J. Anat. (1979), 128. 2, pp. 411-443 With 11 figures Printed in Great Britain

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Proceedings of the Anatomical Society of Great Britain and Ireland NOVEMBER 1978 The Annual General Meeting for the session 1978-9 was held on Friday and Saturday, 24 and 25 November at the Department of Anatomy and Embryology, University College, London. The following are the authors' abstracts of papers:

COMMUNICATIONS 1. Postnatal development of glial cells in the visual cortex of the rat: a qualitative ultrastructural analysis. By R. LUDER, J. G. PARNAVELAS and A. R. LIEBERMAN. Department of Anatomy and Embryology, University College, London (Fig. 1) The development of astrocytes, oligodendrocytes and microglial cells was studied by electron microscopy in the visual cortex of female albino rats 0, 4, 6, 8, 10, 12, 14, 16, 20, 24 days and 3 and 6 months old. To avoid bias in the selection of cells for analysis and to ensure inclusion in the material of cells at all levels of the cortex, the survey was conducted on photographic montages, each comprising a strip of cortex 75 /,tm wide and extending from the pia into the subcortical white matter. Six montages were prepared, from either two or three animals, at every age, i.e. 72 montages in all. Every cell with its nucleus present was categorized. In the mature tissue, glial cell types were identified according to currently accepted criteria (Peters, Palay & Webster, The Fine Structure of the Nervous System, Saunders, Philadelphia, 1976). In the immature specimens individual cells were classed according to sets of common characteristics and the classes related to the probable lineages of the mature cell types. The astrocyte line consisted of astroblasts and immature and mature astrocytes, criteria for the identification of whicb were presented. At birth only glioblasts, including some astroblasts, and immature astrocytes were recognized and these were replaced by more mature cells until at 3 months old only a few immature astrocytes (A, Fig. 1 A) were identified. The oligodendrocyte line comprised 'light', 'medium' (0, Fig. I B, 24 days postnatum) and 'dark' oligodendrocytes (0, Fig. 1 A, 3 months old), as previously described by Mori & Leblond (J. Comp. Neurol. 139, 1970): oligodendroblasts were tentatively identified as cells intermediate in appearance between glioblasts and 'light' oligodendrocytes. 'Light' oligodendrocytes (first recognized around day 6 and most numerous at day 14) were progressively replaced by 'dark' oligodendrocytes, the 'medium' oligodendrocytes representing intermediate forms. The time course of these events was correlated with that of myelination. Microglia could not be identified with certainty before day 6 and many displayed immature cytological features up to about day 20 despite their evident involvement in phagocytic activity during this period (e.g. M, Fig. 1 C, 10 days postnatum). 2. Postnatal development of glial cells in the visual cortex of the rat: a quantitative ultrastructural study. By S. G. POLLARD, J. G. PARNAVELAS and A. R. LIEBERMAN. Department of Anatomy and Embryology, University College, London (Fig. 2) Information on glial cell development in the mammalian C.N.S. is limited and chiefly qualitative. We have attempted to analyse the process systematically and quantitatively in the visual cortex of the rat, using the 72 montages prepared as described in the preceding abstract (Communication 1). For each montage, every cell whose nucleus was present was classified and its position plotted on a map of the cortex. Cells were classified as neurons, glioblasts (in younger animals only; subdivided when possible into astroblasts and oligodendroblasts), astrocytes (mature, immature), oligodendrocytes (dark, medium, light), microglia or pericytes. The relative proportions of each cell type were calculated for each age and for different positions in the cortex, histograms of cell numbers as a function of cortical depth were prepared, examples of which are shown in Fig. 2, and neuron/glia ratios were calculated. Immature astrocytes and glioblasts predominated at birth with the majority situated beneath

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Fig. 2 the pial surface and in the lower portion of the cortical plate. By day 8 most non-neuronal cells could be classified in one of the three main glial cell categories and 80 % were astrocytes or astroblasts. From day 8 onwards, there were obvious concentrations of astrocytes in layers I and VI and a less obvious concentration in layer IV. Oligodendrocytes were first recognized in the deep cortical layers at day 6, when they constituted 4 % of the glial cell population. Thereafter the proportion of oligodendrocytes was steadily raised, and with increasing age these cells were found in greater numbers in the more superficial layers. In the more mature animals concentrations of oligodendrocytes in layers IV and VI were apparent. Significant numbers of microglial cells were first recorded at day 10 (4 % of glial cells) and adult values were reached by day 16. The neuron/glia ratio decreased from 4-54 at birth to 2-31 at 6 months, when 50 % of the glial

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cells were found to be astrocytes, 40 % oligodendrocytes, 8 % microglia and 2 % unclassified glial cells. These findings were discussed in relation to other aspects of cortical development, particularly synaptogenesis and myelination. 3. Efferent connexions of the hyperstriatum ventrale in the chick brain. By P. BRADLEY and G. HORN. Department of Zoology, University of Cambridge (Fig. 3) Recent work has suggested that a region of the chick forebrain, the medial part of the hyperstriatum ventrale (MHV), (Horn, Rose & Bateson, Science, 181, 1973; Bateson, Horn & McCabe, J. Physiol. 275, 1978) is intimately involved in the process of imprinting, through which young chicks learn the characteristics of a visually conspicuous object. An input from the visual pathways to the MHV has been described (Bradley & Horn, J. PhysioL 278, 1978); the present study was undertaken to determine the efferent projections of the region. [3H]leucine was injected into the MHV in 6 one day old anaesthetized chicks. 05 ,u of an [3H]leucine solution (specific activity 112 Ci/mmol; 20 mCi/ml) was injected over a period of 10 minutes, using a 36 gauge needle connected to a microlitre syringe. After recovery the chicks survived 2j days and were then killed. Autoradiographs were prepared from serial wax sections, dipped in Ilford K2 emulsion, exposed for 4 weeks and developed in D19. A further 6 chicks received control injections of [3H]leucine into telencephalic areas adjacent to the MHV, i.e. the hyperstriatum accessorium and the hippocampus. Injections into the MHV resulted in strong labelling over the paleostriatum augmentatum, the archistriatum intermedium and the lateral corticoid zone. Injections into the hyperstriatum accessorium produced labelling over the periectostriatal belt, the hyperstriatum ventrale and a discrete area of the lateral neostriatum as well as revealing a major descending projection to the optic tectum. Injections into the hippocampus resulted in strong labelling of the medial archistriatum (Fig. 3: pH]leucine distribution following injection into the MHV and hippocampus). We conclude that cells in the MHV in addition to sending efferents to the lateral corticoid area also send efferents to the paleostriatum augmentatum, considered to be the avian homologue of the caudate-putamen complex, and to the archistriatum intermedium. These latter two regions are thought to be concerned with the control of movement (Zeier & Karten, Brain Res. 31, 1971). 4. A stereological analysis of the frontal cortex of 30 days old rats undernourished from birth. By Y. M. THOMAS, K. S. BEDI and J. DOBBING. Department of Child Health, University of Manchester There is increasing evidence to show that the developing brain is particularly vulnerable to environmental adversity. For example, undernutrition of rats at specified periods during their brain growth spurt results in permanent deficits in brain weight and brain DNA content (Dobbing, Scientific Foundations of Paediatrics, Heinemann, London, 1974). Only one previous worker (Cragg, Brain 125, 1972) has published data on the number of synapses per neuron in the brains of such rats. The main purpose of the present investigation is to repeat and extend this work

using improved stereological procedures. Rats were undernourished from birth to 30 days, by restriction of the mother's food intake to about half that of control mothers fed ad libitum. Six control and six undernourished male rats were then killed by perfusion with 2-5 % phosphate buffered glutaraldehyde. The brains were removed and weighed. Pieces of frontal cortex from the right cerebral hemisphere of each animal were post-fixed in osmium tetroxide and processed for electron microscopy. Sections 0 5 #sm thick were prepared to include the whole depth of cortex and stained with toluidine blue. The mean diameter and numerical density of the neuronal nuclei in layers II to IV were estimated using stereological methods at the light microscope level. The same blocks were subsequently sectioned for electron microscopy. The mean diameter and numerical density of 'synapses' (i.e. for the purposes of this analysis taken to be thickening of the neuronal membrane) were estimated using appropriate stereological procedures. Undernourished rats had significant deficits (P < 0-001) in mean body and mean forebrain weights. On average they had about 38 % fewer 'synapses' associated with each neuron (P < 0 05). This was probably due to a combination of two factors: a reduced numerical density

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Proceedings of the Anatomical Society of 416 of 'synapses' and an increased numerical density of neurons. Individually, however, neither reached significance at the 5 % level. The mean nuclear and synaptic disc diameters did not differ between groups. These observations confirm some of those made by Cragg.

synapse to neuron ratio in the cerebellar cortex of the developing rat brain. By C. A. DAVIES, K. S. BEDI and J. DOBBING. Department of Child Health, University of Manchester Rats undernourished during the suckling period have smaller cerebella than rats fed ad libitum (Dobbing, Scientific Foundations of Paediatrics, Heinemann, London, 1974). There do not appear to have been any quantitative studies published on the synaptic content of the cerebella of such rats. Rats were undernourished during the first 30 days of postnatal life by restricting maternal food intake to about half that of control mothers fed ad libitum. Six control and six undernourished male rats were killed by perfusion with 2-5% phosphate buffered glutaraldehyde. The cerebellum from each animal was removed and weighed. Pieces of cerebellar cortex were taken from each animal from lobes V and VI, at the junction of the vermis and right cerebellar hemisphere. These were post-fixed in osmium tetroxide and embedded in Araldite. Using stereological procedures at both the light and electron microscope levels the number of 'synapses' (as defined in Communication 4) per neuron in the cerebellar granular layer of these rats was analysed. Mean neuronal nuclear diameters and mean synaptic disc (membrane thickenings) diameters were estimated following application of the Schwartz-Saltykov correction procedure (Underwood, Quantitative Stereology, Addison-Wesley, London, 1971). Undernourished rats had significant deficits of 71 % in mean body weight and 33 % in mean cerebellar weight. They had a 30% deficit (P < 0-01) in the 'synapse' to neuron ratio which was a function of the reduced numerical density of the 'synapses'. The mean nuclear diameters and

5. The effects of undernutrition on the

the mean numerical densities of the neurons did not differ between groups; nor were there any differences in the mean synaptic disc diameters. These data were presented in detail and discussed in the context of the critical periods for synaptogenesis and neurogenesis in the cerebellum. 6.

Regeneration of capillaries following brain damage. By J. MITCHELL (introduced by D. MAYOR). Human Morphology, University of Southampton

In experimental models of brain injury the

damaged blood vessels are able to regenerate. An

important feature of the repair process, which appears to have received little attention, is the mechanism by which the damaged vessels regenerate. The--present study concentrated upon the growth of the intracerebral capillaries within the damaged area, up to 4 weeks after a cold lesion in the mouse parietal cortex. During the second week after injury, capillaries grew into the lesion from the thickened pia. Each vessel was covered by several thin lamellae of pial cells and was accompanied by bundles of collagen fibrils. The intracerebral capillaries which grew into the necrotic zone from the surrounding brain during the 2nd and 3rd weeks after injury, were all surrounded by astrocyte processes. Between the astrocyte and endothelial cell basement membranes there was a pericapillary space which contained pericyte processes and a few collagen fibrils. Prior to the appearance of these capillaries, lacunae lined by basement membrane and surrounded by processes of one or more astrocytes were observed. Each lacuna contained a few collagen fibrils together with cell processes which were similar to the pericyte processes around the regenerating capillaries. Cells resembling immature endothelial cells were occasionally seen within the lacunae. It is proposed that the regenerating capillaries grow through or along the astrocyte lacunae and that the course of capillary regeneration is in this way influenced and guided by the reactive astrocytes.

