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Cell Biology International (2004) 28, 111118 (Printed in Great Britain)
Neuronal primary cilia: a review
Jannon L Fuchs* and Harris D Schwark
Biological Sciences, University of North Texas, Denton, TX 76203, USA
Primary cilia in neurons have often been regarded as rare, vestigial curiosities. However, neuronal cilia are now gaining recognition as ubiquitous organelles in the mammalian brain, raising speculation about what their functions may be. They might have some features tailored for the nervous system and others that serve needs shared by a spectrum of other cell types. Here we review clues from the literature and present new data supporting several possibilities for the significance of neuronal cilia. Our immunocytochemical results show regional heterogeneity in neuronal cilia. Brain regions nearer to the cerebral ventricles had longer cilia, suggesting that they might sense chemicals such as peptides, originating from cerebrospinal fluid. In mutant Tg737orpkmice, most brain regions appeared to be missing cilia. The importance of intraflagellar transport proteins establishes a functional link between neuronal cilia and other primary cilia.
Keywords: Neuron, Brain, Cilium, Intraflagellar transport, sst3.
*Corresponding author. Tel.: +1-940-565-4994; fax: +1-940-565-4136
1 The long history of the discovery of neuronal cilia
Most neuroscientists would be surprised—if not downright skeptical—to hear that primary cilia are a consistent feature of neurons in the mammalian central nervous system. Over a century ago, the preeminent neuroanatomist Ramón y Cajal (Ramón y Cajal, 1995) drew and described in meticulous detail a myriad of neurons without including primary cilia, so how could neurons have them? To this day, cilia are rarely catalogued among the organelles of neurons, despite several demonstrations of their presence in brain, over a period of decades. In this review, “neuronal cilia” refers to solitary cilia—one per neuron—with the hallmark 9+0 circle of microtubule doublets forming the backbone of the ciliary axoneme. This article highlights what is known about primary cilia in the mammalian central nervous system, but it should be pointed out that primary cilia have been described in neurons of the other vertebrate classes, and in peripheral neurons and glia (Del Cerro and Snider, 1967; Grillo and Palay, 1963; Milhaud and Pappas, 1968; Taxi, 1961). In searching for clues to functions neuronal cilia might have, our scope is not limited by cell type or phylogenetic category.
Some ultrastructural studies reported that neuronal cilia are infrequent, for example, in cat spinal cord (Duncan et al., 1963), guinea pig and human retinal ganglion cells (Allen, 1965), rat lateral geniculatenucleus (Karlsson, 1966), and rat ventral tegmental area (Bayer and Pickel, 1990). Because only a small percentage of electron micrographic sections show primary cilia, it is difficult to appreciate their prevalence without systematic, labor-intensive efforts. Nevertheless, neuronal cilia were recognized as common ultrastructural features of granule cells in rat dentate gyrus(Dahl, 1963), major neuron types in rat cerebellum(Del Cerro and Snider, 1967, 1969), immature rat supraoptic nucleus (Lafarga et al., 1980), guinea pig cerebellum, hypothalamus and neocortex (Vigh-Teichmann et al., 1980), some (but not all) types of retinal neurons in cat and rabbit (Boycott and Hopkins, 1984), hamster paraventricular hypothalamic nucleus (Suarez et al., 1985), human neocortex (Mandl and Megele, 1989), and neuropeptide-containing neurons of rat striatum (Wolfrum and Nitsch, 1992). Even as evidence accumulated that neuronal cilia are standard in various species and brain regions, the impression persisted that neuronal cilia in mammals are probably regressive, vestigial, or of otherwise dubious functional consequence (Bayer and Pickel, 1990; Dahl, 1963; Duncan et al., 1963; Peters et al., 1976; Ruelaet al., 1981). This impression was apparently fueled by fragmentary evidence that primary cilia are more characteristic of earlier phylogeny and ontogeny, the belief that lack of the central microtubule pair diagnostic of motile cilia indicates regressive loss of function, and the absence of information about functions of primary cilia.
