GABA (gamma-amino-butyric acid) is the predominant neurotransmitter in the mammalian suprachiasmatic nucleus (SCN), with a central role in circadian time-keeping. We therefore undertook an ultrastructural analysis of the GABA-containing innervation in the SCN of mice and rats using immunoperoxidase and immunogold procedures. GABA-immunoreactive (GABA-ir) neurons were identified by use of anti-GABA and anti-GAD (glutamic acid decarboxylase) antisera. The relationship between GABA-ir elements and the most prominent peptidergic neurons in the SCN, containing vasopressin-neurophysin (VP-NP) or vasoactive intestinal polypeptide (VIP), was also studied. Within any given field in the SCN, approximately 40–70% of the neuronal profiles were GABA-ir. In GABA-ir somata, immunogold particles were prominent over mitochondria, sparse over cytoplasm, and scattered as aggregates over nucleoplasm. In axonal boutons, gold particles were concentrated over electron-lucent synaptic vesicles (diameter 40–60 nm) and mitochondria, and in some instances over dense-cored vesicles (DCVs, diameter 90–110 nm). GABA-ir boutons formed either symmetric or asymmetric synaptic contacts with somata, dendritic shafts and spines, and occasionally with other terminals (axo-axonic). Homologous or autaptic connections (GABA on GABA, or GAD on GAD) were common. Although GABA appeared to predominate in most neuronal profiles, colocalisation of GABA within neurons that were predominantly neuropeptide-containing was also evident. About 66% of the VIP-containing boutons and 32% of the vasopressinergic boutons contained GABA. The dense and complex GABAergic network that pervades the SCN is therefore comprised of multiple neuronal phenotypes containing GABA, including a wide variety of axonal boutons that impinge on heterologous and homologous postsynaptic sites.

We examined the distribution of parvalbumin in the pigeon thymus by light and electron microscopic immunohistochemistry. Tissues were also examined by conventional electron microscopy to determine the ultrastructure of immunoreactive cells. Parvalbumin immunoreaction was located in epithelial cells of the cortex, which formed dense mesh-like structures. Parvalbumin-positive epithelial cells were classified into 2 types. The first comprised elongated cells. In these, the nucleus was spindle-shaped, oval, or triangular, with a slightly irregular contour and contained rich heterochromatin peripherally. The cytoplasm was pale and processes extended laterally or ramified among the surrounding thymocytes. This type of cell formed the majority of immunoreactive cells. The other cell type consisted of polygonal epithelial cells. The nucleus was oval with deep indentations. Euchromatin occupied a large part of the nucleus. The cytoplasm contained numerous cell organelles compared with the elongated type, in particular, electron-dense vacuoles of various sizes and often bundles of tonofilaments. Both types of epithelial cell were interconnected by desmosomes. No secretory granules were found in the cytoplasm of elongated or polygonal cells. These results indicate the presence of heterogeneous group of parvalbumin-immunoreactive epithelial cells and suggest the likelihood of different functional roles for parvalbumin in the pigeon thymus.

The basal ganglia occupy a commanding place in neuroscience research, in clinical neurology and in biomedical education. The paucity of our understanding of the role of the basal ganglia in normal everyday life combined with our more extensive knowledge of their deficiencies in a variety of clinical syndromes is a potent spur to continuing investigation. That some of these neurodegenerative syndromes—such as Parkinson's disease—are already common only heightens the need for insight in the face of a population with increasing expectations of longevity. About a decade ago an explosion of information on the connectivity and immunocytochemistry of forebrain structures gave rise to concepts which have shaped the fabric of basal ganglia theory—‘patch and matrix’, ‘disinhibition’, ‘parallel circuits’. Some of these ideas seemed to facilitate an understanding of the basal ganglia, others to render them more complex and impenetrable. Perhaps unsurprisingly, the work of the last decade has tended towards consolidation and refinement. However, several new developments are receiving attention, many of them related to disorders of the basal ganglia. The realisation that some forms of Parkinson's disease have a genetic determinant is gaining strength. The molecular biology of the dopaminergic synapse on the one hand and of the production of insoluble proteins on the other will clearly influence future research into therapeutic options and neuroprotection. The importance of apoptosis, neural plasticity and free radical formation remains unresolved but these are potential areas of promise. Meanwhile, scanning techniques for brain imaging are allowing real time investigation of the working striatum in normal and disordered humans and animals.