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7. A Golgi study of the neurosecretory neurons in the supraoptic nucleus of the rat. By R. E. J. DYBALL, M. HOWARD and SHEILA K. KEMPLAY. Department of Anatomy, King's College, London Physiological studies of neurosecretory neurons, particularly those in the supraoptic nucleus of the rat, have made a major contribution to our understanding of the neurosecretory process. However, morphological studies have been hampered by-the failure of standard impregnation methods to outline the cells. In this study blocks of rat hypothalamus have been impregnated using the Golgi-Cox technique. The major advantages of this method over the rapid Golgi technique are that a far higher number of neurosecretory cells are impregnated and it allows counterstaining of the sections with neutral red. This means that the outlines of the supraoptic nucleus can be clearly defined. In either transverse or sagittal sections, neurons within the nucleus appear to be predominantly of one type; they have elongated perikarya with a transverse diameter of about 20 #esm, and relatively simple outlines with 2-4 dendritic processes. These dendrites are thick (2 #fm), and bear marked varicosities at intervals along their length, the varicosities may be up to 5 um across. Many of the dendrites are directed ventrolaterally, they rarely extend outside the nucleus or branch. Occasionally spines are seen on both dendrites and perikarya. Axons are thinner than dendrites (1 ,um) and are also often beaded. In some cases (5 out of 21) they appear to emerge from dendrites. They tend to be directed dorsally and medially towards the supraopticoneurohypophysial tract. There is no evidence of recurrent axon collaterals, although branching is sometimes seen. Emerging axons frequently cross the border of the nucleus, but no axons entering the area have been found, which implies that the input to the nucleus is probably

myelinated. 8. Topographical relationship between the pars tuberalis and the ependymal tanycytes of the jird hypophysis: an electron microscope study. By D. K. BHATTACHARJEE, P. CHATTERJEE and R. L. HOLMES. Department of Anatomy, University of Leeds (Fig. 4) The pars tuberalis of the adenohypophysis of the jird, Meriones unguiculatus, extends over the median eminence, ensheathes the infundibular stem except dorsomedially and merges with the pars distalis caudally. It is broken up by the network of the hypothalamo-hypophysial portal vessels into anastomosing cords made up of several cell types. One of these contains randomly distributed spherical granules of variable size up to about 100 nm in diameter or sometimes

larger. Tanycytes line the floor and ventral parts of the walls of the third ventricle overlying the median eminence and send long processes across it to terminate, like many nerve terminals of the tubero-infundibular system, on the outer basal lamina of the portal perivascular space. Such terminals frequently intervene between the nerve terminals and the lamina. They possess abundant microfilaments but few microtubules and also show segments of smooth endoplastic reticulum and characteristic large irregular bodies of variable electron density such as are present in other parts of tanycytes. Nerve terminals show 'neurosecretory' granules and synaptic vesicles, which are not present in tanycytes. Portal vessels run between the median eminence and pars tuberalis but at places the two are in intimate contact. As shown in Figure 4, the granulated cells (C) may be almost completely surrounded by a profusion of nerve (N) and tanycyte (T) terminals. This study extends the observation of similar contacts reported in the monkey (Knowles & Anand Kumar, Phil. Trans. R. Soc. B 256, 1969) to a rodent and, apparently for the first time, substantiates the finding with electron micrographs. The significance of these observations is discussed. 9. The localization of Tamm-Horsfall glycoprotein in the nephrons of the kidney of the Syrian hamster as demonstrated by immunofluorescence and immunoelectron microscopical techniques. By C. L. FOSTER, R. D. MARSHALL*, K. SiKRi, F. BLOOMFIELD* and D. PAULINE ALEXANDERt (introduced by A. D. HOYES). Departments of Cellular Biology, *Chemical Pathology and tPhysiology, St Mary's Hospital Medical School, London In 1951 Tamm & Horsfall described a mucoprotein (TH) of high molecular weight in human urine which inhibited haemagglutination induced by myxoviruses. A similar protein is now known 27

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Fig. 4

to be present as a constituent of normal urine in other mammalian species and there is evidence to suggest that TH is a product of the nephron, from which it is released into the urine. Cultured cells derived from baby hamster kidneys likewise synthesize and release TH into the medium. The normal function of the protein, however, remains obscure. The precise site of production of TH has been a matter of controversy, but recent work using fluorescent antibody techniques suggests that it is associated with the cells of the ascending limb of Henle's loop and the distal convoluted tubule in rats and in man. Following work on the production of TH by baby hamster kidney cells, the adult hamster kidney has now been examined with a view to identifying the intrarenal location of TH. Peroxidase-labelled antibody methods were used at the electron microscope level in addition to fluorescent antibody techniques combined with phase contrast microscopy. The results show a very precise localization of TH in the cells of the ascending limb of Henle's loop and the distal convoluted tubule, except that in the cells of the macula densa TH is virtually absent - an observation not previously reported. Further, this mucoprotein appears to be associated with the plasma membranes of the cells. On the basis of these new observations and the interesting fact that TH appears to be restricted to that part of the nephron believed to possess a low permeability to water and involved in the urine dilution process, it is postulated that the absence of TH from the cells of the macula densa permits them to 'sense' the constituents of the luminal fluid without significantly changing its

composition. 10. The effects of bilateral adrenalectomy on the Tamm-Horsfall glycoprotein of the nephron of the kidney of the Syrian hamster - some pilot experiments. By D. PAULINE ALEXANDER, C. L. FoSTER* and K. SIKRI* (introduced by A. D. HoyES). Departments of Physiology and *Cellular Biology, St Mary's Hospital Medical School, London As described in the preceding abstract (Communication 9), the urinary Tamm-Horsfall mucoprotein (TH) first reported by Tamm & Horsfall in 1951 is, in the hamster, associated with

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the plasma membranes of the cells of the ascending limb of Henle's loop and the distal convoluted tubule, with the notable exception of the macula densa. Therefore the localization of TH appears to be confined to that part of the nephron responsible for the process of urine dilution. As this function is at least in part regulated by adrenal cortical hormones, the effect of adrenalectomy on the distribution of TH has been studied. Total adrenalectomies (which are technically difficult in the hamster) were performed on 8 adult animals and sham operations on 4. Of the former, 6 survived the operation and kidneys were removed at intervals ranging from 4 to 8 days. The sbam-operated animals were killed after 8 days. Fluorescent antibody techniques were applied to tissue sections and these were then examined under an epifluorescence system with a phase-contrast attachment. The results obtained with the sham-operated animals were identical with unoperated controls; the adrenalectomized hamsters, however, (with one exception, where possibly adrenalectomy was incomplete) showed varying degrees of disappearance of TH, initially from the distal convoluted tubule and later from the ascending limb of Henle's loop. These changes were frequently accompanied by the appearance of brilliantly fluorescing casts. These preliminary observations were discussed and data on urinary sodium levels presented. 11. G cells and antral gastrin after stimulation with acetylcholine. By N. J. McC. MORTENSEN, J. F. MoRRIS and C. OWENS. Departments of Anatomy and Surgery, University of Bristol A reduction in the electron density of gastrin (G) cell granules associated with gastrin release has been claimed (Forssmann & Orci, Z. Zellforsch. 101, 1969), but the significance of such changes in a variety of pathological and physiological conditions is unclear. Therefore, we have examined the effect of intragastric acetylcholine (ACh), which stimulates gastrin release, on the ultrastructure of antral G cells and serum and antral gastrin concentrations in rats, using stereology and radioimmunoassay. Matched animals were anaesthetized, a cannula inserted into the antrum, the oesophagus ligated, and either 2 ml of 2 % acetylcholine in 0-1 M sodium bicarbonate buffer at pH 7*5 or 2 ml of distilled water (controls) was instilled into the stomach. After either 30 or 120 minutes a blood sample was taken and adjacent strips of antral mucosa were removed. Radioimmunoassay for gastrin was performed on an extract from one strip and the serum sample; the other strip was prepared for electron microscopy. After both 30 minutes and 120 minutes serum gastrin levels in ACh-stimulated animals were 4 to 5 times higher than in controls (P < 0-01). However, there was no significant change either in the antral gastrin concentration or in the cell content or ultrastructural appearance of the secretory granules and other cytoplasmic organelles associated with hormone synthesis. This is consistent with our estimate that only less than 2 % of the antral gastrin store is released after 120 minutes stimulation with ACh. Such a small change would be undetectable using current methods even if unobscured by concurrent hormone synthesis. By contrast, prolonged fixation of the tissue resulted in a marked increase in the proportion of electron-lucent granules which was similar in both ACh stimulated and control animals. The appearance of G cell granules thus appears to be influenced more by fixation conditions than by the release of gastrin.

12. Morphological studies on the human levator ani muscle. By H. 0. D. CRITCHLEY, J. S. DIXON and J. A. GOSLING. Department of Anatomy, University of Manchester The purpose of this investigation was to determine the histological, histochemical and fine structural features of striated muscle obtained from two different regions of the human levator ani muscle. During surgical exposure of the levator ani, biopsy samples were obtained from 8 female and 2 male patients (age range 31-65 years). Of these, 6(5 female, 1 male) were removed from the levator ani adjacent to the urethra (designated region A). An additional 5 specimens (4 female, 1 male) were obtained from that part of the levator ani lying immediately anterior to the anorectal junction (region B). Part of each specimen was processed for histology and for myosin adenosine triphosphatase (Guth & Samaha, Exp. Neurol. 28, 1970) and succinate dehydrogenase (Nachlas et a!. J. Histochem. Cytochem. 5, 1957); the remainder was prepared for electron microscopy. In both regions, a heterogeneous population of type I and type II fibres was histochemically distinguished. Counts of muscle cells per unit area indicated that in region A, type II fibres 27-2

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constituted 4 % of the population whereas, in region B, they comprised 23 of muscle fibres. In addition, the mean diameters of type (45 5 S.E. ± 08 ,um) and of type II (59 5 S.E. ± 3-4 fibres in region A were significantly larger than the mean diameter of the same types of fibre (type I, 40{2 S.E. ± 6 ,um; type II, 45 8 S.E. ± 1 1 ,um) in region B. Using electron microscopy the structure of striated muscle cells from each region was similar and qualitative assessment of mitochondrial and lipid content and Z disc width did not permit individual muscle cells to be classified either as type I or as type II fibres. These findings were compared with those of other workers and discussed in relation to the function of the human levator ani muscle.