Recently, the ubiquity of neuronal cilia as well as possible clues about their functions, have been revealed by the use of two receptor antibodies that label mainly cilia, with minimal staining of somata or neuropil. In most regions of rat and mouse central nervous system, an antibody to somatostatin receptor subtype 3 (sst3) labels a single primary cilium per neuron (Händel et al., 1999; Schulz et al., 2000; Stepanyan et al., 2003). The antibody's identity was substantiated by demonstrating that it stains HEK-293 cells after transfection with sst3 mRNA, and by showing correspondence between the distribution of sst3 immunoreactivity and sst3 mRNA (Händel et al., 1999). Serotonin-6 receptor immunolabeling was localized to the plasma membrane of neuronal cilia in four brain regions (Brailov et al., 2000; Hamon et al., 1999), one of which also has neuronal cilia with sst3 receptors. As evidence for specificity, immunostained cilia were reduced in number afterintraventricular antisense serotonin-6 receptor mRNA(Hamon et al., 1999). We have found a particularly widespread distribution of immunoreactive neuronal cilia in the rat central nervous system (Fuchset al., 2003; Hughes et al., 2002), using an antibody of uncertain identity, raised against Gα
2 Not all neuronal cilia are alike: regional variations in function?
We observed that for nearly all regions of the rat CNS, each neuron appears to have a solitary cilium(Fig. 1) as visualized by distinctive staining with an antibody raised to an N-terminal peptide from subunit of mouse G11 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Fuchs et al., 2000; Hughes et al., 2002), which is the α-subunit of G
Solitary cilia labeled with an antibody raised to Gα
Tufts of cilia on ependymal cells lining the 4th ventricle as well as cilia on neurons in the central gray area, are stained with DAB-tagged antibody raised to Gα
Neurons from the ventromedial hypothalamus are dual-labeled to illustrate colocalization of the antibody raised to Gα
Regional heterogeneity in cilia length is illustrated by comparing the ventromedial hypothalamic nucleus (A) and the ventral posteromedial thalamic nucleus (B). As in Fig. 1, green is the antibody raised to Gα
3 Primary cilia as sensors
3.1 Information from sensory cilia
Specialized sensory cilia convey a large portion of the stream of information to the vertebrate brain. These cilia are typically nonmotile and are thought to be derived from primary cilia (Dubruille et al., 2002; Menco and Farbman, 1985a). Olfactory cilia are endowed with a sensory transduction kit complete with olfactory receptors, G proteins, adenylyl cyclase, and ion channels that initiate depolarization of the ciliated olfactory neuron. Sensory neurons in acoustico-vestibular systems have slightly modified cilia, either transiently during development or persisting through adulthood (Sobkowicz et al., 1995). Visual systemphotoreceptors are highly modified cilia. The phototransducing outer segment no longer resembles the ancestral cilium. The connecting cilium, which bears the 9+0 pattern of microtubules, shuttles supplies between the inner and outer segments (Liu et al., 1999; Pazouret al., 2002).
Do neuronal primary cilia detect chemical, mechanical, or photic stimuli? It seems evident that cilia accomplish sensory transduction of some sort, as has been proposed over the decades for primary cilia of various cell types. In neuronal cilia, chemosensing is the best supported to date, thanks to the discovery that neuronal cilia have receptors for somatostatin and serotonin, which can serve as neuromodulatory or neurohormonal signals. Localization of the G-proteins and effectorsthat are linked to these receptors in cilia awaits the development of suitable antibodies.
3.2 Sensing similarities with other primary cilia
Neuronal primary cilia as a research topic is quite new, and it is imperative that we build upon what is known about other cilia, especially primary cilia (cf. Primary Cilium Resource website). Neuronal ciliaresemble other primary cilia ultrastructurally, but little is known about similarities in function or molecular composition. In kidney, primary cilia extend into the tubular lumen and bend with the flow of fluid, resulting in elevated intracellular Ca2+levels (Praetorius and Spring, 2001, 2003). Polycystin-2 in the ciliary membrane contributes to Ca2+entry (Nauli et al., 2003). A similar story is unfolding for mouse embryonic peripheral node cells (McGrath et al., 2003), whose nonmotile primary cilia bend with directional fluid movement generated by motile cilia in the neighboring central node region. Again, polycystin-2 in the ciliary membrane apparently is involved in mediating mechanotransduction. The resultant asymmetry in Ca2+elevationswithin the peripheral node region promotes left–right differences in gene expression in the developing organism. Mechanosensing roles have been proposed for primary cilia on osteocytes (Whitfield, 2003) and chondrocytes (Poole et al., 1997, 2001). Mechanosensing roles may be most suited to cilia that are surrounded by fluid. In the central nervous system, mechanical stress-induced effects have been studied in connection with injury and pathology. The cerebrospinal fluid and meninges normally cushion the brain against injury, but it is conceivable that neuronal cilia sense mechanical stimuli from daily activity.