We believe that the time is opportune for a broad review of current thinking on the basal ganglia in health and disease. The following articles are based on presentations given at a Symposium on the Neurobiology of the Basal Ganglia held at Glasgow University in July 1999 as part of the Summer Meeting of the Anatomical Society of Great Britain and Ireland. The invited speakers were chosen to be wide ranging and contributions encompassed evolution, circuitry and receptors of the basal ganglia, striatal remodelling after dopamine loss, striatal functioning in humans with Huntington's disease and in primate models after midbrain fetal transplants, and the genetics of basal ganglia disorders. Short presentations and posters of current results supplemented the main presentations and some are also included amongst these reviews.

In the theoretical analysis of arterial networks the existence of geometric optimisation has long been suggested, although observational studies have not yet fully corroborated these theories. Since this could be due to experimental flaws, the aim of this study was to establish the validity of arterial geometric optimisation using a new experimental design and to assess the influence of ageing. Solid vascular casts of arterial mesenteric branching systems of 8-wk-old and adult dogs (beagles) were used to examine vascular diameters and branching angles, the latter in a manner that allowed optimisation of the line of view, thus minimising distortion errors due to a line of view not normal to the branching plane. Internal and external vessel diameters were found to be in accordance with the theoretical principle of minimum work (8-wk-old internal: r = 0.994; adult internal: r = 0.971; adult external: r = 0.985). Although branching angles were found to be in agreement with basic qualitative principles of arterial branching geometry, the measurements still showed a large amount of scatter and were generally smaller than expected on theoretical grounds, despite the newly designed measuring technique. These branching angles demonstrated small age-related differences. However, when biological cost was considered per bifurcation, surprisingly, guidance towards minimum lumen volume and pumping power with increase in age could clearly be demonstrated (P<0.001). It is concluded that our findings support the existence of a degree of arterial geometric optimisation in favour of minimum lumen volume and pumping power, increasing with age. Future investigations should focus on the biofeedback mechanisms involved.

Mammary glandular tissues and mammary secretions were obtained from sheep at 2–60 d after weaning to study the leucocyte phenotypes associated with mammary involution. From 2–4 d after weaning, neutrophils were the predominant leucocytes in the alveolar and ductal lumina. Lymphocytes were present in the alveolar and ductal epithelium, interalveolar and periductal areas. Most of the lymphocytes in the alveolar and ductal epithelium (IEL) were CD8+, some were CD45R+ and few were CD4+. In the periductal clusters and in the interalveolar areas most of the lymphocytes were CD4+. There was a significant increase (P < 0.05) in the percentages of CD45R+ granulated IEL from 2 to 7 d after weaning, and this paralleled the increase in the percentages of apoptotic cells in the glandular epithelium. By 7–60 d after weaning, most cells within the alveolar and ductal lumina were macrophages followed by predominantly CD8+ lymphocytes. CD8+ lymphocytes were still predominant in the alveolar and ductal epithelium while CD4+ cells were predominant in the interalveolar areas. Very few γδ+ T cells were observed at all the stages examined. The cells in the mammary secretions correlated with those observed in the alveolar and ductal lumina. At the early stages of involution, the neutrophils and macrophages were heavily laden with lipid droplets, casein and cellular debris. The most interesting feature was the presence of cells either with extensive cytoplasmic processes (LCA+ MHC class II+) or cytoplasmic veils (LCA+ MHC class II+CD1+), probably dendritic cells. It is concluded that the cellular constituents of the mammary gland at the latter part of involution may afford the mammary gland more resistance to infection than the lactating gland and the gland at early stages of involution. The CD45R+ IEL may trigger apoptotic cell death in the mammary glandular epithelium during mammary involution.

