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Evolution of the amygdaloid complex in vertebrates, with special reference to the anamnio-amniotic transition

N. Moreno and A. González Department of Cell Biology, Complutense University of Madrid, Spain

Numerous studies over the last few years have demonstrated that the amygdaloid complex in amniotes shares basic developmental, hodological and neurochemical features. Furthermore, homologue territories of all main amygdaloid subdivisions have been recognized among amniotes, primarily highlighted by the common expression patterns for numerous developmental genes. Thus derivatives from the lateral pallium, ventral pallium and subpallium constitute the fundamental parts of the amygdaloid complex. With the achievement of new technical approaches, the study of the precise neuroanatomy of the telencephalon of the anuran amphibians (anamniotes) has been possible. Current embryological, hodological and immunohistochemical evidences strongly suggest that most of the structures present in amniotes are recognizable in these anamniotes. These investigations have yielded enough results to support the notion that the organization of the anuran amygdaloid complex includes subdivisions with origin in ventral pallial and subpallial territories; a strong relationship with the vomeronasal and olfactory systems; abundant intra-amygdaloid connections; a main output centre involved in the autonomic system; profuse amygdaloid fibre systems; and distinct chemoarchitecture. Three main components were characterized: (1) the lateral amygdala, that corresponds with the ventral portion of the ventral pallium, shares many hodological features with the amygdaloid ventropallial derivatives of the basolateral complex of amniotes, such as olfactory inputs, relation with amygdaloid nuclei and the projection to the ventral hypothalamus via the stria terminalis; (2) the medial amygdala which shows pallial and subpallial markers and constitutes the main secondary vomeronasal centre that strongly projects to the ventral hypothalamus, being additionally strongly innervated by peptidergic centres, features presented by amniotes; and (3) the central amygdala which constitutes a subpallial derivative representing the caudal continuation of the striatum and represents the autonomic amygdaloid subdivision as in amniotes, sharing with its counterparts connections with autonomic centres as the nucleus of the solitary tract, or the parabrachial area. Therefore the new ideas about amygdaloid evolution based on recent findings in anamniotes, and specially anurans, strongly supports the notion that basic amygdaloid structures were present at least in the brain of ancestral tetrapods organized following a basic plan shared by tetrapods.

Supported by DGCYT (BFI2003-0375).

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Development and evolution of the claustroamygdaloid complex

L. Puelles and M. Martínez de la Torre Department of Human Anatomy and Psychobiology, University of Murcia, Spain

Development and evolution of the claustroamygdaloid complex has received limited attention in classical and recent neuroembryological literature. The controversy between proponents of either a striatal origin or a cortical origin for the claustrum (among mammals) was never resolved to general satisfaction. Holmgren (1925) proposed an hypopallial origin deep to olfactory cortex, jointly with pallial parts of the amygdala.

Our approach derived from the analysis of gene expression patterns in the developing mouse and chick telencephalon and associated data in other species. It turns out that the classic hypopallium consists of 2 adjacent and similar histogenetic domains, distinct mainly in terms of molecular genetic specification. We named these pallial sectors ‘lateral’ and ‘ventral’ pallium. Evidence has accrued that such partitions exist in frogs, reptiles, birds and mammals (and probably also in fish). The dorsal ventricular ridge of reptiles and birds arises out of these primordia, whereas in mammals the claustroamygdaloid complex appears at the corresponding locus. The molecular codes remain constant, irrespective of important differences in histogenesis and adult morphology (connectivity still needs more data and discussion). The large difference between such sauropsidian and mammalian derivatives is still perceived as a difficulty, jointly with the recently discussed issue whether monotremes actually have a claustrum or not (classical and some recent authors said they don't, though some recent authors hold that the monotreme claustrum exists). We discuss available evidence on this point, including Elliot Smith's pioneering data of 1896 and relate to this our own data on monotreme embryos.

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Comparative aspects of cortical neurogenesis in vertebrates

F. P. Cheung, A. Tavare, A. Pollen and Z. Molnár Department of Physiology, Anatomy and Genetics, University of Oxford, UK

The mammalian neocortex consists of 6 layers in contrast with the reptilian and avian cortices which only have three. These are believed to be equivalent to layers I, V and VI of mammals. In mammals, the majority of cortical cell proliferation occurs in the ventricular (VZ) and subventricular (SVZ) zones. The intermediate zone, cortical plate and marginal zone also contain a few scattered mitotic cells (‘abventricular’ divisions). To estimate the proportions of different forms of divisions in reptiles and birds, we examined the site of proliferation in embryonic turtle (stage 18–25) and chick (E8–15) brains using phospho-histone H3 (H3, a G2 and M phase marker) immunohistochemistry and BrdU (a S-phase marker) pulse-labelling.