,m)

13. The extensor assembly of the opossum (Philander opossum and Didelphis marsupialis marsupialis). By J. M. F. LANDSMEER. Department of Anatomy and Embryology, University

of Leiden, The Netherlands The opossum holds a fairly firm position in concepts of primate development and it owes this position particulary to its grasping hind foot, the latter being "probably the only locomotor specialization characteristic of the whole order Primates" (Cartmill, Primate Locomotion, Academic Press, London, 1974). The hand of this animal is of the divergent-convergent type (Napier, Symp. Zool. Soc. Lond. 5, 1961), a basic or generalized type from which the hand with an opposable thumb can more or less easily be evolved. In view of the fact that arching of the fingers and thumb is a basic functional feature of the human hand - static pinch between index and thumb is the all inclusive example of this property - and as it is known from the human condition (Landsmeer, Atlas of Anatomy of the Hand, Livingstone, London, 1976), the structural basis for this function is found in the extensor assembly and its relationships. In this respect the formation of three tendon bands, through intercrossing of extensor and interosseus fibres has to be mentioned and the relative positions of these bands at the level of the trochlea of the first phalanx. Dissection of hand and foot of two opossum species (Phylander opossum and Didelphis marsupialis marsupialis) yielded the following results. No intercrossing of extensor and interossei can be observed, hence there is no trace of a formation of separate tendon tracts as in man. The extensor assembly over the dorsum of phalanx I is a structure of parallel arranged fibres from one or two extensors and with a contribution from interossei, the latter moderate to almost nothing in the toes, but fairly important in the fingers. One fairly broad tendon passing over the dorsal aspect of the trochlea is attached to the base of phalanxII. This insertion is hidden from view by the terminal tendon which originates from the main tendon so as to become inserted into the third phalanx and into the bed of the claw. A retinacular ligament is present with a most conspicuous oblique band. The appearance and macrostructure of the terminal tendon led us to investigate histologically the presence of elastic tissue in this tendon. An elastic cushion-like formation was also found on the deep aspect of the flexor profundus. This links the profundus tendon from about halfway along the middle phalanx to the neck of the latter. The terminal tendon and this cushion-like structure of the profundus tendon contain elastic fibres, both as marked elastic bands and as scattered fibres of elastic nature. The functional implications of these structural dispositions are quite remarkable. The clawbearing phalanx is held in an elastic system which invariably tends to retract the claw. The strong oblique bands assist in this mechanism so that proximal interphalangeal extension will immediately evoke claw retraction or extension of the third phalanx. Further, it does seem that both the lumbricals and the paratendineal intravaginalflexors (Wheeler Haines, J. Anat. 84, 1950) have a role in the mechanism of claw retraction. 14. Allometry, and dental proportions in fossil hominids. By B. A. WOOD. Department of Anatomy, The Middlesex Hospital Medical School, London There is general agreement that morphological differences exist between 'robust' and 'gracile' australopithecines, but there is debate about the significance of these differences. It has been proposed that the different dental proportions in the two forms are the result of difference in their overall size, but evidence is presented which suggests that this is an unlikely explanation. Dental and cranial measurements were taken on a total of 202 adult skeletons made up of samples from the following taxa; Homo, Gorilla, Pan, Papio, and Colobus. Using logarithmically

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transformed data, the least squares regression coefficients and the reduced major axis were computed for canine base area and molar crown area, using cranial length (glabella-opisthocranion) as the variable representing overall body size. Within the taxa, canine size was, with one exception, always positively allometric with respect to cranial length. Molar crown area on the other hand was consistently negatively allometric. Data from all the non-human primates were pooled to compute 'interspecific' slopes; these also displayed positive allometry for canine size and negative allometry for molar crown area. Thus, using the models of either 'intraspecific' variation or 'interspecific' variation, forms with a larger overall body size would be expected to have relatively larger canines and a relatively smaller molar crown area. When these dental dimensions are compared between the 'robust' and 'gracile' australopithecines, they show a very different pattern of variation. This makes it most unlikely that they represent functionally similar animals, exhibiting only sizerelated differences, and more likely that they represent the results of different adaptive strategies. 15. A study of brain to body size ratios in extant hominoids using skeletal material. By J. BUSH (introduced by W. J. MOORE). Department of Anatomy, University ofLeeds The interpretation of the phylogenetic significance of hominoid fossils is usually based on a comparison of their skeletal features with those of closely related living groups. In the past, much emphasis has been placed on skeletal features reflecting brain size/body weight ratios, but this approach has been hampered by lack of knowledge of differences in skeletal structure between the extant hominoids. This has necessitated making possibly unwarranted extrapolations between skeletal data obtained from fossils and somatic measurements available from man and the apes. The aim here is to describe a quantitative method, together with a statistical analysis, for comparing skeletal variations in man (Homo sapiens), gorilla and chimpanzee such that large differences are located between these species. To do this, each species was divided into eight age groups according to the stage of dental eruption. Then, using an allometric formula, endocranial capacities (a measure of brain size) were plotted against femoral length and width of the femoral shaft. Thus the growth stage, brain size and an attribute related, at least in part, to locomotion were investigated - three factors which are known to differ greatly between man and the African apes. This method accentuates the differences between the human and pongid skeletons. Although the data so far obtained are inadequate for a definitive study, the method described appears to provide the basis of a useful approach to the study of some aspects of hominoid evolution.

16. Arteriovenous anastomoses in the skin of hooded and harp seals (Pinnipedia: Phocidae). By M. M. BRYDEN (introduced by R. J. HARIuSON). Department of Anatomy, University of Cambridge The presence of large numbers of relatively small, simple arteriovenous anastomoses (AVAs) in the skin of Weddell and elephant seals was reported by Molyneux & Bryden (Anat. Rec. 191, 1978). Most AVAs are observed in the superficial layers of the dermis and are similar in position and density over the entire body surface, but this finding is not observed in some other pinniped species (Bryden & Molyneux, Anat. Rec. 191, 1978). In the present comparative study, serial sections of full thickness skin segments from the body and flippers of hooded seals (Cystophora cristata) and harp seals (Pagophilus groenlandicus) were examined, and the structure, density and distribution of AVAs in the skin were observed. In both species AVAs were small, simple structures similar to those described in Weddell and elephant seals. The densities of AVAs were: harp seal pup 1290-1560/cm2, harp seal adult 980-1100/cm2; hooded seal pup 1220-1350/cm2, hooded seal adult 1080-1240/cm2. The mean percentages of AVAs observed at different levels of the skin in both species were: superficial dermis 75 %, deep dermis 20 %, hypodermis 5 %. In neither species was there any appreciable difference in density or distribution of AVAs between body skin and flipper skin. It is concluded that large numbers of AVAs occur superficially in the skin of the entire body and extremities of many, if not all, phocid seal species, and that these structures play an important part in temperature regulation.

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17. The anatomical implications of lacrimal gland atrophy in delphinids. By G. H. WALLER and R. J. HARRISON. Department of Anatomy, University of Cambridge Odontocetes have been credited with a well-developed lacrimal gland since 1827 when Wilhelm Rapp first described this structure in Delphinus (Naturw. Abh. 1, H2, 1827). Many authors have subsequently elaborated on his findings, but others have denied the existence of a lacrimal gland in dolphins. Dissections and combined light and electron microscopic studies were made of the eyelid glands of four species of delphinid (Tursiops, Delphinus, Phocoena, Lagenorhynchus). Although a lacrimal gland primordium develops embryologically in dolphins, a functional lacrimal gland lying external to the palpebral musculature could not be found in adult animals. There is, however, marked hypertrophy of other glandular elements related to the eyelids. These consist of a circumorbitally placed conjunctival gland lying largely in the upper and lower eyelids in close apposition to the conjunctiva, and a Harderian gland lying nasal and slightly ventral to the medial rectus. The ducts open into the conjunctival sac over the whole length of the upper and lower fornices as well as the conjunctival surfaces of both lids. Both glands are of a compound seromucous tubular type. The serous cells are pyramidal with large membrane-bound osmiophilic granules occupying the narrow cell apices. The columnar mucous cells bear characteristically swollen balloon-like cell tips. Secretory granules of variable electron density form clumped masses within the cytoplasm. Direct observation shows copious secretion from the glands and the maintenance of a protective tear film over the cornea under water. These ultrastructural features and the development of a conjunctival gland mass are adaptations which are probably related to the marine environment; since pinnipeds retain a small lacrimal gland these characteristics would appear to be confined to the Cetacea and Sirenia. 18. Factors affecting the hatchability of farm reared turtles. By SARAH E. SOLOMON and T. BAIRD. Departments of Veterinary Anatomy and Chemistry, University of Glasgow The mineralized surface of the turtle egg is deposited in the shell forming region of the 4 m long paired oviducts. This region has several functions, namely shell membrane formation, shell calcification and cuticle production. In each oviduct approximately 60 eggs undergo these processes simultaneously. The calcified aspect of the shell consists of long needle shaped crystals of aragonite which project from the mammillary layer of the shell membranes and are overlain, preceding oviposition by a cuticular membrane which serves to minimise bacterial penetration. This layer is extremely fragile and easily dislodged in handling. Ultrastructural analyses of feral eggs have shown the presence of fungal hyphae within the shell membranes and in extreme cases total disruption of the membranes immediately beneath the 'true shell' has been observed, accompanied by embryonic death. The aragonite modification of calcium carbonate represents the main mode of crystal growth in feral eggs. Scanning electron microscopic analyses and electron diffraction studies of the 'true shell' of some farm reared eggs, however, have revealed the presence of large calcite crystals. In the natural environment such morphological abnormalities are accepted as part of the population control mechanism. Under farmed conditions where the aim is to produce the maximum number of viable hatchlings, the effect of these structural changes must be assessed and measures taken to rectify the situation.

19. The effects of vincristine sulphate on DNA synthesis in the murine fetal thymus. By A. C. RICHES, LINDA MCQUEEN and D. BRYNMOR THOMAS. Department of Anatomy and Experimental Pathology, University of St Andrews Although metaphase arrest agents are widely used for estimating the cell production rate in a tissue, it is important only to use these agents under carefully defined conditions of dose and time. It is also widely assumed that these agents have no effect on other stages of the cell cycle or the rate of entry into metaphase. Kinetic studies on the murine fetal thymus using vincristine sulphate have demonstrated that a higher dose is required for adequate metaphase arrest and that cell production rate measurements must be made within 1 hour after drug administration (Riches, McQueen, Carr & Thomas, J. Anat. 126, 1978). This short period of metaphase arrest contrasts with that found in adult tissues and was thought to be due to the concentration of drug available in the fetus.

