Cell Tissue Res (1992) 267:199-202 Cell&Tissue

Research �9 Springer-Verlag 1992

High frequency of ciliated neuropeptide Y-immunoreactive neurons in rat striatum* Gabriele Wolfrum 1 and Cordula Nitsch 2

i Anatomische Anstalt der Ludwig-Maximilians-Universit~it Mfinchen, Pettenkoferstrasse 11, W-8000 Mfinchen, Federal Republic of Germany 2 Anatomisches Institut der UniversitSA Basel, Pestalozzistrasse 20, CH-4056 Basel, Switzerland

Received June 17, 1991 / Accepted August 29, 1991

Summary. An analysis of the ultrastructure of neuropep- tide Y-immunoreact ive neurons in rat striatum revealed the presence of a cilium in half of the neurons serially sectioned in part, and in a quarter of the neurons ob- served in single sections. It is speculated that the cilium is a developmental remnant, i.e., a sign of the less differ- entiated state of the NPY-containing neurons compared with the other neurons, and that this could explain the plasticity of this type of neuron after lesions.

Key words: Striatum - Neuropeptide Y - Cilium - Plas- ticity - Rat (Wistar)

The occurrence of cilia in neurons of the central neurons system (CNS) was first reported by De Robertis (1956) in retinal receptor cells in the kitten, and by Palay (1961) in the neurosecretory cells of preoptic nucleus of the goldfish. Systematic descriptions of neuronal cilia in rat CNS were carried out by Dahl (1963) in the fascia denta- ta, and by Karlsson (1966) in the lateral geniculate nucle- us. Cilia-bearing neurons have also been described in the human neocortex (Mandl and Megele 1989). In the mammal ian nucleus caudatus/putamen complex, i.e. the striatum, Rafols and Fox (1971/72) observed a few single cilia on spiny neurons of primates, and Chung and Keefer (1976) described cilia in astroglia and in a single neuron of the cat fundus striati.

Here, we report that a high port ion of immunocyto- chemically identified striatal neurons, the neuropeptide Y (NPY)-containing neurons, possess a cilium.

Materials and methods

This study is part of a larger investigation on the ultrastructure and synaptic connectivity of NPY-immunoreactive striatal neu-

* This work is part of the thesis of G. Wolfrum submitted to the Ludwig-Maximilians-Universitfit in partial fulfillment for the re- quirements of a Dr. rer. nat. degree

Offprint requests to: C. Nitsch

rons. Seven male adult Wistar rats, 4 of which, 3 days previously, had received a single injection of the specific neurotoxic agent 6- hydroxydopamine into the left substantia nigra, were perfused with aldehyde fixative (4% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M phosphate buffer). Serial Vibratorne sections were incubated with a rabbit NPY-antiserum (specifications given in Schwartzberg et al. 1990), diluted 1:800 in TRIS-buffered saline (TBS, 0.05 M, pH 7.6), for 48 h. Immunoreaction was carried out with the avidin- biotin-complex method of Hsu et al. (1981) and visualized with diaminobenzidine. Osmicated sections were block-stained with ura- nyl acetate in 70% ethanol, dehydrated in graded ethanol and flat embedded in Epon.

Both single and serial ultrathin-sectioned NPY-immunoreactive neurons were studied. For serial sections, areas containing intensely stained NPY-neurons were trimmed. Ultrathin sections were col- lected until the immunoreactivity faded in the depth of the tissue. It should be noted that, in non-solubilized tissue sections as used for the present study, the immunoreaction only takes place in struc- tures (dendrites or perikarya) open to the surface. Penetration of the antibodies depends on the size of the surface opening that - when large will result not only in good staining, but also in substantial loss of part of the neuron for the serial analysis.

Results and discussion

Of 11 NPY-immunoreact ive neurons cut partially in seri- al sections, 6 possessed a cilium. The origin of the cilium was always located at one pole of the mostly bipolar perikarya near the base of the proximal dendrite. Large Golgi complexes were observed in this area, often in close vicinity to the centriole associated with the basal body of the cilium. In most cases, these Golgi cisternae exhibited intense immunoreact ion product (Fig. 1 b, c), in contrast of the other Golgi complexes in the cyto- plasm distant f rom the centriole (see Fig. 2 a-c). The as- sociation of the cilium with the basal body could be seen in longitudinally sectioned cilia (Fig. 1 b, c). Striated rootlets, often obscured by the immunoreact ion product, radiated into the center of the perikaryon, occasionally turning back towards the plasma membrane (Figs. 1 a, 2b). In cross sections, both basal body and associated centriole had a ring-like structure (Fig. 2d), which has been described as a ring of triplets (Dahl 1963; Allen

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Fig. 1. a Oblique section through the proximal part of a cilium emerging from an NPY- immunoreactive soma. A 9+0 arrangement of tubuli (arrowheads) can be seen. Fine rootlets radiate from the basal part into the cytoplasm (arrows). b, e Serial neighboring sections of a longitudinally cut cilium and its basal body (B). The cilium extends into the neuropil over a distance of about 3 gm and is enclosed by a tubular extension of plasma membrane. The associated centriole (Ce) lies in close proximity to Golgi cisternae (G) showing strong NPY- immunoreactivity. Nearby an non-immunoreactive terminal ( - 7 ) contacts the soma. Bar: 1 gm

