The vertebrate inner ear forms a highly complex sensory structure responsible for the detection of sound and balance. Some new aspects on the evolutionary and developmental origin of the inner ear are summarised here. Recent molecular data have challenged the longstanding view that special sense organs such as the inner ear have evolved with the appearance of vertebrates. In addition, it has remained unclear whether the ear originally arose through a modification of the amphibian mechanosensory lateral line system or whether both evolved independently. A comparison of the developmental mechanisms giving rise to both sensory systems in different species should help to clarify some of these controversies. During embryonic development, the inner ear arises from a simple epithelium adjacent to the hindbrain, the otic placode, that is specified through inductive interactions with surrounding tissues. This review summarises the embryological evidence showing that the induction of the otic placode is a multistep process which requires sequential interaction of different tissues with the future otic ectoderm and the recent progress that has been made to identify some of the molecular players involved. Finally, the hypothesis is discussed that induction of all sensory placodes initially shares a common molecular pathway, which may have been responsible to generate an ‘ancestral placode’ during evolution.

Tongues were removed from rat fetuses on d 16 of gestation (E16) and from newborn (P0) and juvenile rats on d 7 (P7) and d 21 (P21) postnatally for examination by light and transmission electron microscopy. In the fetuses at E16, no rudiments of filiform papillae were visible on the dorsal surface of the tongue. No evidence of keratinisation could be recognised over the entire dorsal lingual epithelium. At P0, rudiments of filiform papillae showed a similar distribution to that seen in the adult, but had a more rounded appearance. The columnar structure of cells in the epithelium, with the different degrees of keratinisation as observed in the mature adult, was indistinct, but a keratinised layer was clearly located at the tip of each filiform papilla. In juveniles at P7, the filiform papillae on the anterior part of the tongue were long and slender, and the anterior and posterior cell columns of the filiform papillae and the interpapillary cell columns were clearly distinguishable. In juveniles at P21, the structure of filiform papillae was identical to that in the adult. These results indicate that, in rats, the morphogenesis of filiform papillae advances in parallel with keratinisation of the lingual epithelium from just before birth to a few weeks after birth.

Spermatogenesis in the platypus (Ornithorhynchus anatinus) is of considerable biological interest as the structure of its gametes more closely resemble that of reptiles and birds than marsupial or eutherian mammals. The ultrastructure of 16 steps of spermatid development is described and provides a basis for determining the kinetics of spermatogenesis. Steps 1–3 correspond to the Golgi phase of spermatid development, steps 4–8 correspond to the cap phase, steps 9–12 are the acrosomal phase, and steps 13–16 are the maturation phase. Acrosomal development follows the reptilian model and no acrosomal granule is formed. Most other features of spermiogenesis are similar to processes in reptiles and birds. However, some are unique to mammals. For example, a thin, lateral margin of the acrosome of platypus sperm expands over the nucleus as in other mammals, and more than in reptiles and birds. Also, a tubulobulbar complex develops around the spermatid head, a feature which appears to be unique to mammals. Further, during spermiation the residual body is released from the caudal end of the nucleus of platypus sperm leaving a cytoplasmic droplet located at the proximal end of the middle piece as in marsupial and eutherian mammals. Other features of spermiogenesis in platypus appear to be unique to monotremes. For example, nuclear condensation involves the formation of a layer of chromatin granules under the nucleolemma, and development of the fibrous sheath of the principal piece starts much later in the platypus than in birds or eutherian mammals.

The peptidergic and noradrenergic innervation of rat and human thymus was investigated by immunohistochemistry at the light and electron microscopical level (avidin-biotin-complex, sucrose-phosphate-glyoxylic-acid, and immunogold techniques). The distribution of noradrenergic neural profiles, and positive immunoreactivity for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY) is described in female rats during ageing, and in human children. In the neonatal rat thymus, the arteries and septa are well supplied by fine varicose nerves. In older animals (2 wk–1 y) the number of septa and blood vessels increase and consequently also the innervation. No nerves were found in the cortex. Apart from the innervation of the septal areas, immunoreactivity for CGRP and TH was present in thymic cells. Except for the young rats (neonatal–14 d), all rats showed CGRP positivity in subcapsular/perivascular epithelial cells (type 1 cells). All rat thymuses also contained a few TH positive cells in the medulla, which could only be confirmed as epithelial cells (type 6 cells) in children. Type 1 cells in the human thymus were not CGRP positive, but as in the rat, there were similar TH positive cells in the medulla. It was concluded that in addition to nerves containing CGRP, noradrenaline or dopamine, epithelial cells also contain these transmitters. They could therefore act on different cells (compared with neural targets) in a paracrine manner.