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The effects of vincristine sulphate on DNA synthesis were examined by monitoring the uptake of 1251-iododeoxyuridine in the fetal and maternal thymus. On day 15 following the appearance of a vaginal plug, CD1 mice were injected intravenously with either vincristine sulphate (5 mg per kg body weight) or the diluent fluid as a control. After 30 minutes a second intravenous injection of 1251-iododeoxyuridine was administered and the fetal thymuses and maternal thymus dissected after 1 hour. The tissues were fixed in alcoholic Bouin's fluid and then transferred to four changes of 70 % alcohol to remove the unbound radioactivity (Riches, Gore, Docherty & Littlewood, J. Anat. 121, 1976). The counts per fetal thymus were markedly depressed in the vincristine sulphate injected group, whereas the counts per mg in the maternal thymus were not depressed. This would seem to account for the short accumulation period of metaphases observed in the fetal thymus. Only the G2 cohort of cells enter metaphase. Vincristine sulphate appears to exert a differential effect on DNA synthesis in fetal compared to maternal thymus and thus has to be used under defined time conditions to estimate cell production rates in fetal thymus. 20. Changes in the lymphoid system of the rat during the second half of pregnancy. By I. K. THOMAS and J. M. MCLEAN. Department of Anatomy, University of Manchester In Eutherian mammals with haemochorial placentation the established placenta is a vascularized allograft, since the fetal chorion is bathed in maternal blood. Successful reproduction represents a major immunological paradox since the maternal immune system must protect the allogeneic conceptus from rejection while retaining the capacity to deal with any other antigenic challenge. Since the norm is a successful outcome to pregnancy, it has been suggested that this is due to some form of immunological isolation of the fetus from the mother or the induction in the mother of a specific unresponsiveness to the paternal complement of transplantation antigens. Since the hallmark of the specific immune response is lymphocyte proliferation within the host's lymphoid tissue, evidence of such a response was sought in this study. Fifteen groups, each of 10 SpragueDawley female rats, were used. One group was virgin animals, the others were mated and later killed at 2 day intervals between day 10 and 22 of inbred and outbred pregnancies. The iliac lymph nodes, which drain the uterus, the popliteal lymph nodes, spleen, thymus, liver and uterus were all removed and weighed. Cell suspensions from weighed portions of the lymphoid tissues were prepared to estimate their total and differential white cell counts using a Coulter counter. Amongst other findings the results showed the conceptus to be a significant stimulus to lymphocyte proliferation within the iliac lymph nodes with a consequent increase of weight and total white cell count. The most interesting observation however was the significant falls that occurred in all parameters immediately before parturition during outbred pregnancy. Progressive thymic involution was also observed during pregnancy and was associated with diminished proliferative activity and a net loss of lymphocytes from the thymus. These changes were more significant during outbred than inbred pregnancy. 21. Studies on lymphopoiesis in fetal thymic organ cultures in the presence of various agents. By U. SINGH (introduced by T. E. BARLOW). Department of Anatomy, University of Newcastle upon Tyne The development of immunocompetent T lymphocytes depends upon the entry and subsequent processing of prethymic 'stem' cells by the thymus. However, the exact nature of this process is not fully understood. It may result either due to physical cell to cell interaction between the lymphoid 'stem' cells and the non-lymphoid (epithelial) components of the thymus or as a result of interaction with its secretory products. Experiments using thymus grafts in thymectomized animals indicate that the need for the prethymic stem cells to enter the thymus for this process is perhaps obligatory for the generation of heterogeneous populations of T lymphocytes (Stutman, Yunis & Good, J. Exp. Med. 132, 1970). In our earlier studies, where we observed increases in the expression of differentiation antigen (Singh & Owen, Euro. J. Immunol. 6, 1976) on early thymocytes following treatment with various agents in suspension cultures outside the thymus, no increase in functional competence in assays such as reactivity to mitogens or mixed lymphocyte reactions of these cells was observed (unpublished observations). These observations

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suggest that during early stages of ontogeny of thymocyte maturation, the presence of precursor cells within the microenvironment of thymus is essential. In this investigation an attempt has been made, firstly to study the phenomenon of cellular interaction within the fetal thymus and secondly to alter the chemical nature of the microenvironment. This was carried out by growing 14 days old fetal thymic explants for over one week in serum free medium, but in the presence of various agents in order to observe any change in the pattern of lymphopoiesis and functional competence of cells so generated within the altered microenvironment. The results of such studies demonstrate that though on histological examination lymphoid cells appear in contact with epithelial cells and in many cases even concentrated in the areas where epithelial cells are present, no specialized regions of physical contact or interaction such as microvilli or gap junctions are observed on E.M. examination and a clear intracellular space is seen between them. On the other hand treatment of organ culture with phenylephrine, an alpha adrenergic agonist which raises cGMP, but not isoproterenol or PGE1, which raise cAMP levels, causes increased lymphopoietic activity (with little change in the epithelial cells) as observed by incorporation of 125IUdR, cell yields and autoradiographic studies. In view of these results and the results of our parallel study demonstrating the existence of adrenoceptors on fetal thymocytes during ontogeny (Singh, Millson, Smith & Owen, Eur. J. Immunol. in the Press, 1978) the possible significance of secretory products of epithelial cells, acting at a short range within the thymic microenvironment, surface receptors and cyclic nucleotides as the intracellular mediators of thymocyte maturation will be discussed. 22. A preliminary communication on the weights of human thymus glands at post mortem. By MARION D. KENDALL, H. R. M. JOHNSON*, JAYANTI SINGH and M. SLATERt. Departments of Anatomy, *Morbid Anatomy and tCommunity Medicine, St Thomas's Hospital Medical School, London The thymus glands from 574 autopsies were dissected out and estimates were made of wet weight, lean dry weight, lipid weight and water weight using chloroform extraction techniques in a soxhlet apparatus. The weights were correlated with data on the cause of death and organ conditions as described at post mortem. Inevitably there was a strong sampling bias towards older persons. Regression analyses, that made allowances for age and body weight, indicated that the variation resulting in increased thymus wet weights, dry weights and lipid weights was associated with persons in whom a cardiovascular condition was the cause of death or a contributory cause of death. Similarly, variation giving lower thymus weights was associated with persons who died while suffering from carcinomas or kidney diseases. The lean dry weight (protein and carbohydrate content) was more complex. Over 35 % of the persons over 55 years of age died with a thymus gland having a lean dry weight similar to that found in children and young persons. There is no clear correlation in this group with the cause of death.

23. Observations on the capillaries and perivascular spaces in the human thymus. By JAYANTI SINGH (introduced by MARION D. KENDALL). Department of Anatomy, St Thomas's Hospital Medical School, London The thymus gland has a vital role in the development and maintenance of immunological responses yet the formation of antibodies in the thymus itself, under normal conditions, has not been observed. The existence of a blood-thymus barrier that prevents the passage of antigen into the thymus parenchyma has been postulated, and morphological evidence of its presence in the human thymus has been reported. In the present study, thymic tissue was obtained from 96 patients undergoing open heart surgery. The tissues were processed for light and electron microscopy using conventional methods. The cortex of the thymus was exclusively supplied by capillaries with larger vessels elsewhere. The vascular lumen was often separated from the thymus parenchyma by the vascular endothelium and its basal lamina, some pericytes, a perivascular space containing collagen fibres and cells, and epithelial-reticular cells with their basal laminae. This does suggest the presence of a blood-thymus barrier, but a detailed study of the vascular complex revealed that, in places, the perivascular space communicated directly with the thymic parenchyma. This was found less often

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in the cortex than in the regions of the cortico-medullary junction and medulla where such communications were invariably present. Despite this, none of the thymus glands examined showed the presence of germinal centres, or unusually high numbers of plasma cells. Another equally important observation of this study was the demonstration of the presence of fenestrated capillaries in the cortex and medulla. The presence of fenestrations was independent of the morphological structure of the surrounding perivascular space and does not appear to have been previously reported in man. The significance of their presence in the thymus is unknown although their relationship to endocrine functions of the gland should be considered as they are closely associated with epithelial-reticular cells.

24. The occurrence of thymic erythropoiesis in birds, a rodent and man. By MARION D. KENDALL. Department of Anatomy, St Thomas's Hospital Medical School, London Previous work has established that the lobes of the thymus gland in birds are capable of producing large numbers of erythrocytes that enter the circulation after times of physiological stress. These periods, often of 'low condition', may be induced naturally by nest-building, by production of eggs, by moult, or by artificially inducing haemorrhage. However, since birds may not be phylogenetically on the main line of vertebrate evolution, it was thought possible that there might be basic differences between the lymphomyeloid systems of birds and mammals although studies of the avian thymus at the ultrastructural level did not support this. Recent work, reported here, shows that the thymic environment in a small wild rodent (Clethrionomys glareolus), and in man can support thymic erythropoiesis. Whilst the potential for erythropoiesis in laboratory mice has been reported before, it has not been previously observed in man. These observations raise the important question of stem cell origin. The findings in man suggest that the stem cells could enter the thymus via the perivascular space either along the septa or from the blood vessels. In the rodent, the small foci of erythropoiesis are widely separated, often scattered throughout the cortex, perhaps suggesting a local stem cell origin. This question can only be resolved by further work. 25. In vitro studies on the endoderm of young chick embryos. By G. W. IRELAND, N. P. BRETLAND, MARGARET ROE and RUTH BELLAIRS (introduced by T. A. QUILLIAM). Department of Anatomy and Embryology, University College, London The young chick blastoderm possesses at least four different types of endoderm (Sanders, Bellairs & Portch, J. Embryol. exp. Morph. 46, 1978). The hypoblast is the ventral ofthe two layers at the pie-primitive streak stage, and it gives rise to the extra-embryonic endoderm of the yolk sac stalk. The definitive endoblast becomes inserted into the hypoblast at the primitive streak stage and subsequently forms the embryonic endoderm of much of the gut. The junctional endoblast arises at the caudal end of the primitive streak and probably contributes to the gut. The endoderm of the germ wall (or area opaca) gives rise to the extra-embryonic endoderm of the yolk sac. This paper is concerned with the relationships that exist between these four endodermal tissues when they are grown in culture. Embryos were taken from White Leghorn eggs incubated for 5-24 hours and the tissues dissected in Tyrode's solution. Explants were grown on glass coverslips in sitting-drop cultures using a medium of Earle's 199 and calf serum. Time-lapse cinemicrography was used to record individual cell interactions. The various tissues showed a differential ability to attach to glass. In addition, whereas the hypoblast always formed a monolayer the germ wall showed multilayering as in vivo. The junctional endoblast explants were sometimes monolayered and sometimes multilayered. In confronted cultures, the cells of different tissues could usually be readily identified by their distinctive morphology. The results of confrontations between hypoblast and definitive endoblast have already been reported (Sanders, Bellairs & Portch, J. Embryol. exp. Morph. 46, 1978). No difference could be detected in the behaviou rof hypoblast to germ wall of different ages or to germ wall taken from different locations in the blastoderm. In confrontation with other tissues the hypoblast sheet tended to migrate around them and was frequently dissociated. No barrier regions formed between germ wall and hypoblast or between germ wall and junctional

endoblast.

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26. Changes in the distribution of granulated metrial gland cells in the pregnant mouse uterus in the first half of pregnancy. By I. J. STEWART (introduced by D. BULMER). Human Morphology, University of Southampton In the pregnant mouse uterus, granulated metrial gland cells are scattered throughout the endometrial stroma by day 3 of pregnancy. Following implantation on day 4 there is a large increase in the number of granulated metrial gland cells in the decidua basalis of each implantation site followed later, from day 8 of pregnancy, by the formation of the metrial gland in the mesometrial triangle. In an attempt to follow more closely the changing pattern of distribution of these cells, an autoradiographic study, using tritiated thymidine as tracer, was carried out to plot the relative positions of proliferating and non-proliferating granulated metrial gland cells. Mice between day 4j and day 10 of pregnancy were given a single intraperitoneal injection of tritiated thymidine (2 ,tCi/g body weight) 1 hour before killing. Glycol methacrylate transverse sections (1 ,um) through the centre of implantation sites were prepared for autoradiography and the position of both labelled and unlabelled granulated metrial gland cells was plotted. In addition, uteri from animals between day 1 and day 4 of pregnancy and areas of interconceptual uterus from animals between day 4j and day 10 were examined but were not prepared for autoradiography. On days 3 and 4 of pregnancy, prior to implantation, granulated metrial gland cells appeared to be randomly distributed throughout the endometrial stroma. Following implantation they had disappeared from the interconceptual stroma by day 6, disappearing initially from those regions of the uterus most distant from the implantation sites. They were not found in the antimesometrial region of the implantation sites after day 6. Proliferation of granulated metrial gland cells appeared to occur initially throughout the decidua basalis but at later stages of pregnancy proliferating cells were no longer found in the lateral and antimesometrial areas of the decidua basalis and by day 9 were found only in the mesometrial portion of the compact zone. By day 8, however, there were proliferating granulated metrial gland cells in the mesometrial triangle and the number of these increased throughout the remainder of the period of study. The sequence of these changes was illustrated and related to the changing morphology of the mouse uterus in pregnancy.