The accessory olfactory bulb (AOB) is the primary target of the sensory epithelium of the vomeronasal organ (VNO), and thus constitutes a fundamental component of the accessory olfactory system, which is involved in responses to behaviour-related olfactory stimuli. In this study we investigated the characteristics of the AOB, VNO, vomeronasal nerves (VNNs) and caudal nasal nerve (CdNN) in pigs and sheep, species in which olfaction plays a key behavioural role both in the neonatal period and in adulthood. The patterns of staining of the AOB by the Bandeiraea simplicifolia and Lycopersicon esculentum lectins were the same in the 2 species, whereas the Ulex europeus and Dolichos biflorus lectins gave different patterns. In both species, lectin staining of the AOB was consistent with that of the VNNs, while the CdNN did not label any of the structures studied. The entire sensory epithelium of the pig was labelled by Ulex europeus and Lycopersicum esculentum lectins, and all 4 lectins used labelled the mucomicrovillar surface of the sensory epithelium in sheep.

Retinal microglia were selectively and sequentially labelled in different layers of the retina of postnatal rats following a single intravenous injection of the fluorescent dye, rhodamine isothiocyanate (RhIc). The fluorescent cells were doubly immunostained with OX-42 and ED-1 antibodies that recognise complement type 3 (CR3) receptors and macrophage antigen, respectively. RhIc was first detected in the retinal blood vessels 5 min after injection. At 1 h, a variable number of microglia in the inner layers of the retina, namely, the nerve fibre and ganglion cell layers appeared to emit weak fluorescence. Labelled microglial cells in the inner nuclear and outer plexiform layers were not detected until 1 and 2 d had elapsed following RhIc injection. The number of labelled retinal microglia was progressively increased with time, peaking at 4 d after RhIc injection. The frequency of RhIc labelled cells also increased with age, with the largest number of cells occurring in 7-d-old rats but declined thereafter. In 11 d or older rats, RhIc was confined to the retinal blood vessels. It is concluded that when injected into the circulation, RhIc could readily gain access into the retina tissues due to an inefficient blood-retina barrier in early postnatal stages. It became impeded with maturation of the blood-retina barrier, which was established between 11 and 13 d of age. RhIc that inundated the retinal tissues was thoroughly sequestered by the resident microglial cells. It is therefore suggested that the latter could play a protective role against serum-derived substances that may be deleterious to the developing retina.

The basal ganglia (BG) have received much attention during the last 3 decades mainly because of their clinical relevance. Our understanding of their structure, organisation and function in terms of chemoarchitecture, compartmentalisation, connections and receptor localisation has increased equally. Most of the research has been focused on the mammalian BG, but a considerable number of studies have been carried out in nonmammalian vertebrates, in particular reptiles and birds. The BG of the latter 2 classes of vertebrates, which together with mammals constitute the amniotic vertebrates, have been thoroughly studied by means of tract-tracing and immunohistochemical techniques. The terminology used for amniotic BG structures has frequently been adopted to indicate putative corresponding structures in the brain of anamniotes, i.e. amphibians and fishes, but data for such a comparison were, until recently, almost totally lacking. It has been proposed several times that the occurrence of well developed BG structures probably constitutes a landmark in the anamniote-amniote transition. However, our recent studies of connections, chemoarchitecture and development of the basal forebrain of amphibians have revealed that tetrapod vertebrates share a common pattern of BG organisation. This pattern includes the existence of dorsal and ventral striatopallidal systems, reciprocal connections between the striatopallidal complex and the diencephalic and mesencephalic basal plate (striatonigral and nigrostriatal projections), and descending pathways from the striatopallidal system to the midbrain tectum and reticular formation. The connectional similarities are paralleled by similarities in the distribution of chemical markers of striatal and pallidal structures such as dopamine, substance P and enkephalin, as well as by similarities in development and expression of homeobox genes. On the other hand, a major evolutionary trend is the progressive involvement of the cortex in the processing of the thalamic sensory information relayed to the BG of tetrapods. By using the comparative approach, new insights have been gained with respect to certain features of the BG of vertebrates in general, such as the segmental organisation of the midbrain dopaminergic cell groups, the occurrence of large numbers of dopaminergic cell bodies within the telencephalon itself and the variability in, among others, connectivity and chemoarchitecture. However, the intriguing question whether the basal forebrain organisation of nontetrapods differs essentially from that observed in tetrapods still needs to be answered.

