J. Cell Sci. 36, 175-199 (i977) 175 Printed in Great Britain INTERCELLULAR JUNCTIONS IN THE CENTRAL NERVOUS SYSTEM OF INSECTS NANCY J. LANE, HELEN LE B. SKAER AND LESLEY S. SWALES A.R.C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology, Downing Street, Cambridge CBz 3EJ, England SUMMARY The intercellular junctional complexes in the central nervous system (CNS) from a variety of insect species have been examined by thin-sectioning and freeze-fracturing techniques. Of particular concern has been the fine-structural basis of the blood-brain barrier observed to be present in the outer perineurial layer around the avascular insect CNS. The basis of this has been found in the form of tight junctions (zonulae occludentes) present both in sections and in replicas of the perineurium. In the latter, they appear as one or two simple linear ridges, lying parallel to the outer surface, which occasionally display overlapping. The complex geometry of the interdigitating perineurial cells apparently permits such a relatively simple series of ridges to function as a barrier, since tracers are found not to penetrate beyond this level into the underlying nervous tissue. Such evidence is supported by microprobe X-ray analysis of lanthanum-incubated tissues, the perineurium compared with the glia-ensheathed axons show- ing the presence and absence of lanthanum, respectively. Possible physiological mechanisms that could operate 'in vivo' to maintain the blood-brain barrier are also considered. Other intercellular junctions such as desmosomes, septate junctions and gap junctions are found in the perineurial layer too, the last exhibiting EF particle plaques and PF pits. Glia-glia junctions also occur in some insect species; they include desmosomes, inverted gap junctions and occasional tight junctions. Septate, gap and tight junctions are also found on the membranes of tracheoles penetrating the CNS. Short, ridge-like elaborations and other particle arrays are found on the PF of the axon surfaces and the significance of these structures is discussed. INTRODUCTION A wealth of detail has recently been forthcoming on the fine structure of intercellular junctions as revealed by tracer studies and freeze-fracture techniques (see reviews by McNutt & Weinstein, 1973; Overton, 1974, and Staehelin, 1974). The great bulk of these observations, however, has been made on vertebrate material. Those invert- ebrate tissues which have been investigated include a variety of different epithelial tissues (Noirot & Noirot-Timoth6e, 1967; Danilova, Rohlenko & Bodryagina, 1969; Gilula, Branton & Satir, 1970; Leik & Kelley, 1970; Flower, 1971, 1972; Gilula & Satir, 1971; Hudspeth & Revel, 1971; Hand & Gobel, 1972; Satir & Fong, 1972; Noirot-Timothee & Noirot, 1973, 1974a, b; Flower & Filshie, 1975; Skaer, Berridge & Lee, 1975), the nervous system of insects (Gemne, 1969; Skaer & Lane, 1974), Correspondence to: Dr Nancy J. Lane, Department of Zoology, Downing Street, Cambridge CB2 3EJ, England. 176 N. J. Lane, H. le B. Skaer and L. S. Swales other arthropod tissues (Gilula, 1972, 1975; Johnson, Herman & Preus, 1973; Johnson, Quick, Johnson & Herman, 1974; Peracchia, 1973a, b, 1974) and tunicate heart (Lorber & Rayns, 1972). These reports show that invertebrate junctions, especially those of insects, differ from those of vertebrates in a number of features. In particular, with the exception of tunicates, the tight junctions or zonulae occludentes appear either to be missing (Satir & Gilula, 1973; Noirot & Quennedy, 1974) or are only rarely to be found (Skaer & Lane, 1974). In an attempt to look in more detail into the question of the existence of tight junctions in arthropods and to elucidate further the features of the junctional complexes in the insect nervous system, an extensive study of tissues from a variety of insects has been undertaken in this investigation. The avascular central nervous system of insects was chosen for examination since it has been shown to possess a blood-brain barrier (see, for example, Treherne & Pichon, 1972; Lane, 1974; Treherne, 1974); the existence of such a barrier suggests the liklihood that the outer ensheathing cell layer, the perineurium, represents the site of an occluding structure of some kind. A variety of earlier studies on thin sections of insect nervous tissues (for a review see Lane, 1974) indicate that the perineurium possesses a number of different junctional types, including septate junctions, desmosomes and gap junctions. In addition the existence of perineurial tight junctions or zonulae occludentes has been shown in thin-sectioned material (Lane, 1972, 1974; Lane & Treherne, 1972, 1973; Leslie, 1973; McLaughlin, 1974; Skaer & Lane, 1974; Lane, Leslie & Swales, 1975 a). Five genera of insects were