27. Ultrastructure of human placental syncytiotrophoblast in relation to steroid hormone synthesis. By D. M. HAMBIDGE (introduced by A. D. HOYES). Department of Cellular Biology and Histology, St Mary's Hospital Medical School, London Mitochondria, with cristae which appear as circular profiles on section and which have been interpreted as being tubular or vesicular in form, have been described in cells active in steroid hormone synthesis, such as those in the human adrenal cortex, but have only been reported in the syncytiotrophoblast of the human placenta in early gestation (Wynn, Eur. J. Obs. Gyn. Repro. Biol. 5, 1975). To obtain further evidence on the distribution of such mitochondria in the placenta, blocks of tissue were taken from close to both the basal and chorionic plates of placentae fixed by perfusion through the umbilical artery, as soon as possible after delivery, with 3 % glutaraldehyde in 0-1 M sodium cacodylate buffer at 37 °C and at a pressure of 100 mmHg, and then processed for electron microscopy. The mitochondria in syncytiotrophoblast from near the basal plate of placentae from 4 early second trimester hysterotomy abortions exhibited the ultrastructure described by Wynn. Approximately 65 % of the mitochondrial profiles contained tubovesicular cristae and the mean number of such cristae in 239 of these profiles was 2-72 (range 1-8, S.E. ± 011). In the region close to the chorionic plate, the mitochondrial cristae were difficult to define, and in contrast to the mitochondria in the syncytiotrophoblast near the basal plate, their matrix was of low electron density. Syncytiotrophoblast from near both the basal and chorionic plates of placentae from 4 normal, spontaneous vaginal deliveries at term, and of a placenta from an elective caesarian section at 39 weeks gestation, also contained mitochondria with tubovesicular cristae. In all, 42-45 % of the mitochondrial profiles contained these cristae in a matrix of medium electron density. The mean number of tubovesicular cristae in 265 of these mitochondria was 2-03 (range 1-6, S.E. ± 0 07).

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If the association between mitochondrial cristae ultrastructure and steroid hormone synthesis is real, then these observations are compatible with the synthesis of placental steroid hormones in the syncytiotrophoblast of placentae both in early and late gestation. 28. The development of the human atrioventricular heart valves. By F. A. W. VAN GILS (introduced by A. OPPENHEIMER-DEKKER). Department of Anatomy and Embryology, University of Leiden, The Netherlands The atrioventricular valves are usually said to be derived from the endocardial cushions. In order to get a more detailed insight into this developmental process, serial sections of embryonic and fetal hearts were studied, as well as some postnatal material. In early embryonic stages the endocardial cushions, embedded in the surrounding myocardium, form the most conspicuous structures at the atrioventricular transition and appear to have a valve function. In this transitional area, these cushions become connected with the tissue in the atrioventricular sulcus and have the same loose appearance. In later embryonic and in early fetal stages, an undermining process of the myocardium around the cushions, together with an increase in the amount of collagen fibres on the peripheral side of these cushions, results in a valvular apparatus. This consists of a mostly collagenous cylinder and extends from the atrioventricular sulcus to larger, apical trabeculae (the precursors of the later papillary muscles). On its inner apical side, this collagenous cylinder is covered with the cushion tissue. In later fetal stages the cushion tissue will be found at the very apical part of the collagenous cylinder. The development as described above, is not the same in all parts; the central parts, i.e. septal tricuspid leaflet and aortic mitral leaflet, do not maintain a close connexion with the atrioventricular sulcus. In their formation, two aspects are of vital importance: firstly the posterior ingrowth of the interventricular septum and secondly the remaining continuity between the apical portions of cushion tissue and a portion which stays during the whole development at the basal part of the cusp. Our observations suggest that endocardial cushion tissue in the early embryonic heart acts as a valve and that in later stages, a new valvular apparatus comes into being. This is in direct continuity with atrioventricular sulcus tissue and (in the central cusps) with the forementioned basal portion of cushion tissue. This basal portion in the central valves contributes to the anulus fibrosus, whereas the apical cushion tissue will transform into the so-called 'noduli Albini'. 29. The sinus venosus of the adult avian heart - present or absent? By P. R. VASSALL-ADAMS. Department of Anatomy, Charing Cross Hospital Medical School, London Many writers describe the adult avian heart as having a fifth chamber - the sinus venosus which is listed in the Nomina Anatomica Avium, 1975. To elucidate the anatomy of this chamber, hearts of adult birds from seven different species were examined by dissection, by light microscopy, and by making injection replicas. The birds were the ostrich, chicken, turkey, magpie, pigeon, starling and herring gull. When the right atrial cavity of the hearts of all species was examined two large valves were seen to project into the cavity of the atrium from its posterior wall. The large systemic veins emptied into the space between these two valves so that the inferior vena cava entered the middle part of the space, the right superior vena cava the upper part, and the left superior vena cava the lower part. The valves were bilaminar muscular structures formed by inflexions of the atrial-caval junction. Thus, the outer muscle layer of the valves was continuous with atrial muscle while the inner muscle layer of the valves was continuous with the muscle of the appropriate vein. It was noteworthy that the inferior vena cava itself was a very large vein (about four times the size of either of the superior venae cavae) forming a distensible muscular cavity which linked the right atrium and the liver. Where, then, is the sinus venosus? At best it may be argued that the atrial space between the valves should be regarded as a sinus venosus, but, if so, it is an extremely ill-defined entity. It is therefore suggested that the term sinus venosus be abandoned with reference to the adult avian heart. This will ensure accurate description of the location of structures at the atrial-caval junction (e.g. the sinu-atrial node), the description of which can be related to a specific vessel rather than to an ill-defined cavity.

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30. Electron microscopic observations on hypertrophic secondary cartilage in the embryonic chick skull. By J. T. KARASHANI. Department of Anatomy and Histology, University of Dar es Salaam, Tanzania A study of the fine structure of secondary (adventitious) cartilage in the squamosal bone of chick embryos at 12-14 days of incubation was made with a view to elucidating the morphological changes associated with the gradual 'replacement' of this type of cartilage by bone. Particular attention was paid to the hypertrophic chondrocytes in the 'transitional zone' where provisional calcification is taking place. On morphological criteria, three types of cells are distinguishable at the calcification front: (1) Type A cells retain the features of hypertrophic chondrocytes, with somewhat fragmentary organelles scattered in a rarefied granular cytoplasm. The rough endoplasmic reticulum consists of narrow tubular cisternae containing fibrillar electron-dense material in sharp contrast to the pale background, while occasional dense bodies and lipid droplets may be found. There is a large amount of fibrillogenesis to be seen around these cells. (2) Type B cells resemble osteoblasts with an abundant organelle content: a profuse juxtanuclear Golgi complex, an array of rough endoplasmic reticulum with moderately dilated cisternae and numerous mitochondria. (3) Type C cells show signs of degeneration, with disintegration of organelles and rupture of the plasma membrane. The appearances suggest that many hypertrophic chondrocytes with type A and B features remain viable throughout the mineralization process until they are completely enveloped by a calcified collagenous matrix which is morphologically indistinguishable from that of bone. It is suggested that such chondrocytes may in this way be incorporated into bony tissue and assume the role of bone cells.

This work was done during the tenure of a World Health Organization Fellowship at the Queen's University of Belfast. 31. The formation of autonomic neuromuscular junctions in vitro. By G. BURNSTOCK. Department of Anatomy and Embryology, University College, London Time lapse cinematography is employed to show that autonomic nerves form long-lasting, intimate, functional relationships with smooth muscle cells cultured from potentially densely innervated tissues, but not with smooth muscle cells from potentially sparsely innervated tissues or with fibroblasts. Neurotransmitter receptor sites are not the same as the recognition sites. Recognition sites do not appear to distinguish between adrenergic or cholinergic nerves, although there may be a later readjustment mechanism that leads to degeneration of foreign fibres. Some speculations are made about the mechanisms of recognition and the possible events occurring during the 1 hour period of palpation before long-lasting junctions are formed. Once neuromuscular junctions are formed, further long-lasting associations with nerve fibres are inhibited. Whether this rejection is due to switching off of recognition or due to the production of a specific compound by the host cell is not known. Formation of neuromuscular junctions delays de-differentiation of muscle cells in culture, but accelerates the formation of muscle effector bundles and

gap-junctions. 32. A study of intramural autonomic nerves in relation to a large bowel anastomosis in the rat. By E. S. KIFF, M. G. LORD and J. A. GOSLING. Departments of Surgery and Anatomy, University of Manchester Following procedures involving the surgical excision of part of the human large bowel, a successful anastomosis cannot be guaranteed despite meticulous operative technique. Whilst it is known that muscle fibres can cross an intestinal anastomosis during the post-operative phase, little is known about the response of the intramural autonomic nerves to this operation. In the present investigation we have examined, using an histochemical technique, the arrangement of intramural nerves at the site of large bowel anastomoses at selected times during the post-operative period. In 24 Sprague-Dawley rats, the distal colon was transected, preserving the marginal artery, and a single layer inverting anastomosis created. The animals were allowed

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to resume their normal diet post-operatively Four rats were killed at each of the following post-operative times; 3 days, 7 days, 2 weeks, 4 weeks, 2 months and 4 months. The anastomotic area and the colon for 1 cm on either side was removed from each animal, placed on a tissue holder and frozen in 2-methylbutane cooled in liquid nitrogen. Cryostat sections 15-20 ,um in thickness were prepared and processed according to Gomori's technique for tissue cholinesterases. Nonspecific cholinesterase was inhibited using TIPA and acetyl cholinesterase, using the inhibitor