Apoptotic cells of the human growth plate have not previously been demonstrated in situ. We have investigated the distribution of apoptotic cells in costosternal growth plates and bone of premature infants aged 4–11 d with a gestational age of ∼ 26 wk. In addition, we investigated the immunolocalisation of apoptosis-related proteins within the growth plates and associated bone. A proportion of late hypertrophic chondrocytes and osteocytes within newly formed primary spongiosa showed evidence of highly fragmented DNA. The incidence of osteocyte apoptosis decreased as the distance from the chondroosseous junction increased. Tissue transglutaminase (tTG) expression was associated with apoptosis of osteocytes and hypertrophic chondrocytes. In contrast the presence of tTG was demonstrated in osteoblasts and bone lining cells but it did not colocalise with evidence of apoptosis. The anti-apoptotic gene product Bcl-2 was absent from the growth plate but was present in osteocytes. Visual assessment indicated a greater occurrence of the protein in cells occupying regions of low apoptosis. P53 was not demonstrated in the growth plate or bone. These findings would indicate that human growth plate chondrocytes appear to show little provision for ensuring cell longevity. In contrast osteocyte apoptosis appears negatively correlated with the skeletal distribution of Bcl-2 protein in the human infant, implying a potential selective vulnerability in individual cells. Lack of Bcl-2 and the high incidence of osteocyte apoptosis in the more rapidly remodelling bone of the human infant suggest a potential role of osteocyte apoptosis in the remodelling process.

The medial cortical surface of the mandible can be involved by tumour infiltration from the floor of the mouth. A detailed study of spread via accessory foramina through the edentulous alveolar crest has been previously undertaken, but no similar study has been carried out for the medial surface. In order to gain further appreciation of the mode of tumour spread, a study of the number and distribution of accessory foramina on the medial mandibular surface was performed on 89 mandibles. The number of foramina varied greatly from specimen to specimen. In the ascending ramus above the inferior dental foramen, 3 mandibles showed no foramina; the condylar section possessed the greatest proportion followed by the sigmoid and the coronoid. On the rest of the medial surface below the inferior dental foramen, all specimens showed at least 1 accessory foramen; the greatest concentration was in the middle third along the path of the inferior dental canal, followed by the upper third and the lower third section. Accessory foramina were repeatedly present at certain dedicated sites. The medial facing wall of the inferior dental foramen was found to be the commonest dedicated site (98.3%) followed by foramina on either side of the genial tubercles (71.9%), the digastric fossa (71.9%) and the median foramen above the genial tubercles (64%). The findings of this study are in keeping with the current observation that the lower border is least commonly involved in tumour spread. In view of the presence of accessory foramina along the inferior dental canal and especially on the medial facing wall of the inferior dental foramen, it is imperative to preclude tumour spread in this region prior to undertaking the conservative rim resection procedure. Medial to the symphysis the alveolar mucosa dips down almost to the level of the dedicated foramina in the vicinity of the genial tubercles. As a general rule the attached muscle forms a barrier to tumour spread except in the later stages, however, in irradiated mandibles resistance to spread has been previously reported to be diminished. Under these circumstances, it is possible that the numerous accessory foramina reported in this study could facilitate a direct pathway into the cancellous bone.

Models of basal ganglia function are described which encapsulate the principal pathophysiological mechanisms underlying parkinsonian akinesia on the one hand and abnormal involuntary movement disorders (dyskinesias) on the other. In Parkinson's disease, degeneration of the nigrostriatal dopamine system leads to overactivity of the ‘indirect’ striatopallidal projection to the lateral (external) segment of the globus pallidus. This causes inhibition of lateral pallidal neurons, which in turn project to the subthalamic nucleus. Disinhibition of the subthalamic nucleus leads to abnormal subthalamic overactivity and, as a consequence, overactivity of medial (internal) pallidal output neurons. Dyskinesias, such as are observed in Huntington's disease, levodopa-induced dyskinesia and ballism, share mechanistic features in common and are associated with decreased neuronal activity in both the subthalamic nucleus and the medial globus pallidus.