studied in the present work in an attempt to cover a range of species and to include both hemimetabolous and holometabolous types. As preliminary reports predicted (Skaer & Lane, 1974; Lane, Skaer & Swales, 19756) the tight junctions of insects are among those which display differences from vertebrate junctions; their distribution and structural features, together with their possible physiological role in the insect central nervous system, are discussed. MATERIALS AND METHODS The insects used included adult specimens of the cockroach, Periplaneta americana, the locust, Schistocerca gregaria, the stick insect, Carausius morosus, the moth, Manduca sexta, and the blowfly, Calliphora erytlirocepliala. The tissue examined was from the central nervous system, including ganglia and interganglionic connectives from the abdominal nerve cord of the first 4 species, and thoracic and cephalic ganglia from the blowfly. In all cases the tissues were exposed by a dorsal incision, and flooded with fixative. The nervous tissue was then dissected out and placed in fresh fixative solution for the duration of the fixing time. Conventional fixation and embedding The tissues were fixed in glutaraldehyde, ranging from 2 to 3-5 %, in one of two buffers, phosphate or cacodylate, pH 6-9-7-4. The osmolality of these buffers was adjusted to approxi- mate to that of the haemolymph of the insect being fixed. This was achieved by varying the concentration of buffer and by adding suitable concentrations of sucrose. In some cases Karnovsky's fixative (Karnovsky, 1965) was employed. After fixation, which took place at room temperature (R.T.) for 30 min to 2 h, the tissues were washed in several changes of buffer to which appropriate concentrations of sucrose were added. Treatment with buffered Intercellular junctions in the insect CNS 177 1 % osmium tetroxide plus sucrose followed for about 60 min at R.T. The tissues were then stained en bloc with 2 % uranyl acetate, usually dissolved in sodium hydrogen maleate buffer, pH 60. The material was rinsed before and after this staining treatment with a sodium hydrogen maleate buffer wash at pH 52. Dehydration through an ascending series of ethanols, and propylene oxide ensued, followed by embedding in Araldite. Ultrathin sections were cut on an LKB Ultrotome III, stained with lead citrate and uranyl acetate, and examined in a Philips EM300. Lanthanum incubation The nervous tissues were treated with lanthanum in one of two ways: (1) Unfixed material was incubated 'in vivo' in i, 5 or 10 mM lanthanum in appropriate Ringer solution for 10 min to 2 h. This allows the uptake of lanthanum to occur under 'physiological' conditions. The lanthanum solution was pipetted out from around the dissected tissues and replaced with glutaraldehyde made up in phosphate buffer which precipitates the lanthanum. The tissue was then dissected out and placed in fresh fixative solution after which it was treated in the con- ventional fashion as outlined above. (2) Tissue was fixed for 16 h with a solution of 3 % glutaraldehyde in 01 M cacodylate buffer plus 0-2 M sucrose to which 1 % colloidal lanthanum hydroxide, prepared from lanthanum nitrate, had been added. This was followed by buffer washes, treatment with osmium tetroxide in collidine buffer pH 7-2 and en bloc staining with uranyl acetate, with lanthanum added to the solutions used at each of these stages. This tech- nique permits elucidation of the details of the external membrane surfaces (ES) composing the various junctional complexes.* A variety of thin sections, incubated in lanthanum at different concentrations for varying periods of time, were examined by electron-probe microanalysis. The analysis was carried out in an EMMA-4 analytical microscope. Lanthanum analyses were performed at an accelerating voltage of 40 kV with a probe current in the neighbourhood of 20 nA. Freeze-fracturing The nervous tissue was frozen either unfixed or fixed, more frequently the latter, unfixed preparations mainly being examined to check that no fundamental differences existed between specimens prepared in the two ways. If fixed, tissue was dissected out in fixative in the usual way and then placed in fresh fixative solution at R.T. for a total of 30 min. The fixative used was glutaraldehyde in phosphate or cacodylate buffer, as described earlier. The tissue was then washed in several changes of buffer before being placed for 30 min at R.T. in 20 % glycerol in a suitable buffer wash. The tissues were subsequently placed in copper holders, mounted in a yeast paste. Tissue and holder together were rapidly frozen in liquid Freon 22 cooled with liquid nitrogen. The material was placed in a Balzers freeze-etching device (BA 360M), and fractured at— 100 °C. Shadowing with a tungsten-tantallum