In turtle only a few scattered abventricular H3-immunoreactive cells were found outside the VZ, the majority of the H3-immunoreactive cells were located in the VZ throughout the entire turtle brain. VZ cell proliferation peaks at stage 18 and 20, before an increase of abventricular proliferation at stage 23 and 25. In the cortex, however, abventricular proliferation at any given stage never exceeded 17.5% ± 2.5% of the total division.

In chick, similarly to turtle, most H3-positive cells were located in the VZ. However a secondary proliferative zone lying above the VZ, the SVZ was also observed in the mesopallium and nidopallium, but not the hyperpallium (homologue of the dorsal cortex). The SVZ was most prominent at E8. By E12 this organisation was no longer seen, and by E15 only few scattered H3-immunoreactive abventricular divisions were detected.

Preliminary data on BrdU pulse-labelling revealed that the distribution of BrdU-positive cells was slightly different from the distribution of H3-immunoreactive divisions in turtle. After 2 h of BrdU incubation, a larger proportion of BrdU-positive cells were found in the dorsal cortex. The cell fate of these newly generated cells is currently being investigated.

Our study suggests that the presence of SVZ is unique for mammalian dorsal cortex. The mechanism enabling subventricular divisions might have been the major driving force behind the evolution of the 6-layered neocortex in mammals.

This work is supported by MRC (G300200).

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Molecular mechanisms involved in the migration of cortical interneurons

J. G. Parnavelas Department of Anatomy, University College London, UK

The molecular mechanisms that guide the migration of the GABA-containing interneurons from their origin in the ganglionic eminence in the ventral telencephalon into the cortex are the subject of intensive investigations at present. We have investigated the role of the LIM-homeodomain gene, Lhx6, which has been localized in neurons of the medial ganglionic eminence (MGE), including those destined for the developing cortex. We performed loss of function studies for Lhx6 in mouse brain slices and dissociated MGE neuronal cultures using Lhx6-targeted siRNA. We found that silencing Lhx6 impeded the migration of interneurons into the cortex, although it did not obstruct their dispersion within the ganglionic eminence. Blocking Lhx6 expression in dissociated neurons taken from the MGE did not interfere with the production of GABA in these cells. These results indicate that Lhx6 does not specify the neurochemical identity of interneurons, but regulates their migration to the cortex.

Studies by Marin and colleagues (2001) have suggested that cortical interneurons express neuropilin (Npn) receptors that enable them to respond to chemorepulsion produced by class 3 semaphorins in the striatal mantle. Their data further suggested that the repulsive activity of semaphorins in the developing striatum creates an exclusion zone for migrating interneurons and channels them into adjacent paths, leading to the formation of their migratory routes into the cortex. We have been investigating the role of Slit signalling in interneuron migration by examining the forebrains of Robo1 and Robo2 knockout mice generated by targeted deletion. We have found that Robo1 is required to keep the cells originating in the MGE clear of the striatum on their way to the cortex. However, it has been reported that neurons avoid the striatal area in Slit1/Slit2 double mutant mice, indicating that this may be a Slit independent event. Taken together, these observations suggest that both Npn/Sema and Robo1 signalling are required to steer interneurons around the striatum and into the cortex. Our analysis has also shown that more interneurons migrate into the cortex of Robo1 null mice. Our recent proliferation studies in MGE dissociated cultures have shown that the increase in interneuron numbers in the cortex is due, at least in part, to increased proliferation in the MGE.

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Evidence of NMDA-mediated calcium signalling in NG2-expressing glia

N. Hamilton, A. Nishiyama and A. M. Butt Institute of Biomedical and Biomolecular Science, School of Pharmacy and Biomedical Sciences, University of Portsmouth, UK