1965). The exact configuration, however, was obscured by the impaired tissue preservation because of the im- munoreaction. The typical ring formed by 9 elements could be recognized only when the cilium had reached the surface of soma where it lay in a protrusion of the cytoplasm (Fig. 2e). In contrast to the situation with glial cilia (Dahl 1963; Chung and Keefer 1976), neuronal cilia in our material were never situated in an invagina- tion of the plasma membrane. They directly emerged from the protursion of the perikaryal cytoplasm (Fig. 2 e, f) to project into the neuropil (Fig. 2g, h), where they could be followed for a distance up to 3 gm (Fig. I c). No specialized contacts to other cells or cell elements were found. The cilia tapered off distally and ended ap- parently blindly among other cell processes. During their extraperikaryal course, one filament (the exact structure in doublets is not recognizable) was dislocated to the center of the cilium, resulting in the 8 + 1 arrangement (Fig. 2i). Thus, as described by Dahl (1963) and Allen (1965) for neuronal cilia, a 9 + 0 organization in the cyto- plasmic and proximal part of the cilia was found in our material; this changed to a 8 + 1 pattern in the periphery of the cilia. More than 1 cilium and 1 centriole were never found per neuron, in agreement with Karlsson (1966).

We had the impression that large numbers of NPY- containing neurons possessed a cilium. We not only found cilia in half of the neurons cut at least partially in serial sections, but also in (or associated with) a quarter of those NPY-positive perikarya observed in single ran- domly collected sections (in 11 out of 37 NPY-positive neurons). In the striatal neuropil analyzed in this investi- gation, we also observed two cilia that were sectioned peripherally and that could not be assigned to a specific cell. It could be argued that our attention was focused on NPY-immunoreactivity thus resulting in a bias of

Fig. 2. a--e Serial sections of the peripheral part of a bipolar NPY- immunoreactive perikaryon bearing a cilium. NPY-immuno- reactivity is found in patches in the cytoplasm, concentrated around neurofilaments and the outer membrane of mitochondria. Characteristically, several large dense-core vesicles (see enlargement in fl) with immunoreaction product are distributed in the cytoplasm. Rectangular areas are further magnified in d, f and h Bar: 1 gm. d The basal body (B) lies near the cellular surface. The associated centriole (Ce) is located deeper in the cytoplasm and is surrounded by peripheral profiles of the Golgi cisternae (G) that, in this section, are not immunoreactive, as are other Golgi cisternae at some distance from the centriole (section 1); dcv dense-core vesicles, e The cilium (Ci) is located just beneath

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the plasma membrane. A circular arrangement of 9 tubuli can be recognized, as can the peripheral portion of the centriole (Ce) in the cytoplasm (section 7). f The outer segment of the cilium (Ci), at its point of protrusion, is engulfed by the cellular membrane. Arrows show rootlets radiating into the cytoplasm (section 9). g The cilium (Ci) is still attached to the perikaryon

and contains 9 peripherally located tubuli (section 11). h The cilium (CO lies free in the extracellular space. A 9 + 0 arrangement is still present (section 15). i A distal section of the cilium; its tubuli are rearranged, so that 8 peripheral tubuli surround a nearly central one. The ciliary membrane has an irregular appearance (section 26). Bar: 1 gm

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our analysis. However , cilia were rarely noted in other ul t ras t ructural studies o f s t r ia tum (Rafols and Fox 1971/ 72; Chung and Keefer 1976), and in our own parallel s tudy on GABAerg i c neurons in rat s t r ia tum (in prepa- rat ion) cilia were never observed. Thus, we would like to p ropose that possession o f a cilium is a characterist ic feature o f striatal NPY-c on t a i n i ng neurons. NPY- im- munoreac t ive cells in other brain areas were no t yet in- vestigated in this respect.

A l though the a m o u n t o f material that we analyzed at the ul t ras t ructurat level is insufficient to make any quant i ta t ive assessment, it should be ment ioned that no apparen t difference was noted in the f requency o f ciliat- ed N P Y - n e u r o n s in control rats vs. rats subjected to 6 -hydroxydopamine- induced dopaminerg ic denervat ion. Thus, our s tudy has no relat ionship whatsoever to the repor t o f Mi lhaud and Pappas (1968) where a massive increase in ciliation was described in cat brain nuclei after a 6-day- long injection o f the m o n o a m i n e oxidase inhibitor pargyline [in a dosage 70 times higher than the oral dose r ecommended for humans ( G o o d m a n and Gi lman 1975)].

Striatal N P Y - n e u r o n s are aspiny medium-sized inter- neurons (Smith and Parent 1986; Kowall et al. 1987). On denervat ion o f the str iatum, they show a certain de- gree o f plasticity as indicated by their apparen t increase in number after lesions o f the dopamine -p roduc ing cells o f the substant ia nigra (Kerkerian et al. 1986) or after cortical ablat ion (Kerker ian et al. 1989). Neurona l cilia m a y have a sensory funct ion (Vigh-Teichmann et al. 1976a, b; Kemali and Gioffre 1978) or they m a y repre- sent functionless vestigial structures f rom earlier devel- opmen t (Ruela et al. 1981; Mar re ro et al. 1987). The hypothesis o f Palay (1961), i.e., tha t neuronal cilia are survivors o f those existing in the primitive neuroepi the- lium, seems especially relevant for our purely circum- stantial speculat ion tha t N P Y - n e u r o n s have arrested their differentiat ion at a somewha t earlier stage than the more mature , non-ci l iated striatal neurons, and therefore have preserved the potent ial to react and adap t to changes in their local envi ronment as, e.g., p roduced by different types o f deafferentat ion.

Acknowledgements. We thank Dr. J. Unger for the NPY-antibody. This study was supported in part by DFG (SFB 220, TP C5) and by Swiss Nationalfonds (31 .-25292.88).

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