To date, there appear to have been no detailed and clear descriptions of the nerve plexuses and their subdivisions in the intestine of cattle. In this study, the enteric nervous system in the jejunum and ileum of 12 1-y-old calves was examined using neurofilament protein and vasoactive intestinal peptide immunohistochemistry in wholemounts and paraffin sections combined with staining of paraffin and historesin sections with haematoxylin and eosin. The main organisation of the plexuses was similar to that of the pig, horse and man with external and internal submucous plexuses being morphologically distinct, with further subdivisions of the internal submucous plexus into the external and internal subplexuses. However, in contrast to pig, horse and man, the submucous layer was firmly attached to the inner circular muscle layer. The myenteric plexus was well developed with large ganglia, and primary and secondary nerve strands. Its main axis was oriented parallel to the outer longitudinal smooth muscle; large ganglia and primary nerve strands fused to form complex ganglia, and 2 types of tertiary nerve strands were observed. Antibodies to neurofilament proteins and vasoactive intestinal peptide revealed adendritic, pseudouniaxonal or multiaxonal type II neurons only in the myenteric and submucous plexuses. This appears to be the first report of the identification of isolated uniaxonal, multidendritic type IV neurons in the mucous pericryptal plexus. The new information presented here provides further evidence for the existence of anatomical and functional differences between the external and internal submucous plexuses and for supporting the nomenclature proposed earlier.

The gross and histological appearance and the distribution of T and B lymphocytes and plasma cells are described for lymphoid tissues obtained from 15 platypuses. The spleen was bilobed and surrounded by a thick capsule of collagen, elastic fibres and little smooth muscle. White pulp was prominent and included germinal centres and periarterial lymphoid sheaths. Red pulp contained haematopoietic tissue. A thin lobulated thymus was located within the mediastinum overlying the heart. The cortex of lobules consisted of dense aggregates of small and medium lymphocytes, scattered macrophages and few reticular epithelial cells. In the medulla, Hassall's corpuscles were numerous, lymphocytes were small and less abundant, and reticular cells were more abundant than in the cortex. Lymphoid nodules scattered throughout loose connective tissue in cervical, pharyngeal, thoracic, mesenteric and pelvic sites measured 790±370 μm (mean±S.D., n = 39) in diameter, the larger of which could be observed macroscopically. These consisted of single primary or secondary follicles supported by a framework of reticular fibres. Macrophages were common in the germinal centres. The platypus had a full range of gut-associated lymphoid tissue. No tonsils were observed macroscopically but histologically they consisted of submucosal follicles and intraepithelial lymphocytes. Peyer's patches were not observed macroscopically but histologically they consisted of several prominent submucosal secondary follicles in the antimesenteric wall of the intestine. Caecal lymphoid tissue consisted of numerous secondary follicles in the submucosa and densely packed lymphocytes in the lamina propria. Bronchus-associated lymphoid tissue was not observed macroscopically but was identified in 7 of 11 platypus lungs assessed histologically. Lymphoid cells were present as primary follicles associated with bronchi, as aggregates adjacent to blood vessels and as intraepithelial lymphocytes. The distribution of T lymphocytes, identified with antihuman CD3 and CD5, and B lymphocytes and plasma cells, identified with antihuman CD79a and CD79b and antiplatypus immunoglobulin, within lymphoid tissues in the platypus was similar to that described in therian mammals except for an apparent relative paucity of B lymphocytes. This study establishes that the platypus has a well-developed lymphoid system which is comparable in histological structure to that in therian mammals. It also confirms the distinctiveness of its peripheral lymphoid tissue, namely lymphoid nodules. Platypus lymphoid tissue has all the essential cell types, namely T and B lymphocytes and plasma cells, to mount an effective immune response against foreign antigens.