The ultrastructure and size distributions of collagen fibrils in Glisson's sheath were investigated in the rat liver to analyse the mechanical environment around the fibrils and their possible cells of origin. Glisson's sheath was found to contain 2 populations of collagen fibrils with different diameters and distinct localisations, namely fibroblast-associated and bile epithelium-associated. Fibroblast-associated collagen was composed of fibrils arranged in bundles and constituted the majority of the collagen in Glisson's sheath. Bile epithelium-associated collagen was represented by small dispersed groups of fibrils just beneath the basement membrane of the bile duct. The basement membrane of the bile duct was frequently reduplicated into a few or as many as 10 layers of laminae densae, with scattered collagen fibrils between these laminae. The diameters of the fibrils of both groups of collagen increased in relation to the calibre of the bile duct, whereas at any given place in Glisson's sheath bile epithelium-associated collagen fibrils had a smaller diameter compared with those of the fibroblast-associated fibrils. The increment in fibril diameter along the bile duct is considered to be correlated with the increase in mechanical stress acting on Glisson's sheath. The difference in diameter between the 2 populations as well as the incorporation of fibrils between the laminae densae of the basement membrane of the bile duct supports the view that the bile epithelium-associated collagen is produced by the epithelial cells of the bile duct, thus having a different origin from that of fibroblast-associated collagen. These findings provide the first evidence that the epithelial cells of the interlobular bile duct produce fibril-forming collagen. Furthermore, it is suggested that cholestasis stimulates the epithelial cells of interlobular bile duct to increased synthesis of fibril-forming collagen that is also produced by these cells under physiological conditions.

Studies of tetraploid[harr ]diploid (4n[harr ]2n) mouse chimaeras have demonstrated unequal contributions of 4n cells to different tissues of the midgestation conceptus. Such a pattern has also been reported in chimaeras as early as E3.5d, which show an enhanced contribution of 4n cells to the mural trophectoderm (Everett & West, 1996). In this study, sectioned 4n[harr ]2n and 2n[harr ]2n control chimaeric blastocysts were digitised and reconstructed in 3 dimensions (3-D). The 3-D images revealed only limited mixing of cells from the 2 contributing embryos of individual blastocysts in both chimaera groups. Consequently, the distribution pattern of the 2 cell types was dependent on the spatial relationship between the orientation of the blastocyst and the boundary between the 2 clusters of cells. The distribution patterns observed were not strikingly different for 4n[harr ]2n and 2n[harr ]2n chimaeras, each showing some transgenic positive cell contribution in all 3 identifiable developmental lineages. It was notable, however, that in all 4n[harr ]2n blastocysts at least some 4n cells were located adjacent to the blastocyst cavity. Such a consistent pattern was not evident in 2n[harr ]2n chimaeras. This study has demonstrated the value of 3-D reconstructions for the analysis of spatial relationships of 2 cell populations in chimaeric mouse blastocysts.

The expression of carbonic anhydrase (CA) in the quail metanephros was investigated during embryonic development. The immunohistochemical localisation of the isoenzymes CAII and CAIII was compared with the distribution of enzyme activity visualised by a histochemical cobalt-precipitation procedure. The developmental profile of CA activity was also evaluated by means of a biochemical method. The occurrence of a moderate and diffuse CAII immunostaining from the first developmental appearance of the metanephros anlage testified to an early expression of carbonic anhydrase. This finding is discussed in relation to the involvement of the enzyme in the morphogenetic mechanisms leading to the establishment both of cell polarity and epithelial phenotype. CA expression in the renal sites that are positive in adults proved to be developmentally regulated. In the collecting duct system, enzyme activity could not be identified until the time of hatching. No CA was detected at any stage examined at the sites where, in adults, enzyme occurrence has previously been interpreted as a membrane-associated CA isoform. The differentiating renal tubules displayed no CAIII immunoreactivity. It can be argued that the bulk of the enzyme activity in the embryonic metanephros is due to the cytosolic isoenzyme CAII.