Other possible sensory functions that neuronal cilia and other primary cilia might have include monitoring osmolarity or pH. The cilium's small volume relative to surface area could allow cilia to change volume quickly in response to small changes in osmolarity; also, there is precedence for osmoreception in sensory cilia of C. elegans (Tobin et al., 2002). Extracellular pH can be affected by momentary reduction of O
4 Primary cilia as responders
Sensing in cilia is useful insofar as the end result is an adaptive response. The cilia of sensory cells transduce stimulus energy into changes in ion permeability, which in turn translates as changes in membrane potential. But how can a tiny cilium get the attention of a large neuron? Based on the information that olfactory cilia—an average of 11 per neuron in rat (Menco and Farbman, 1985b)—can trigger action potentials in an olfactory receptor neuron, it seems likely that a single primary cilium could confer upon a neuron enough bias in membrane potential to affect neuronal firing rate. The small diameter of the cilium predicts high input resistance and a large membrane potential for a given current. Electrical signals generated in the relatively short cilia might diminish less than some dendritic postsynaptic signals en route to the soma. The cilium's narrow geometry also seems ideal for retaining second messengers such as Ca2+. In neuronal soma and axon terminals, Ca2+transients are typically ephemeral, subject to immediate diffusion and a battery of strategies for defending baseline Ca2+levels. Levels of Ca2+and other second messengers in the cilium might be uniquely sustainable, resulting in prolonged changes in ionic conductances and other cellular effects.
Neurons are already exquisitely chemoreceptive. Their elaborate dendritic trees locally sense specific chemicals such as neurotransmitters, neurotrophins, hormones, and ions—so wouldn't neuronal cilia be extraneous? While synaptic membrane is dedicated to communication with other neurons, the ciliary membrane could survey the external milieu. Additionally, and perhaps most unique to the nervous system, select neuronal groups might be recruited in concert through endocrine and paracrine receptors common to their cilia, leading to specific physiological and behavioral responses.
Cilia might help protect neurons from sustained high-frequency firing, which can jeopardize a neuron's energy reserves or produce excitotoxic cell death. Over-excitation is believed to contribute to neuronal cell death in neurodegenerative diseases and other stressful conditions. If cilia can sense effects of vigorous neural activity such as altered levels of glutamate, K+, CO
5 Cilia in development
The relationship between cilia and cell proliferation is a topic of long-standing interest (Fonte et al., 1971; Ho and Tucker, 1989). Cilia emerge from basal bodies, which originate as centrosomes, which in turn organize the mitotic spindle. Precursor cells resorb cilia just before mitosis and regrow them afterwards. It is tempting to consider that cilia may mediate influences from the extracellular milieu on cell proliferation and differentiation. Ca2+levels are implicated in mediating many signals that influence whether a cell shall divide, die, or differentiate. The cilium is strategically situated to pass along such signals from its location adjacent to the basal body/centriole, close to the Golgi apparatus and not far from the nucleus.