284C51. The results showed that enzyme containing nerves were absent from the anastomotic line and for a short distance on either side, throughout the first 4 post-operative weeks. Two months after operation, nerves were discerned traversing the anastomotic site in both longitudinal and circular layers. These findings were discussed in relation to some of the facto. rsaffecting healing at an intestinal anastomosis. 33. Horseradish peroxidase studies on the guinea-pig inferior mesenteric ganglion in vivo. By F. A. H. AL-KHAFAJI, D. MAYOR, P. ANDERSON and J. MITCHELL. Human Morphology, University of Southampton The histochemical localization of horseradish peroxidase within the inferior mesenteric ganglia of young adult Hartley guinea-pigs was studied by light and electron microscopy at periods between 30 minutes and 30 hours after an intravenous injection of the enzyme. Thirty minutes after injection, dense peroxidase reaction product was seen within the blood plasma, in pinocytotic vesicles within the endothelial cells, in the spaces between adjacent endothelial cells and throughout the extracellular space within the ganglia. This suggests that the peroxidase can readily penetrate the walls of the blood vessels within the ganglion. After 30 minutes, the enzyme had also penetrated into the spaces between satellite cells and their associated neurons and SIF cells (small, intensely fluorescent, granulated cells). Evidence was found to suggest that neurons, SIF cells and satellite cells were taking up the enzyme, often within coated vesicles. The neuronal perikarya contained some vesicles labelled with peroxidase reaction product, but such vesicles were less frequently observed in the SIF cells. Macrophages exhibited dense reaction product, both diffuse in their cytoplasm and within vesicles. The macrophages were less strongly labelled after 6 hours or 9 hours than they were at 30 minutes. Six hours after injection there was little reaction product within the blood plasma and the extracellular spaces, but many more peroxidase-positive vesicles were present within the neurons and SIF cells. The neurons appeared to contain more peroxidase-labelled vesicles than the SIF cells after 6 hours, but this situation was reversed in ganglia examined 9 hours or longer after

injection. In ganglia examined 19 hours after injection the peroxidase vesicles within the neurons and SIF cells were less numerous and smaller in size than those at 6 or 9 hours, and by 30 hours after injection, no neurons or SIF cells were found to contain peroxidase reaction product in their cytoplasm. The significance of these results with regard to horseradish peroxidase tracer studies was discussed. 34. Quinacrine uptake into peripheral neurons By A. D. HoYES, PAULINE BARBER and L. ETHRIDGE*. Departments of Anatomy and *Cellular Biology and Histology, St Mary's Hospital Medical School, London Recent fluorescence microscopical studies on the gut (Olson et al. Cell Tissue Res. 171, 1976) have suggested that uptake of quinacrine into peripheral neurons occurs preferentially into cells employing ATP or substance P as a transmitter. To obtain further evidence on the uptake of quinacrine into peripheral neurons, fluorescence microscopical studies were undertaken on the peripheral ganglia of rats given 10 mg/kg quinacrine i.p. 24 hours previously. In the small intestine, fluorescence was seen in occasional nerve fibres and in many of the perikarya of neurons in the myenteric plexuses. In the perikarya, the drug was concentrated in discrete granules in the cytoplasm. Large numbers of similar granules were seen in the perikarya of the neurons in the superior cervical and other sympathetic ganglia, the pelvic ganglion and both the nodose ganglion and the lumbar and sacral dorsal root ganglia. The distribution of

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fluorescence in dorsal root ganglia incubated in acridine orange, which is taken up into lysosomes, corresponded closely to that observed after quinacrine treatment. Ultrastructural examination showed that, compared to ganglia from control animals, after quinacrine treatment a substantially greater proportion of the lysosomes in the dorsal root and pelvic ganglia contained electron-dense lamellae. Typically the lamellae were also twice as wide as most of those found in normal dorsal root ganglion cell lysosomes and frequently exhibited regular transverse striations. In animals injected with quinacrine 24 hours after preganglionic section of the pelvic nerves, fluorescent granules could still be seen in the perikarya of the pelvic ganglion cells. Numerous fluorescent granules were also observed in the proximal segments of the cut nerve fibres, and ultrastructural examination confirmed that this was associated with the presence in the axons of large numbers of dense bodies with the features of lysosomes. 35. The response of Schmidt-Lanterman incisures to nerve crush. By M. N. GHABRIEL and G. ALLT. Departments of Anatomy and Neurological Studies, The Middlesex Hospital Medical School, London (Fig. 5) Several investigators, including Ranvier and Cajal, have noted that in response to nerve crush Schmidt-Lanterman incisures initially undergo dilatation and subsequently become the sites at which the myelin sheath is segmented into ovoids. We have demonstrated by electron microscopy that it is the peri-incisural myelin which undergoes dilatation (Fig. 5 B, arrows) by a separation of myelin lamellae at the intraperiod line, while this myelin at other incisures is initially unaffected (Fig. 5 A). The incisures themselves remain intact with either moderate or no widening of the intraperiod line gap. The cytoplasmic spiral at the incisure persists (Fig. SC, arrows), even within fibres segmented into ovoids. Webster (Ann. N. Y. Acad. Sci. 122, 1965) reported an increase in the number of incisures during the early phase of Wallerian degeneration. The increase appeared to facilitate the formation of myelin ovoids. In view of more recent studies which reveal the regular and elaborate fine structure of incisures, it seems theoretically unlikely that new incisures would be synthesized and intercalated into the myelin sheath during an essentially degradative response such as Wallerian degeneration. We have therefore re-examined quantitatively the changes in incisures in the rat sural nerve following a proximal crush, using a magnification of x 1000 and counting a total of 3564 incisures in a total of 358 internodes from teased nerve fibres. There was no significant difference between the number of incisures at 12 hours and 24 hours after crush, compared with normal fibres. Regression lines relating the number of incisures to fibre diameter were found not to differ significantly between the three groups. It seems likely that previous reports which have described an increase in the number of incisures have misinterpreted the data. Incisures become more readily identified and counted, especially at lower magnifications, as the peri-incisural myelin undergoes progressive dilatation in response to injury.

36. Observations on the Schwann cell plasma membrane using freeze-fracture. By P. H. ABRAHAMS, A. DAY and G. ALLT.* Departments of Anatomy and *Neurological Studies, The Middlesex Hospital Medical School, London (Fig. 6) Electron microscopic studies of the Schwann cell are numerous, but only recently has the freeze-fracture technique added a new dimension to our understanding. We have found that the outer Schwann cell plasma membrane and the subjacent outer layer of the Schwann cell cytoplasm are organized into irregular circumferential bands. These thickened bands (Pb) as seen on the P face (Fig. 6A, C), which contain numerous mitochondria and vesicles, correspond to the concavities of the E face (Fig. 6B, Eb). The plaque-like areas (Epl) of the E face are regions of the Schwann cell plasma membrane where there is little outer Schwann cell cytoplasm. Associated with the cytoplasmic bands are numerous micropinocytotic vesicles or caveolae (Fig. 6A, B, C, arrows) which are absent on the thin cytoplasmic regions (Mugnaini et al. J. Neurocytol. 6, 1927). The nerve fibre size and differentiation may correlate with the concentration of vesicles and formation of cytoplasmic bands. Zonulae occludentes in the long axis of the mylinated nerve fibre have recently been studied (Tetzlaff, Cell Tissue Res. 189, 1978). Occasionally, we have found on large diameter fibres, a form of puncta occludens associated with channelling of the Schwann cell cytoplasm (Fig. 6A, asterisk).

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At the node of Ranvier the Schwann cell cytoplasm terminates as microvilli which are well demonstrated by freeze-fracture and can be seen to interdigitate with the microvilli of the adjacent Schwann cell but are separated by intervening nodal gap substance. These results are compatible with those of previous freeze-fracture studies and again demonstrate the value of this technique in determining membrane surface topography. Direction of shadowing is indicated by arrows in the bottom left of each micrograph.

DEMONSTRATIONS D.1. Scanning electron microscopy of the developing heart in mouse embryos. By M. H. KAUFMAN and V. NAVARATNAM. Department of Anatomy, University of Cambridge Scanning electron microscopy is a particularly suitable method for illustrating the three dimensional changes which take place in the morphology of the developing heart, and consequently facilitates understanding of the early events associated with the formation of this organ. Mouse embryos were isolated in phosphate buffered saline (pH 7-3) between the morning of the 9th day (at about the 3-4 somite stage) and midday on the 11th day of gestation; the day on which a vaginal plug was found is designated the first day of pregnancy. Embryos were dissected free of their membranes and fixed in 2% glutaraldehyde in 0 1 M sodium cacodylate buffer. After about 2 hours the embryos were transferred to 0-1 M sodium cacodylate buffer containing 10 g sucrose/100 ml. After a period of 3-7 days the thoracic wall was dissected away to expose the heart. Embryos were then transferred through a graded acetone series to 100% acetone, critical point dried, sputter coated with gold, and subsequently viewed in a Cambridge S600 scanning electron microscope. On the morning of the 9th day, when embryos are at about the 3-4 somite stage, the two endocardial heart tubes meet in the mid-line and commence fusion to form a single median heart tube. Shortly thereafter, at about the 6-7 somite stage, the heart enlarges and bulges ventrally, and the first deviation from bilateral symmetry is seen. Between the 7-10 somite stages the heart becomes C-shaped. These and subsequent stages of development are illustrated by appropriate low resolution scanning electron micrographs. Higher resolution scanning micrographs of the surface morphology of the various regions of the developing heart tube are also illustrated. To complement the scanning electron micrographs, representative histological sec!pons through the heart region at similar stages of development are also presented. D.2. Scanning electron microscopy of cell movements from chick embryo grafts. By MARJORIE A. ENGLAND and JENNIFER WAKELY. Department of Anatomy, University of Leicester (Fig. 7) Studies of cell movement in vitro have previously been based on the use of glass or plastic substrates. Recent reports however have described the use of a cellular substrate prepared by culturing dissociated cells on glass or plastic until a monolayer of cells has formed. The experimental cells can then be introduced on to the monolayer and their movements noted. The present study reports on the use of an in vivo cellular substrate with all its normal cellular contacts, junctions and extracellular materials intact over which the movement of outgrowing cells from a graft of chick embryo Hensen's node has been studied. Host substrates were prepared by surgically removing area opaca endoderm from stage 4 chick embryos mounted as for New culture. Grafts of Hensen's node minus the endoderm layer were surgically removed from donor stage 4 embryos and transferred to the prepared area opaca host ventral ectoderm. Initially the graft ectoderm layer curled ventrally towards the host ectoderm layer. However following re-incubation at 37 5 °C the graft ectoderm cells formed an epithelial ball and the mesoderm layer sandwiched between the graft ectoderm and host ectoderm moved radially away from the graft ectoderm ball as an entire layer (Fig. 7). The exposed surface of the outgrowth was covered with flap-like processes and the underlying mesoderm cells in contact with one another had a normal appearance. Those mesoderm cells contacting the host substrate and moving away from the graft, had a tapered leading edge with several filopodia closely associated with the substrate. Their main cell body bulged behind the leading edge and numerous small filopodia were present in mid-air. The tapered trailing edge also had numerous filopodia. 28 A NA 128

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These in vivo morphological findings are consistent with previously reported studies of in vitro cell movement. The patterns of cell shape associated with cell movement are similar to those described by Rajamaran et al. (Exp. Cell Res. 96, 1974). D.3. The distribution and patterns of basement membrane in the early chick embryo. By JENNIFER WAKELY and MARJORIE A. ENGLAND. Department of Anatomy, University of Leicester (Fig. 8) Previous studies on the distribution of basement membrane in the chick embryo have described its first appearance randomly distributed in the early area pellucida (stage 3) (Low, Anat. Rec. 152, 1967). By transmission electron microscopy a narrow sheet of basement membrane was present by stage 4 except in the primitive streak and Hensen's node. Several authors have suggested that this basement membrane sheet has a role of contact guidance of cell movement in the embryo but they have been unable to locate a pattern of basement membrane in embryos stages 3-4 (Trelstad, Hay & Revel, Devel. Biol. 16, 1967). We have found by scanning electron microscopy that at stages 3-4 the basement membrane has two appearances, a fine fibrillar meshwork and thicker fibrous threads. The heavier fibrils are present as an arc at the anterior area pellucida edge with the primitive streak as a central reference point (Fig. 8). This arc tapers to form fine fibrils on either side, on a level with Hensen's node, and then continues posteriorly. Parallel bands of fine fibrils arranged roughly concentrically are present between this outer arc and the primitive streak. Alcian blue staining at varying concentrations of MgCl (Pearse, Histochemistry, Theoretical and Applied, Churchill, London, 1968) has confirmed the presence of associated glycosaminoglycans which were mainly nonsulphated but around the area opaca margins they were partly sulphated. The formation and role (if any) of these patterns of basement membrane is unclear at the present time.