The basal ganglia are a group of subcortical nuclei involved in a variety of processes including motor, cognitive and mnemonic functions. One of their major roles is to integrate sensorimotor, associative and limbic information in the production of context-dependent behaviours. These roles are exemplified by the clinical manifestations of neurological disorders of the basal ganglia. Recent advances in many fields, including pharmacology, anatomy, physiology and pathophysiology have provided converging data that have led to unifying hypotheses concerning the functional organisation of the basal ganglia in health and disease. The major input to the basal ganglia is derived from the cerebral cortex. Virtually the whole of the cortical mantle projects in a topographic manner onto the striatum, this cortical information is ‘processed’ within the striatum and passed via the so-called direct and indirect pathways to the output nuclei of the basal ganglia, the internal segment of the globus pallidus and the substantia nigra pars reticulata. The basal ganglia influence behaviour by the projections of these output nuclei to the thalamus and thence back to the cortex, or to subcortical ‘premotor’ regions. Recent studies have demonstrated that the organisation of these pathways is more complex than previously suggested. Thus the cortical input to the basal ganglia, in addition to innervating the spiny projection neurons, also innervates GABA interneurons, which in turn provide a feed-forward inhibition of the spiny output neurons. Individual neurons of the globus pallidus innervate basal ganglia output nuclei as well as the subthalamic nucleus and substantia nigra pars compacta. About one quarter of them also innervate the striatum and are in a position to control the output of the striatum powerfully as they preferentially contact GABA interneurons. Neurons of the pallidal complex also provide an anatomical substrate, within the basal ganglia, for the synaptic integration of functionally diverse information derived from the cortex. It is concluded that the essential concept of the direct and indirect pathways of information flow through the basal ganglia remains intact but that the role of the indirect pathway is more complex than previously suggested and that neurons of the globus pallidus are in a position to control the activity of virtually the whole of the basal ganglia.

The arterial supply of the digits of the forelimb of the Bactrian camel is described. The arteries supplying the digits were the palmar metacarpal and common palmar digital arteries III. The palmar metacarpal artery III was the continuation of the deep medial proximal metacarpal branch which was derived from the medial branch of the radial artery. It gave rise to a nutrient branch, medial branch, lateral branch and distal perforating palmar branch at the proximal end of the distal sixth of the cannon bone (fused third and fourth metacarpal bones). The common palmar digital artery III was the continuation of the median artery, which divided into medial and lateral branches. The medial branch of common palmar digital artery III which occasionally arose from the axial palmar proper digital artery III, after giving rise to the axial proximal proximal phalangeal branch, divided into the axial and abaxial palmar proper digital arteries III. The axial palmar proper digital artery III gave off the dorsoaxial distal proximal phalangeal, dorsoaxial proximal middle phalangeal, dorsoaxial distal middle phalangeal, palmoaxial middle phalangeal, palmoaxial distal phalangeal, dorsoaxial distal phalangeal branches, coronal artery and some digital tori branches. The abaxial palmar proper digital artery III gave rise to the abaxial proximal proximal phalangeal, dorsoabaxial distal proximal phalangeal, dorsoabaxial middle phalangeal, palmoabaxial middle phalangeal, palmoabaxial distal phalangeal, dorsoabaxial distal phalangeal branches, coronal artery and some digital tori branches. The lateral branch of the common palmar digital artery III in its origin, course, branching pattern and supply in the fourth digit was similar to the medial branch of common palmar digital artery III in the third digit.

In this review, the value of functional imaging for providing insight into the role of the basal ganglia in motor control is reviewed. Brain activation findings in normal subjects and Parkinson's disease patients are examined and evidence supporting the existence for functionally independent distributed basal ganglia- frontal loops is presented. It is argued that the basal ganglia probably act to focus and filter cortical output, optimising the running of motor programs.