In early fetal development, neurogenic precursors—radial glia and many astrocytes in the subventricular zone—have a primary cilium which projects into the ventricular lumen (Tramontin et al., 2003). Few details are known about the development of neuronal cilia in fetal stages (Cohen et al., 1988; Cohen and Meininger, 1987). Interestingly, they emerge transiently from olfactory neurons in prenatal development before being replaced by specialized olfactory cilia (Menco and Farbman, 1985a). Neuronal cilia were also described in some regions of early postnatal rat brain (Del Cerro and Snider, 1967, 1969; Lafarga et al., 1980). We have observed rapid appearance of Gα
6 Neuronal cilia depend on intraflagellar transport; clinical implications
We recently found that most neuronal cilia appeared to be absent in mutant Tg737orpkmice, suggesting that the intraflagellar transport protein polaris (IFT88), which is necessary for ciliogenesis in various cell types (Pazour et al., 2000; Yoder et al., 2002), is also important for neuronal cilia. Again, heterogeneity is evidenced by the observation that while many brain regions in IFT88-deficient mice lacked sst3-immunoreactive cilia, some brain regions retained shortened cilia, including much of the olfactory bulb, layer 2 of the piriform cortex, and all layers of neocortex. There is evidence that intraflagellar transport proteins are involved in active maintenance of cilia, through a dynamic balance between synthesis and breakdown of the ciliary axoneme (Baker et al., 2003; Marshall and Rosenbaum, 2001). The importance of intraflagellar transport proteins (Rosenbaum and Witman, 2002; Sloboda, 2002) in neurons of these mice supports the proposal that neuronal cilia are actively maintained and have fundamental properties in common with other primary cilia. The mutant brains were clearly hydrocephalic, as previously reported by Taulman et al. (2001). A lack of ependymal cilia to move the cerebrospinal fluid has often been assumed to cause hydrocephalus, although this causal relationship is in dispute (Roth et al., 1985, 1988). The next challenge is to unravel the sequelae of ciliary defects. Recent awareness that neurons have cilia calls for a reevaluation of clinical cases involving brain cysts and other neurological abnormalities that accompany symptoms of defective motile or nonmotile cilia in other tissues. We would expect to see individual differences in the constellations of neurological symptoms depending on which cilia-related genes are affected, especially in view of the heterogeneity of gene expression among neuronal cilia.
We thank our students who participated in the research, particularly Rhome Hughes for his morphometric study of neuronal cilia. Those currently working on the Tg737orpkmouse brains include Ankur Patel, Rajin Shahriar, Suman Pasapuleti, and Weilan Zuo. We are grateful to Dr Gregory Pazour for providing the Tg737orpkbrains. This research was supported by NIMH MH41865 (JLF), NIH NS41891 (HDS), and grants from the University of North Texas.
Baker SA, Freeman, K, Luby-Phelps, K, Pazour, GJ, Besharse, JC. IFT20 links kinesin II with a mammalian intraflagellar transport complex that is conserved in motile flagella and sensory cilia. J Biol Chem 2003:278:34211-8
Bayer VE, Pickel, VM. Ultrastructural localization of tyrosine hydroxylase in the rat ventral tegmental area: relationship between immunolabeling density and neuronal associations. J Neurosci 1990:10:2996-3013
Boycott BB, Hopkins, JM. A neurofibrillar method stains solitary (primary) cilia in the mammalian retina: their distribution and age-related changes. J Cell Sci 1984:66:95-118
Brailov I, Bancila, M, Brisorgueil, MJ, Miquel, MC, Hamon, M, Verge, D. Localization of 5-HT6 receptors at the plasma membrane of neuronal cilia in the rat brain. Brain Res 2000:872:271-5
Dale N, Roberts, A, Ottersen, OP, Storm-Mathisen, J. The morphology and distribution of ‘Kolmer-Agduhr cells’, a class of cerebrospinal-fluid-contacting neurons revealed in the frog embryo spinal cord by GABA immunocytochemistry. Proc R Soc Lond B Biol Sci 1987:232:193-203