The pulmonary arterial smooth muscle cell (SMC) cytoskeleton was studied in tissue from 36 piglets aged from within 5 min of birth to 21 d of age, and in 8 adults. An additional 16 piglets were made pulmonary hypertensive by exposure to hypobaric hypoxia (50.8 kPa) for 3 d. In conduit intrapulmonary elastic arteries α, β and γ actin, the 204, 200 and 196 kDa myosin heavy chain (MHC) isoforms and vinculin were localised by immunohistochemistry. The total actin content, the proportion of monomeric to filamentous α and γ actin and changes in the proportions of the MHC isoforms were determined biochemically. Dividing SMCs were localised and quantified using Ki-67. We found a transient reduction in immunohistochemical expression of γ actin, 204 kDa MHC isoform and vinculin at 3 and 6 d in the inner media, associated with a transient increase in Ki-67 labelling. The actin content also decreased at 3 and 6 d (P < 0.05), but there was a postnatal, permanent increase in monomeric actin, first the α then the γ isoform. The relative proportions of the MHC isoforms did not change between birth and adulthood in elastic pulmonary arteries but in muscular arteries the 200 kDa isoform increased between 14 d and adulthood. Pulmonary hypertension prevented both the immunohistochemical changes and the postnatal burst of SMC replication and prevented the transient postnatal reduction in actin content. These findings suggest that rapid remodelling of the actin cytoskeleton is an essential prerequisite of a normal postnatal fall in pulmonary vascular resistance.

The formation of crossveins in Drosophila was an important early case study in understanding the role of the environment in the development and evolution of morphological structures. More recent work has shown that signalling processes play a crucial role in the formation of crossveins in Drosophila and that the interaction of a heat shock factor, Hsp90, with components of signal transduction pathways may account for the sensitivity of these structures to environmental perturbations. A new model for the development of crossveins is presented that divides the formation of crossveins into 3 separate stages. First, the number and placement of the crossveins is determined by signalling along the proximal-distal axis of the wing. This signal may involve the cdc42 gene product and the Jun-N-terminal Kinase signal transduction pathway. Then, during the second stage, an inductive signal from the dorsal wing epithelium is sent to the ventral wing epithelium at locations specified by the first signal. The second signal appears to involve the BMP-like signalling pathway. Finally, in the third stage, a domain of vein competent cells is defined by the signalling from the EGF-receptor-Map Kinase signal transduction pathway, and the exact location of the veins is eventually determined within that domain by Notch-Delta signalling interactions. By altering components of these 3 stages, insects can independently regulate the presence or absence, the number and placement, and the thickness and flexibility of the crossveins. This capacity for the modulation of crossvein structure in many different ways may have contributed to the evolution of different modes of insect flight.

To evaluate whether the lines occasionally detected on clinical magnetic resonance (MR) images are genuine hippocampal layers, a formalin fixed hippocampal specimen was scanned using T2 weighted sequences at 7 Tesla (voxel dimensions 0.064×0.064×1 mm) and at 1.5 Tesla (voxel dimensions: 0.156×0.156×1 mm) and compared with the results of histological examination. In addition, a healthy volunteer was scanned with a T2 weighted sequence at 1.5 Tesla (voxel dimensions: 0.469×0.469×2 mm). On 7 Tesla images hippocampal layers and the granule cell layer of the dentate were visible. On 1.5 Tesla images of the specimen, the hippocampal layers were again identified, but the granule cell layer of the dentate was not detectable. On 1.5 Tesla images of the hippocampus in vivo, 3 layers could be distinguished in the hippocampus on some slices. These mainly represented the alveus, pyramidal cell layer and stratum radiatum. A dark line consisting of a few pixels possibly represented the dentate gyrus. Our results show that the lines occasionally detected on clinical MR images are likely to be real hippocampal layers. However, the resolution currently used in clinical imaging (typically 0.469×0.469×2 mm or lower) is not sufficient for the detection of all hippocampal layers. For the reliable detection of all hippocampal layers on MR images an increase by a factor of approximately 20 would be necessary.