The thigh muscles of New Zealand White (NZW) rabbits are frequently used in experimental surgery, particularly for evaluation after reinnervation or ischaemia. Although histomorphometric analyses are regularly performed, morphological data for untreated thigh muscles in previously unoperated animals are not available. Specimens from the rectus femoris (RF), vastus medialis (VM) and adductor magnus (AM) muscles from both thighs were harvested in 7 untreated rabbits and were processed for histomorphometric evaluation. The right RF and VM were harvested in a further 5 rabbit hindlimbs after experimental denervation and reinnervation of the contralateral RF and subsequently processed for histomorphometric analysis. Muscle fibre type distribution, diameter and connective tissue content were evaluated on serial transverse cryosections reacted for ATPase and NADH tetrazolium reductase activity and statistical analysis was performed. In all untreated animals RF revealed the highest proportion of type I muscle fibres (right: 8.4±4%, left: 11.4±4.9%), whereas VM showed the highest percentage of IIa fibres (right: 31.9±5.5%, left: 28.3±7.8%) and AM the highest proportion of IIb/d fibres (right: 80.5±8.6%, left: 84.4±6.3%). Fibre type distribution and diameter in rabbits after contralateral experimental operations revealed a statistically significant difference from the data obtained in bilaterally untreated animals. Knowledge of the morphology of untreated muscles is fundamental to the understanding of changes induced by intervention to the ipsi and/or contralateral thigh muscles.

The vocal ligaments insert at the anterior and posterior commissures of the larynx. These structures fulfil biomechanical functions, balancing the different elastic moduli of tendon, cartilage or bone and undergo age-related changes that may be responsible for voice changes with increasing age. The aim of this study was to analyse the insertion structures of the vocal ligaments by means of macroscopic, histological, immunohistochemical and electron-microscopic methods and to draw conclusions from age-related structural changes on a functional basis. Investigations were carried out on the larynges of 22 males and 15 females (aged 1–95 y). In adolescence, the insertion zone of the vocal ligament tendon, a dense network of connective tissue rich in sulphated glycosaminoglycans at the thyroid cartilage, is characterised by a layer between tendon and cartilage comparable to fibrocartilage. The insertion zone lacks a perichondrium. Collagen fibrils of the vocal ligament tendon penetrate directly into the thyroid cartilage. In the insertion area, the chondrocytes are surrounded by collagen fibrils, which show positive reactivity to antibodies against type I and type III collagen. Sulphated glycosaminoglycans are integrated between the collagen fibrils. In the area of the posterior glottis, elastic cartilage rests like a cap on the hyaline base of the arytenoid cartilage. There is no distinctive border between the structures. With increasing age, ossification of the laryngeal skeleton occurs, involving hyaline cartilage at the posterior glottis and hyaline and fibrocartilage at the anterior commissure. At the same time, a loss of sulphated glycosaminoglycans is observed inside the vocal ligament tendon. Advanced ossification of the laryngeal skeleton, particularly in the area of the commissures, an increasing loss of glycosaminoglycans in the vocal ligament tendon and changes in the elastic tissue reduce the elastic modulus between tendon, cartilage and bone, thus ‘stiffening’ the insertion zones, which could be one factor among others favouring voice changes with advancing age.

Previous studies have shown that chronic administration of oestrogen during postnatal rat development dramatically reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. In the present study we analysed if this response is due to a direct and selective effect of oestrogen on the uterine noradrenaline-containing sympathetic nerves, using the in oculo transplantation method. Small pieces of myometrium from prepubertal rats were transplanted into the anterior eye chamber of adult ovariectomised host rats. The effect of systemic chronic oestrogen treatment on the reinnervation of the transplants by noradrenaline-containing sympathetic fibres from the superior cervical ganglion was analysed on cryostat tissue sections processed by the glyoxylic acid technique. In addition, the innervation of the host iris was assessed histochemically and biochemically. The histology of the transplants and irises was examined in toluidine blue-stained semithin sections. These studies showed that after 5 wk in oculo, the overall size of the oestrogen-treated transplants was substantially larger than controls, and histology showed that this change was related to an increase in the size and number of smooth muscle cells within the transplant. Chronic oestrogen treatment did not provoke trophic changes in the irideal muscle. Histochemistry showed that control transplants had a rich noradrenergic innervation, associated with both myometrium and blood vessels. Conversely, in oestrogen-treated transplants only occasional fibres were recognised, showing a reduced NA fluorescence intensity. No changes in the pattern and density of innervation or in the total content of noradrenaline of the host irises were detected after chronic exposure to oestrogen. We interpreted these results to indicate that the effects of oestrogen on uterine noradrenaline-containing sympathetic nerves are neither selective or direct, but result from an interaction between sympathetic nerve fibres with the oestradiol-primed uterine tissue. A potential effect of oestrogen on the neurotrophic capacity of the uterus is discussed.