D.4. Palate formation in the ferret studied by scanning electron microscopy. By A. GULAMHUSEIN and MARJORIE A. ENGLAND. Department of Anatomy, University of Leicester (Fig. 9) Palatal shelves from ferret embryos from 27 to 29 days of gestation were examined by whole mount, thick sectioning and scanning electron microscopy prior to and during secondary palate formation. At day 27 the palatal shelves were small and widely separated but the anterior margins of the shelves in the presumptive area of initial fusion were slightly convex. Rugae markings were present on each shelf. The premaxilla was a flattened region anteriorly placed. The epithelial surfaces of the shelves were composed of cells with distinct perimeters and occasionally short microvilli were present. The convex shelf margins continued to enlarge and by day 28 the palatal shelves had come together in the mid-line and fusion proceeded both anteriorly and posteriorly. Initial fusion did not immediately include the triangular premaxilla area (Fig. 9). In the immediate area of fusion the epithelial surface of the shelves was composed of cells with distinct cell perimeters while laterally the cell perimeters were indistinct. Numerous microvilli covered the cell surface, but even in the area of fusion no ruffles or recognized features associated with cell movement were present. A large amount of debris was found in the immediate area of fusion. Waterman (Anat. Rec. 180, 1974) has associated this in the human embryo with cell

death and desquamation. By day 29 the hard palate had fused but the posterior soft palate and uvula had not yet contacted each other. The region of fused hard palate was clearly marked by elongated, overlapping cells. These results are similar to those described by Waterman et al. (Anat. Rec. 180, 1974) in the human and in the mouse (Anat. Rec. 176, 1973). D.5. The developing ferret tongue from days 27 to 29 studied by scanning electron microscopy. By MARJORIE A. ENGLAND and A. GULAMHUSEIN. Department of Anatomy, University of Leicester (Fig. 10) The formation and closure of the secondary palate in the ferret occurs mainly between days 27 and 29. In other species, the tongue is known to have a crucial role in palatal closure (Ferguson, J. Anat. 124, 1977). In the ferret the developing tongue is closely associated with the developing 28-2

436

Proceedings of the Anatomical Society of

Fig. 9

palate and the embryology of this region is similar to that described for other species. On day 27 the palatal shelves were widely separated and the tongue bulged convexly into the space between them. The grooved apex of the tongue fitted into a depression in the lower lip (Fig. 10). At the base of the tongue there were approximately 4 or 5 circumvallate papillae forming a roughly Vshaped region. Along the margins and on the dorsal surface, numerous fungiform papillae were present as enlarged regions composed of approximately 16 cells raised above the surface of the tongue. The regions between the fungiform papillae consisted of cells whose perimeters were clearly outlined by numerous microvilli. The ventral surface of the tongue was smooth in appearance. On day 28 the palatal shelves met in the mid-line and started to fuse. The apex of the tongue was no longer present in the depression in the lip. The tongue was flat and elongated, resting on the lower lip and protruding slightly from the mouth. On the dorsal surface of the tongue impressions from the palatal rugae were present. In addition to the circumvallate and fungiform papillae, the parallel rows of the filiform papillae were present radiating from the median furrow. D.6. A comparison of vertebral body heights and widths in normal infants and in those with myelomeningocele. By R. G. LENDON and D. NAIK. Department of Anatomy, University of Manchester and Department of Radiology, Northern General Hospital, Sheffield It has been suggested that by comparison with controls, the lumbosacral region in infants with myelomeningocele is usually greatly shortened and that the cervical region is slightly increased in length (Forbes & Barson, Dev. Med. Child. Neurol. Suppl. 32, 1974). To test this hypothesis, we have measured vertebral body heights and widths from anteroposterior radiographs obtained post mortem from 20 newborn children dying with myelomeningocele and, in

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Fig. 10 addition as controls, 10 'normal' infants dying from unrelated conditions. All children were 7 days post partum or less. The results showed that the means (± 1 standard deviation) of the vertebral body heights measured on the radiographs from the children with myelomeningocele approximated closely to the values obtained from the normal cases. At no vertebral level was there a statistically significant difference between the two groups although there was a slight reduction of heights in the sacral region of myelomeningocele cases. However, variation was obtained between the groups with regard to vertebral body widths which were on average narrower in the mid and lower thoracic region of the myelomeningocele cases but only at Tll was this difference significant (P = 0039 for 2 tailed t-test). Vertebral body width was increased slightly in the upper cervical region, but the cervical body heights were not increased. These results indicate that vertebral body heights in the lumbosacral region of myelomeningocele infants are not markedly shortened as has been suggested. There is also no evidence that the upper cervical vertebrae have an increased height although they were insignificantly wider in myelomeningocele infants compared with controls. D.7. Some observations on the ultrastructure of the macula densa of the distal convoluted tubule of the kidney of the Syrian hamster. By C. L. FOSTER and K. SIKiu (introduced by A. D. HOYES). Department of Cellular Biology, St. Mary's Hospital Medical School, London During the course of a study on the localization of Tamm-Horsfall glycoprotein within the nephron of the kidney of the Syrian hamster, the opportunity was taken of examining the ultrastructure of the macula densa and the neighbouring region of the distal convoluted tubule. Interest in this structure stems from the notion that it may constitute a mechanism for

438

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monitoring sodium and chloride levels in the urine flowing past it. In an early account, McManus (Quart. J. Med. Sci. 85,1945), using light microscopical methods, claimed an infranuclear position for the Golgi elements in the cells of the macula densa in the rabbit and the cat, as well as an absence of a basement membrane in this region. This aroused great interest, since it suggested a reversed polarity of the cells with the possibility of some secretory product being transmitted to the renin-producing cells which are in close proximity. The results of ultrastructural studies on the hamster macula densa in general concur with the recent work of others on the rat, the mouse and the human, and support the view that macula densa cells differ in a number of important respects from those of the rest of the distal convoluted tubule. Among these differences may be mentioned the predominantly circular and elliptical profiles of the mitochondria which are much more widely scattered in the cytoplasm than in the distal convoluted tubule. The basal folds of the plasma membrane, unlike those in the distal convoluted tubule, are restricted to the basal region of the cell, where small numbers of dense membrane-bounded (?secretory) granules are sometimes found. The Golgi complexes, however, were observed in a variety of situations ranging from supranuclear to infranuclear, suggesting that this organelle may not occupy a constant position - even perhaps in the same cell. In this respect, the hamster appears to differ from the mouse, the rat and man - the species on which most of the recent work has been done. D.8. Identification of the cholinergic cortical projection from the diagonal band. By ZAINEB J. SELIM and P. R. LEWIS. Physiological Laboratory, University of Cambridge (Fig. 11) There is now strong evidence for a cholinergic innervation to the rat cortex arising from the basal forebrain. Much of the pathway had been followed by a histochemical technique for acetylcholinesterase (AChE) but the cells of origin could not be identified. This has now been achieved by combining the technique for AChE with the recently developed method for tracing pathways utilizing axonal transport of horseradish peroxidase (HRP). The combined procedure used has been described elsewhere (Lewis & Selim, J. Physiol. 284, 1978). Among the cells that are visibly labelled at the light microscope level by injections of HRP into occipital cortex are groups of large multipolar neurons in the areas of the basal forebrain which Shute & Lewis (Brain 90, 1967) suggested gave rise to cholinergic cortical afferents. Cells from the most rostral group, in the diagonal band, were examined by the combined technique at the electron microscopic level. Only a small proportion of cells in the diagonal band shows labelling with HRP. Those cells that become labelled almost all stain for AChE and most belong to one cell type which has several distinctive morphological features and stains very heavily for AChE (Fig. 11). Staining for the two enzymes is easily distinguished because the HRP is confined to lysosome-like particles (Fig. 11, L) whereas the AChE is concentrated along linear structures. The cells are rich in rough endoplasmic reticulum (Fig. 11, ER) which extends far along the dendrites and everywhere is heavily stained for AChE. In places the reticulum is in an unusual condensed form (Fig. 11, CER) without attendant ribosomes. Another very characteristic feature of these cells is the heavy and continuous staining for AChE at the plasma membrane (Fig. 11, P) over the whole cell body and along the dendrites. It seems certain that these large intensely AChE-positive cells are the source of the cholinergic innervation to the occipital cortex. D.9. In vitro effect of capsaicine on the axons in the subepithelial nerves of the rat ureter. By A. D. HoYEs and PAULINE BARBER. Department of Anatomy, St. Mary's Hospital Medical School, London It has recently been suggested (Hoyes et al. J. Anat. 119, 1975) that the morphologically distinct type of vesicle-containing axon, which forms the principal component of the subepithelial nerves of the rat ureter, is involved in pain perception. The unsaturated fatty acid, capsaicine, is a potent stimulator of peripheral pain afferents, and at high dose levels, produces a long-lasting and apparently specific de-sensitization of these afferents to noxious chemical stimuli (Jancso et al. Brit. J. Pharmacol. 31, 1967). To obtain evidence on the early effects of capsaicine on the axons in the subepithelial nerves of the rat ureter, their ultrastructure in specimens of ureter, incubated in buffer for 15 minutes and then exposed to capsaicine at a concentration of 20 mg/100 ml for 5 minutes before fixation in

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In the control specimens, the mean diameter of the axons in the nerves was 339 0 nm (S.E. ±

13v8 nm). In specimens treated with the vehicle, the diameter of the axons had increased to 422@8 nm (S.E. ± 27-52 nm; P < 0'02, paired t-test), and in the capsaicine-treated specimens, it was 592@0nm (s.E. ±1495 nm; P < 0001, paired t-test). 1P8% of the axonal profiles in the micrographs contained numerous closely packed small clear vesicles, and subjective evaluation indicated that axons of this type were unaffected by treatment with capsaicine. In the remaining axons, treatment with capsaicine was accompanied by disappearance of the microtubules and, as in ureters exposed to acute mechanical stimuli (Hoyes & Barber, J. Anat. 122, 1976), by an increase in the number of large dense-cored vesicles and a reduction in the number of small clear vesicles/ 100 axonal profiles. Figures obtained by correction of estimates of the numerical density of the large dense-cored vesicles for the changes in axonal diameter also indicated that treatment with capsaicine was accompanied by a considerable increase in the number of such vesicles in the axons. Measurement of their profile diameter showed that there was at the same time a small increase in the size of the vesicles. We are indebted to the Medicinal Research Centre of Beecham Pharmaceuticals for financial support for this work.