Dubruille R, Laurencon, A, Vandaele, C, Shishido, E, Coulon-Bublex, M, Swoboda, P. Drosophila regulatory factor X is necessary for ciliated sensory neuron differentiation. Development 2002:129:5487-98
Ferjoux G, Bousquet, C, Cordelier, P, Benali, N, Lopez, F, Rochaix, P. Signal transduction of somatostatin receptors negatively controlling cell proliferation. J Physiol Paris 2000:94:205-10
Hamon M, Doucet, E, Lefevre, K, Miquel, MC, Lanfumey, L, Insausti, R. Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5-HT6 receptors. Neuropsychopharmacology 1999:21:68S-76S
Karlsson U. Three-dimensional studies of neurons in the lateral geniculate nucleus of the rat. I. Organelle organization in the perikaryon and its proximal branches. J Ultrastruct Res 1966:16:429-81
Liu X, Udovichenko, IP, Brown, SD, Steel, KP, Williams, DS. Myosin VIIa participates in opsin transport through the photoreceptor cilium. J Neurosci 1999:19:6267-74
Mandl L, Megele, R. Primary cilia in normal human neocortical neurons. Z Mikrosk Anat Forsch 1989:103:425-30
Marshall WF, Rosenbaum, JL. Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control. J Cell Biol 2001:155:405-14
Menco BP, Farbman, AI. Genesis of cilia and microvilli of rat nasal epithelia during pre-natal development. I. Olfactory epithelium, qualitative studies. J Cell Sci 1985:78:283-310
Menco BP, Farbman, AI. Genesis of cilia and microvilli of rat nasal epithelia during pre-natal development. II. Olfactory epithelium, a morphometric analysis. J Cell Sci 1985:78:311-36
Pazour GJ, Baker, SA, Deane, JA, Cole, DG, Dickert, BL, Rosenbaum, JL. The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance. J Cell Biol 2002:157:103-13
Pazour GJ, Dickert, BL, Vucica, Y, Seeley, ES, Rosenbaum, JL, Witman, GB. Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene Tg737, are required for assembly of cilia and flagella. J Cell Biol 2000:151:709-18
Poole CA, Jensen, CG, Snyder, JA, Gray, CG, Hermanutz, VL, Wheatley, DN. Confocal analysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and aortic smooth muscle cells. Cell Biol Int 1997:21:483-94
Poole CA, Zhang, ZJ, Ross, JM. The differential distribution of acetylated and detyrosinated alpha-tubulin in the microtubular cytoskeleton and primary cilia of hyaline cartilage chondrocytes. J Anat 2001:199:393-405
Praetorius HA, Spring, KR. . Ramón y Cajal S. Histology of the nervous system of man and vertebrates. Vols I and II (History of Neuroscience, No. 6). Translated by Swanson N, Swanson L. New York: Oxford, 1995.
Schulz S, Händel, M, Schreff, M, Schmidt, H, Höllt, V. Localization of five somatostatin receptors in the rat central nervous system using subtype-specific antibodies. J Physiol Paris 2000:94:259-64
Sobkowicz HM, Slapnick, SM, August, BK. The kinocilium of auditory hair cells and evidence for its morphogenetic role during the regeneration of stereocilia and cuticular plates. J Neurocytol 1995:24:633-53
Stepanyan Z, Kocharyan, A, Pyrski, M, Hubschle, T, Watson, AM, Schulz, S, Meyerhof, W. Leptin-target neurones of the rat hypothalamus express somatostatin receptors. J Neuroendocrinol 2003:15:822-30
Suarez I, Fernandez, B, Perez-Batista, MA, Azcoitia, I. Ciliated neurons in the paraventricular nuclei in old hamsters. J Submicrosc Cytol 1985:17:351-6
Taulman PD, Haycraft, CJ, Balkovetz, DF, Yoder, BK. Polaris, a protein involved in left-right axis patterning, localizes to basal bodies and cilia. Mol Biol Cell 2001:12:589-99
Tobin D, Madsen, D, Kahn-Kirby, A, Peckol, E, Moulder, G, Barstead, R. Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron 2002:35:307-18
Tramontin AD, Garcia-Verdugo, JM, Lim, DA, Alvarez-Buylla, A. Postnatal development of radial glia and the ventricular zone (VZ): a continuum of the neural stem cell compartment. Cereb Cortex 2003:13:580-7
Yoder BK, Tousson, A, Millican, L, Wu, JH, Bugg, CE, Schafer, JA. Polaris, a protein disrupted in orpk mutant mice, is required for assembly of renal cilium. Am J Physiol Renal Physiol 2002:282:F541-52
Received 30 September 2003; accepted 4 November 2003doi:10.1016/j.cellbi.2003.11.008