The timing of the first appearance of lymph follicles and germinal centres in various lymph nodes, and the ways in which numbers of these and IgM-synthesising cells increase within the nodes, were investigated in male and female C57Bl/6N mice aged from 4 d to 16 wk. The lymphoid organs examined were the Peyer's patches, spleen, somatic (submandibular, deep cervical, brachial, axillary, inguinal and popliteal) and visceral (mesenteric and lumbar) lymph nodes. Primary follicles appeared in most somatic lymph nodes 6 d after birth. The number of follicles per node then increased rather sharply in larger lymph nodes and slowly in smaller nodes, up to 28 d of age, reaching a level which varied according to the location of the node. Thereafter, the number of follicles in the somatic lymph nodes increased only slightly to moderately, reaching a peak or plateau at 8–12 wk. In the mesenteric (ileocaecal) nodes, primary follicles first appeared at 12 d, then increased linearly during the suckling period and after weaning to reach a plateau at 8 wk of age. Germinal centres appeared in the submandibular and mesenteric nodes at 28 d and their numbers increased consistently in the latter, while remaining low in the former. The impact of possible ‘natural’ exogenous antigen stimulation of the various lymph nodes was estimated from the presence of IgM-synthesising cells and germinal centres. Differences between the patterns of age-dependent changes in the numbers of lymph follicles observed in the somatic and mesenteric lymph nodes during their ontogeny are discussed in relation to differences in the magnitude of the exogenous antigen stimulatory effect. We also found that the variations in the numbers of lymph follicles produced in somatic lymph nodes at different locations during the first 28 d after birth reflected differences in the dimensions of the body regions drained by a particular somatic lymph node at this stage of development.

A study was carried out on the discomalleolar ligament by dissection of adult human cadavers. The ligament corresponds to the most internal portion of the superior lamina of the temporomandibular joint capsule. It extends from the posterointernal portion of the temporomandibular joint disc, penetrates the petrotympanic fissure and reaches the malleus of the middle ear. Because of its morphology and anatomical arrangement the discomalleolar ligament should be considered as an intrinsic ligament of the temporomandibular joint and distinguished from the tympanic portion of the sphenomandibular ligament (anterior ligament of the malleus).

Visual system abnormalities are commonly encountered in the fetal alcohol syndrome although the level of exposure at which they become manifest is uncertain. In this study we have examined the effects of either low (ETLD) or high dose (ETHD) ethanol, given between postnatal days 4–9, on the axons of the rat optic nerve. Rats were exposed to ethanol vapour in a special chamber for a period of 3 h per day during the treatment period. The blood alcohol concentration in the ETLD animals averaged ∼ 171 mg/dl and in the ETHD animals ∼ 430 mg/dl at the end of the treatment on any given day. Groups of 10 and 30-d-old mother-reared control (MRC), separation control (SC), ETLD and ETHD rats were anaesthetised with an intraperitoneal injection of ketamine and xylazine, and killed by intracardiac perfusion with phosphate-buffered glutaraldehyde. In the 10-d-old rat optic nerves there was a total of ∼ 145000–165000 axons in MRC, SC and ETLD animals. About 4% of these fibres were myelinated. The differences between these groups were not statistically significant. However, the 10-d-old ETHD animals had only about 75000 optic nerve axons (P < 0.05) of which about 2.8% were myelinated. By 30 d of age there was a total of between 75000–90000 optic nerve axons, irrespective of the group examined. The proportion of axons which were myelinated at this age was still significantly lower (P < 0.001) in the ETHD animals (∼ 77%) than in the other groups (about 98%). It is concluded that the normal stages of development and maturation of the rat optic nerve axons, as assessed in this study, can be severely compromised by exposure to a relatively high (but not low) dose of ethanol between postnatal d 4 and 9.

Chasmagnathus granulatus is a hyper-hyporegulating crab that inhabits changing habitats of salinity in Brazil, Uruguay and Argentina. Since the gills are the main sites for active ion transport in crabs, the adaptive changes in the gill epithelium occurring under different conditions of salinity were studied by means of morphological and morphometric analysis, and immunohistochemical identification of cell proliferation (BrdU technique). In anterior (1–3) gills the epithelium thickness from crabs acclimatised to 12, 34 and 44 g/l ranged from 1.27 to 2.46 μm, with no significant change during acclimatisation, thus denoting a respiratory function. Medial (4–5) gill epithelium was slightly thicker in extreme salinities, but these differences were not statistically significant. In contrast, epithelial thickness of the posterior (6–8) gills increased significantly up to 8.10 μm (dorsal zone of gill 8) both in hyper- and hyposaline media compared with seawater. The dark areas measured in gill 8 treated with AgNO3 revealed putative ion transporting tissue, especially at 12 and 44 g/l, corresponding to the zones of higher epithelial thickness. Hence these areas seem to participate both in hyper- and hyporegulation. Proliferating cells labelled with BrdU almost never occurred in the gills/salinity combinations studied during the initial 48 h of transfer from seawater to hyperconcentrated or diluted media, thus suggesting an increase in cell size rather than cell proliferation.