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D.10. Distribution of electron-dense deposits in the ureteric nerves after incubation in strontiumcontaining solutions. By A. D. HOYES and PAULINE BARBER. Department of Anatomy, St Mary's Hospital Medical School, London It has recently been shown (Hoyes & Barber, J. Anat. 124, 1977) that, when specimens of ureter are incubated for 30 minutes in a calcium-free Krebs buffer containing 10 mM-SrCl2 and are then fixed in phosphate-buffered osmium tetroxide, numerous small electron-dense deposits are present in the interval between the membranes of the mitochondria of the smooth muscle cells. To obtain evidence on the distribution of such deposits in the ureteric nerves, the same procedure was applied to a further series of specimens of adult rat ureter. Ultrastructural examination revealed the presence in the nerves of deposits which were approximately the same size, but much less numerous than those in the muscle cell mitochondria. In the Schwann cells, the deposits were mostly situated on the plasma membrane, and were present in only small numbers between the membranes of the mitochondria. No deposits were found in the vesicles of the axons in the submucous plexuses, and they were only occasionally present on the plasma membranes of the axons. Except in axons in which there was a major reduction in the density of the axoplasm, there were also only a few small deposits in the mitochondria. In axons with terminals packed with small vesicles, deposits were also present on the plasma membrane and in the interval between the membranes of the mitochondria. In the terminals of the axons in the arteriolar perivascular plexuses, which are known to be predominantly adrenergic (Hoyes et al. Invest Urol. 14, 1976), electron-dense deposits were also found in some of the small vesicles. Some of these deposits resembled those present on the plasma membranes and in the mitochondria, but the possibility that they consisted mainly of other components of the vesicles cannot be excluded. D.11. Quantitative studies in neuroanatomy. I. Studies on the spatial distribution of microtubules. By N. T. JAMES. Department of Human Biology and Anatomy, University of Sheffield The existence of microtubules in neurons (neurotubules) has been known since their observation by Palay (J. biophys. biochem. Cytol. 1, 1956). They are thought to extend for the whole length of many axons and are a constituent of all neurons (Dustin, Microtubules, Springer, Berlin, 1978). It is probable that they play a role in maintaining the shape of long cytoplasmic extensions and also in providing a transport mechanism for the movement of various organelles and granules along axons and dendrites. In transverse sections of axons and dendrites, the microtubules do not seem to be randomly distributed. The present study was carried out to determine whether the microtubules possess a quantifiable non-random spatial distribution. Since the microtubules are relatively small in diameter (- 25 nm) and never seem to occur in densities greater than 450 per um2 of neuronal cytoplasm, they seem to be an ideal population for analysis using previously described nearest neighbour methods (James, J. Anat. 122, 1976). In nearest neighbour analyses of the random distribution of objects on a plane surface, the mean distance between each object and its nearest neighbour (iT) is given by the term 1 / 4-7T -_ = 21D S.E. v 47TND'

where N and D are the total number of objects counted and their number per unit area respectively. Usually, tests of randomness are effected by calculating F from known values of D and comparing it with experimentally determined values of f. Significant differences indicate either a clumped or an hyperdispersed population. The ratio of the experimentally measured value to the theoretically calculated value for the random state yields an Index of Dispersion (Id). For a random population Id would possess the value of one, whilst in a contagious population its value would be less than one. Values of Id greater than one indicate the presence of an hyperdispersed population in which the individuals of the population are spaced further from each other than if they were randomly distributed. The values for Id obtained for the distribution of microtubules in the present study were consistently greater than one and usually lay in the range 1P45 to 1P68. Some mechanism responsible for the maintenance of this spatial pattern must necessarily be postulated.

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D.12. Differential alterations in the sizes of muscle fibre types during compensatory hypertrophy. By N. T. JAMES. Department of Human Biology and Anatomy, University of Sheffield Compensatory muscle hypertrophy can readily be studied following the surgical removal of synergistic muscles. The present preliminary investigation was carried out on 6 mice to determine the responses of type I, intermediate and type II fibres in the extensor digitorum longus (EDL) muscle following the removal of the tibialis anterior muscle (James, J. Anat. 122, 1976). Hypertrophic EDL muscles were obtained from animals 116 days after operation. Contralateral muscles were used as controls. Fresh frozen transverse sections of the muscles were stained using enzyme histochemical techniques in order to identify the different types of muscle fibre. The sizes of the muscle fibres were measured using simple morphometric point counting techniques. The results are presented in Table 1. In general, it was found that type I, intermediate and type II fibres increased in the cross sectional areas by 18 %, 30 % and 42 % respectively. Surgically-induced hypertrophy is a convenient method for studying hypertrophy though its mechanism is not understood and the observable changes probably differ markedly from exerciseinduced hypertrophy. The findings in the present study are of interest in that variations in the fibre type composition of muscles could markedly influence the degree of observed hypertrophy. The reason for the greater response of type II fibres is not clear although it is possible that increase in size can be more readily achieved by relatively anaerobic fibres.

Table 1 Animal no.

Type I fibres ( Sm2+ S.E.)

1 2 3 4 5 6

723-1 +54 3

444'6±27-4 610-7± 35.4 547-6±27*4 542-9±31-4 556.9±43 9

1 2 3 4 5 6

781-6±30-2 697'3 ±43-3 870-5± 62-0 491*4±37-4 575-6± 34.9 1010*9±66-7

Intermediate fibres (Um2 ± S.E.)

Type II fibres (C4m2 ± S.E.)

Control 15491 ± 50 5 13034 ± 65*0 1624-0± 53 0 1223-8± 67*3 1258-9±47-6 1258-9± 62*3 Hypertrophic 2265*1±97 3 1670-8± 87-8 2323-6±210-1 1310-4±94-1 1432-1 ± 67-6 2150*5+ 158*2

1778-4± 38-7 1949-2±77-6 2204'3 ± 61 8 1638-0±40 0 1488-2±43-3 2021*8±48*8

2761*2±84-9 2295-5± 81*2 3582*5 ± 218*3 2111 ±108-1 1773-7± 80-3

3830-6±146-0

D.13. Anisotropic stereology. VII. A comprehensive model of mitochondrial membranes in skeletal muscle fibres. By N. T. JAMES and G. A. MEEK. Department ofHuman Biology and Anatomy, University of Sheffield The mitochondria in skeletal muscle fibres are not randomly distributed. Their long axes tend to lie parallel with the long axes of the muscle fibres and their contained cristae tend to be orientated transversely. The surface membranes and the cristae of mitochondria each consist of random surface elements and also non-random planar or linear surfaces. Consequently these membranes can be analysed using stereological techniques specially derived for analysing partially orientated surfaces in 3D space (Underwood, Quantitative Stereology, Addison-Wesley, London, 1970). Models for estimating separately the various components of the external surfaces (James & Meek, J. Anat. 118, 1974) and also the internal cristae (James & Meek, J. Anat. 126, 1978) have previously been used. In the present quantitative study of muscle mitochondria, a comprehensive model allowing for the isometric, planar and linear surfaces of both the external surfaces of mitochondria and their cristae has been used.

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Transversely and longitudinally orientated electron micrographs of mitochondria were prepared from skeletal muscle fibres. A linear test grid consisting of parallel lines was superimposed on each micrograph and the number of intersections made by either surface membranes or cristae per unit length of test line were counted. Values for three parameters, (PL),, (P,),, and (PL)I were obtained. (PL)1 and (PL)II are the number of intersections per unit length of test line on longitudinal sections when the test lines were lying perpendicular and parallel to the long axes respectively. Counts for (PL)I were obtained by superimposing the test grid on transversely orientated sections. The isometric, planar and linear components of the mitochondrial membranes are given by the terms 2(PL),I, (PL)± - (PL)I and P {(PL)I - (PL)JI) respectively. The total surface area of mitochondrial membranes (Sv) is given by the relation S = (PL)1+ (2- fP)(PL)u + (a7- 1)(PL);. Some typical values for mitochondrial membranes in muscle fibres of the pectoralis major muscle of the pigeon were demonstrated. Some methods of determining the relative orientation of the various components of the partially orientated mitochondrial membranes were also demonstrated.

D.14. Anisotropic stereology. VIII. The derivation of an index of contiguity for analysing the distribution of fibre types in skeletal muscle. By N. T. JAMES. Department of Human Biology and Anatomy, University of Sheffield Most mammalian skeletal muscles consist of an heterogeneous population of type I, intermediate and type IL muscle fibres. In transverse sections of whole muscles the different types of fibre seem to form distinct patterns related to the functional activity of the muscle. In the present study, a formula for quantifying the degree of contiguity between the fibre types has been formulated which can be used to measure these patterns. The contiguity of a fibre type can be defined as the fraction of the total interface area between fibres which is shared by fibres of that type. The conventional formulae for evaluating the contiguity of three dimensional test objects (Underwood, Quantitative Stereology, Addison-Wesley, London, 1970) are not applicable to muscle fibres on account of their marked anisotropy. The interface areas between fibres (S) have been measured in muscle sections using the relation S = 7T.PL /um2 -m' (i.e. area of interface per unit length of muscle). PL is the number of intersections of fibre profiles per unit length of test line contained in a superimposed test grid. The relation is similar to that used for estimating length per unit area. Let type I, intermediate and type II fibres arbitrarily be termed R, I and W. Let the interface areas (S) between the fibres of each type be termed SRR, SII and SWw and the interfaces between fibres of different types be termed SRI, SIW and 5WW. An index of continguity for R fibres (CR) can then be defined by the term: SRR SRR + SRI + SRW

Whilst the index of contiguity for W fibres (Cw) can be defined by the term: sww SRW+SIW+SWWw

Values for CR and Cw are presented for normal and hypertrophic skeletal muscles. Slight changes are found in the pattern of fibre types in hypertrophic muscles. D.15. Microscope stage with multiple co-ordinate memory. By W. K. TAYLOR and H. MAMIGONIANS (introduced by T. A. QUILLIAM). Department of Electronic and Electrical Engineering, University College, London Random access memories (RAMs) are ideally suited to the problem of storing the positions of points of interest on a microscope slide. Any selected area of the specimen is automatically divided into small squares that are identified in the memory by their X and Y quantised coordinate numbers and also by an address counter that stores an order-of-selection number. A specially constructed stage driven by X and Y stepping motors replaces the usual hand manipulated microscope stage and movement is remotely controlled during the writing phase by

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push buttons or centre-off switches that may be foot operated to leave the hands free. In one mode of operation the co-ordinate origin X = 0, Y = 0 is first chosen and a set-zero co-ordinate button is pressed. The smallest square dimension is determined by the stepping motor step size and a micrometer screw drive. At low power the squares can be made larger by using multiples of the smallest step for the sides of the squares. The set-zero co-ordinate operation also sets the memory address counter to zero. If examination of the slide reveals an interesting feature the microscopist simply presses a 'write' button when the feature is central within the field of view (and also within the selected boundary area) at co-ordinates X1 Y1 say. The write button has two effects. Firstly, it causes the co-ordinates X1 and YL to be stored at address zero in an XRAM and a YRAM respectively. Secondly, it increases the RAM address counter by one after a short delay. This process is repeated for each new feature up to a maximum of 128 for the particular RAM employed. Fortapplications that require automatic measurement, such as timedependent spectral transmission of light through cells or time lapse photography, a clock is used to cycle repeatedly through the set of features so that each is examined automatically at specific times.

Proceedings of the Anatomical Society of Great Britain and Ireland. November 1978. Abstracts.

J. Anat. (1979), 128. 2, pp. 411-443 With 11 figures Printed in Great Britain 411 Proceedings of the Anatomical Society of Great Britain and Ireland...
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