and neuronal phenotype variability.
The findings can be summarized as the following Table 2. DAB-based immunolabeling also shows that several interneuron populations in subcortical structures, e.
Table 2. Semi-quantification of sortilin immunoreactivity in major neuroanatomical regions and neuronal populations in the adult human brain. Data regarding the whole brain expression pattern of sortilin in mammalian species are limited to date.
An early study, which used a different antibody from the ones used here, reported the distribution of sortilin IR in the rat central nervous system Sarret et al. According to the rat study Sarret et al. Thus, the labeling in the cerebral neocortex and paleocortex is mostly distinct in layers II and V pyramidal neurons.
In the hippocampal formation, labeled neurons appear to be mainly interneuron-like and are distributed in the s. In the cerebellar cortex, the somata and dendrites of Purkinje cells exhibit heavy labeling, whereas other cellular elements show little labeling. Thus, there appear to have substantial difference in the regional and cellular expression of sortilin in rodent brain relative to humans as revealed in the current study.
Notably, in a recent publication used the same goat antibody as in the current study Boggild et al. In comparison, sortilin IR is much limited in more caudal brain levels including the cerebellum Boggild et al. Another recent study also shows a greater sortilin expression in the forebrain than hindbrain in mice, with the cerebellum displayed fairly low labeling Johnson et al.
The morphometric analysis establishes that among amygdalar and neocortical neurons, those in larger somal size tend to have stronger sortilin expression.
The relatively large-sized PV neurons contain granule-like sortilin-IR, with a higher sortilin colabeling seen in those in CA1 relative to the neocortex, likely because hippocampal PV neurons are often relatively large while cortical PV neurons are more variable in size. Sortilin expression is generally low or minimal in CB cortical neurons that are much smaller than principal neurons Yan et al.
However, it should be noted that cerebellar Purkinje cells and OB mitral cells are large-sized neurons in the corresponding regions, and are GABAergic and glutamatergic, respectively, while neither exhibits strong sortilin IR. The GABAergic nature of Purkinje cells might be a reason for their lack of impressive sortilin expression. Purkinje cells and mitral cells also appear quite unique relative to other neurons as they do not express the common neuronal marker, NeuN Herculano-Houzel and Lent, It is of interest to note that a recent study shows segregated localizations of sortilin and SorCS2, another member of the vps10p family, in mouse brain during embryonic to postnatal development.
Thus, the regionally segregated expression of vps10p family proteins might also account for a low expression of sortilin among the neurons in the cerebellum and OB.
The observation of somal size as a factor correlating to the amount of sortilin expression might be of functional relevance. Recent cell biology and in vivo studies have increasingly recognized the role of sortilin for protein sorting, trafficking and homeostasis in neurons Allard et al.
Neurons are unique relative to other bodily cells most prominently as they generate and transmit electronic impulses to communicate between each other. This physiological property is carried out by structurally specialized cellular apparatus, dendrites and axons, which can extend far away from their parent somata. Larger somal size means a greater cell surface for ligand-receptor interaction as well as the operation and maintenance of membrane-associated molecular systems responsible for signaling functions.
Thus, based on its assumed role in transmembrane signaling and intracellular protein trafficking in neurons, one can expect that larger neurons may require a higher supply of sortilin to carry out the above cellular functions. Further, because sortilin is primarily expressed in the somata, dendritic processes and spines, this VPS10P member appears to mainly support protein sorting in the somatodendritic, including postsynaptic, compartments, but may not play a significant role in axonal protein transportation.
As denoted in the Introduction, NT has been considered a drug target for some neurological and psychiatric conditions, with efforts being taken to develop pharmaceutically usable agonists and antagonists to its receptors, including sortilin as NTR3 Lazarus et al.
This has also raised discussion of pharmacological manipulation of sortilin as a disease modifying strategy, regardless whether it involves neurotensin signaling. The current mapping study indicates that sortilin is enriched in many neuronal populations of the human brain, such as the cerebral principal neurons as well as subcortical dopaminergic, noradrenergic and cholinergic neurons. However, the broad expression of sortilin in multiple neuronal populations as seen in the human brain also raises a concern of substantial neurological side-effects.
Therefore, in future development and evaluation of centrally and peripherally acting drugs for some specific diseases or clinical conditions Baxendale et al. In summary, the present study has extended a regional and cellular mapping of sortilin expression in the adult human brain through immunohistochemical characterization. Sortilin immunolabeling occurs broadly in cortical and subcortical structures, with large-sized neurons exhibiting heavier labeling relative to smaller ones.
JM and X-XY contributed to neuropathological evaluation. X-XY contributed to funding acquisition, experimental design and manuscript composition. The sponsors have no role in the design of this work; collection, analysis, and interpretation of the data; writing of manuscript; or the decision to submit this manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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