The movement of the carpal bones in gripping was clarified in the giant panda (Ailuropoda melanoleuca) by means of macroscopic anatomy, computed tomography (CT) and related 3-dimensional (3-D) volume rendering techniques. In the gripping action, 3-D CT images demonstrated that the radial and 4th carpal bones largely rotate or flex to the radial and ulnar sides respectively. This indicates that these carpal bones on both sides enable the panda to flex the palm from the forearm and to grasp objects by the manipulation mechanism that includes the radial sesamoid. In the macroscopic observations, we found that the smooth articulation surfaces are enlarged between the radial carpal and the radius on the radial side, and between the 4th and ulnar carpals on the ulnar side. The panda skilfully grasps using a double pincer-like apparatus with the huge radial sesamoid and accessory carpal.

The structure and vascular pattern of the human tibialis anterior tendon was investigated using injection techniques, light and transmission electron microscopy and immunohistochemistry. From the well vascularised peritenon, blood vessels penetrate the tendon tissue and anastomose with a longitudinally oriented intratendinous network. The distribution of blood vessels within the tibialis anterior tendon was not homogenous. The posterior part of the tendon had a complete vascular network that extends from the musculotendinous junction to the insertion at the first metatarsal and medial cuneiform bones. In the anterior half, the tissue was avascular in a zone with a length of 45–67 mm. This zone was covered by a single layer (∼30 μm) of oval shaped cells. Transmission electron microscopy showed that these cells have the characteristics of chondroid cells. This region was stained by Alcian blue at pH 1 which indicates a high concentration of acid glycosaminoglycans and immunohistochemical staining for chondroitin-4-sulphate, chondroitin-6-sulphate and aggrecan was positive. However, immunostaining for the typical cartilage specific type II collagen within this zone was negative. The location of the avascular zone corresponds to the region where the tibialis anterior tendon wraps around the superior and inferior retinacula which serve as fibrous pulleys. This is the region where most spontaneous ruptures of the tibialis anterior tendon occur. The presence of fibrocartilage within gliding tendons is a functional adaptation to compressive and shearing forces. In contrast to reports from the literature about the structure of gliding tendons wrapping around a bony pulley, the gliding zone of the tibialis anterior tendon has only a narrow layer of chondroid cells and proof of type II collagen is lacking.

Although the 3 genera of living lungfish have different-shaped adult tooth plates, their larval stages have similar patterns of development. The sequence in the pattern of initiation of teeth and their modification through ontogeny in Neoceratodus hatchlings provides a developmental model for fossil hatchling tooth plates (smallest 1–2 mm) recovered as 3-dimensional dentitions from Andreyevichthys. This Late Devonian lungfish demonstrates that these also have a similar dentition pattern and suggests strongly conserved developmental processes. We postulate that a specific pattern of development, derived within lungfish, has been conserved in extant forms through evolution from the earliest known lungfish. The most basal early dipnoan, Diabolepis speratus, is also known from juveniles with tooth plates formed in this pattern.

The lungfish pattern is in marked contrast to the typical linear rows of teeth with lingual replacement for each tooth position, characteristic of most osteichthyan and chondrichthyan dentitions. Uniquely for lungfish, teeth are only added to the lateral ends of the radial rows in the palatal and lingual dentition and are consolidated into dental plates without loss through shedding. It is proposed that this tooth pattern is set up from primordial teeth at the patterning stage of the dentition, one in each dentate region of the larval jaws. Although in post-Devonian lungfish marginal dentate bones are absent in the adult, in both the fossil and extant hatchling, teeth are present and function on some of the marginal bones. This pattern of development and loss is described and we conclude that in both forms it is also based on a radial pattern of successive tooth initiation. We propose that this ontogenetic pattern constrained the phylogenetic pattern of adult form, through evolution of dipnoan dentitions from 360 MYBP until the present. The universality amongst dipnoans and the implications for such a conserved constraint in the developmental module for the dentition is discussed.

Peripheral nerve transection induces significant changes in neuropeptide expression and content in injured primary sensory neurons, possibly due to loss of target derived neurotrophic support. This study shows that neurotrophin-3 (NT-3) delivery to the injured nerve influences neuropeptide Y (NPY) expression within dorsal root ganglia (DRG) neurons. NT-3 was delivered by grafting impregnated fibronectin (500 ng/ml; NT group) in the axotomised sciatic nerve. Animals grafted with plain fibronectin mats (FN) or nerve grafts (NG) were used as controls. L4 and L5 DRG from operated and contralateral sides were harvested between 5 and 240 d. Using immunohistochemistry and computerised image analysis the percentage, diameter and optical density of neurons expressing calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP) and NPY were quantified. Sciatic nerve axotomy resulted in significant reduction in expression of CGRP and SP, and significant upregulation of VIP and NPY (P<0.05 for ipsilateral vs contralateral DRG). By d 30, exogenous NT-3 and nerve graft attenuated the upregulation of NPY (P<0.05 for NT and NG vs FN). However, NT-3 administration did not influence the expression of CGRP, SP or VIP. The mean cell diameter of NPY immunoreactive neurons was significantly smaller in the NT-3 group (P<0.05 for NT vs FN and NG) suggesting a differential influence of NT-3 on larger neurons. The optical densities of NPY immunoreactive neurons of equal size were the same in each group at any time point, indicating that the neurons responding to NT-3 downregulate NPY expression to levels not detectable by immunohistochemistry. These results demonstrate that targeted administration of NT-3 regulates the phenotype of a NPY-immunoreactive neuronal subpopulation in the dorsal root ganglia, a further evidence of the trophic role of neurotrophins on primary sensory neurons.

Associated skeletons, which are specimens preserving more than one body part from the same individual, are especially important for taxonomic and functional analyses. This study concentrates on the subset of associated skeletons which preserve the reciprocal surfaces of a joint. It uses laser scanning to explore whether the shapes of the reciprocal surfaces of a joint of an individual are significantly more congruent than the surfaces of randomly-matched pairings taken from the same species. Laser scanning was used to capture the distal articular surface of the left tibia of OH35 and the trochlear articular surface of the talus of OH8, both from Bed I, Olduvai Gorge, Tanzania. The degree of congruency between those articular surfaces was tested against the congruency of the talocrural joint of AL 288-1 (Australopithecus afarensis), and the congruency of both associated and randomly-matched talocrural joints of modern humans, chimpanzees and gorillas. The results suggest that OH35 and OH8 do not come from the same individual and may not come from the same species. Although this analysis leaves open the taxonomic affinity of OH35, it demonstrates the potential of laser scanning for capturing 3D data in palaeoanthropology. It also demonstrates the potential for using the relative congruency of reciprocal joint surfaces as a test of the likelihood that isolated limb bones are components of a single individual.

We examined numerous ovarian follicles from 32–35 d virgin mice by transmission electron microscopy and light microscopic immunohistochemistry. No macrophages were seen, but various stages of apoptotic granulosa cells were encountered. Presumably a granulosa cell or its debris in an advanced stage of apoptosis was destined to be phagocytosed by adjacent normal-looking granulosa cells. Other granulosa cells of normal appearance were seen in the region of the zona pellucida in contact with and apparently phagocytosing atrophic oocytes. Such granulosa cells were characterised by the presence of gap junctions with other cells and frequently contained annular gap junctions in the cytoplasm. To confirm the lack of involvement of macrophages in the process of follicular atresia and elimination, specially prepared ovarian sections were incubated with antimouse macrophage monoclonal antibodies (F4/80, Mac-1, Mac-2). None of the follicles examined showed positive immunoreactivity with these antibodies. Atretic follicles may shrink and eventually disappear from the ovary as a result of repeated apoptosis and phagocytosis by granulosa cells. There is no evidence for the presence or involvement of macrophages in the atretic follicles, at least in prereproductive mice as examined.

A fluorescence confocal microscopy technique was employed to obtain subsurface images of nerve and microvascular structure in the vas deferens and colon of the living rat. The use of dual labelling with vital dyes and 2-channel confocal acquisition allowed differentiation of microscopic structure at both low and higher magnification. Characteristic staining patterns of nerves and blood vessels were repeatedly obtained in each tissue, suggesting the potential of this technique for studying morphological changes associated with surgical procedures and/or models of neuronal or vascular pathology.