A novel class of glia have been identified in the adult CNS by their expression of the NG2 chondroitin sulphate proteoglycan (CSPG). These NG2-glia form exquisite contacts with neurons at synapses in grey matter and nodes of Ranvier in white matter, suggesting they serve an unresolved function in neuron-glial signalling. Here we have examined glutamate-mediated calcium signalling in the optic nerves of mice in which NG2-glia express DsRed. Mice, aged postnatal day (P) 10–16 were humanely killed by lethal injection of pentobarbitone, according to Home Office guidelines. Optic nerves were isolated intact and loaded with calcium sensitive dye Fura-2, prior to calcium imaging. Nerves were maintained alive in oxygenated artificial cerebrospinal fluid and the effects of bath applied agents tested. Glutamate evoked an increase in intracellular calcium in identified NG2-glia. The response to glutamate was increased by cyclothiazide and inhibited by NBQX, indicating a prominent role for AMPA-type ionotropic glutamate receptors (iGluR). In addition NMDA evoked a robust calcium signal that was blocked by the NMDA-receptor antagonist AP5. The results provide evidence that glutamate-evoked calcium signalling in NG2-glia is mediated by both iGluR and NMDA receptors. Notably the optic nerve is a white matter tract that does not contain neuronal cell bodies or synapses. Thus, this study provides further evidence of neurotransmitter signalling along axons.

Niki Hamilton was in receipt of an Anatomical Society PhD Studentship.

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Dogmas in anatomy and neuroanatomy: the central projections of the rat's recurrent and superior laryngeal nerves

A. Pascual-Font, E. Maranillo, A. Merchán, T. Vázquez, J. R. Sañudo and F. J. Valderrama-Canales Department of Human Anatomy and Embryology i Complutense University of Madrid, Spain

Laryngeal nerves contain the fibres that control the laryngeal function. These studies were carried out on the rat with the purpose of broadening the knowledge of the functional components. Previous studies of the real origin of the fibres conveyed by the recurrent laryngeal nerve (RLN) and the superior laryngeal nerve (SLN) are few and in disagreement. None of those studies were developed using biotinylated dextrane amines (BDA), a powerful tool for tracing neural pathways. The aim of our study was to identify using BDA, in the rat, the nuclei of real origin of the fibres of the RLN and the SLN, knowing in this way the functional components of these nerves. The study was carried out in 33 adult male Sprague-Dawley rats, applying the BDA into the lesioned RLN (7 left side, 3 right side), SLN (5 left side, 7 right side), or contralaterally in both nerves (9). For the RLN the results obtained in all the animals showed that the rat RLN does not contain afferent fibres, whereas the efferent fibres originated within the ipsilateral nucleus ambiguus (NA). For the SLN the results obtained in all the animals shown that the rat SLN carries efferent fibres originated within the ipsilateral NA and dorsal nucleus of the vagus (DNV), and that afferent fibres reach the tractus solitari and the nucleus tractus solitari. So in the rat, the RLN seems to contain exclusively efferent fibres, it probably being the SLN nerve which conveys the afferent fibres, together with efferent fibres from the NA and DNV.

This work was supported by the Spanish FIS PI 030035.

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The cellular prion protein in the cow brain: relationship with human nvCJD

M. Cuadrado, A. M. Irujo, B. Paternain, F. J. Moleres and J. L. Velayos Department of Anatomy, School of Medicine, University of Navarra, Pamplona, Spain

The appearance of bovine spongiform encephalopathy (BSE) in the United Kingdom had many economic implications for farmers. Additionally the finding that it could be transmitted to humans (nvCJD) via consumption of BSE-contaminated beef products raised concerns on its safety for human consumption. Neuropathological examination of BSE-affected cattle has revealed a prominent alteration in the brainstem, suggesting that this is the primarily affected brain area. Based on the protein-only hypothesis, the presence of the cellular isoform of the prion protein (PrPC) is necessary for the establishment of BSE. Thus we have studied the expression and localization of PrPC in 15 brains of a breed of cows with a high incidence for BSE in Navarra (Spain). The brains of these animals were negative for prions, as confirmed by routinely used diagnostic procedures for BSE screening. We have observed that the expression of PrPC follows a rostrocaudal shift throughout the cow brain, being more abundant in rostral areas such us the cerebral cortex and hippocampus. Immunohistochemical staining also revealed that PrPC is present in several areas of the brainstem such us the dorsal motor nucleus of the vagus, the hypoglossal nucleus and the inferior olives. The presence of PrPC in the vagus and the hypoglossal nuclei might explain a retrograde spread of prions in cows from the gastrointestinal tract via vagus or pneumogastric (cranial nerve X) and hypoglossal nerves (cranial nerve XII) to those areas of the brainstem. From those nuclei, prions would spread through internal brain connections to rostrally located structures within the brain of cows.

Supported by PIUNA and BMH4-CT96-856. FJM is supported by a FPU grant 2003–5033.

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Lewy-body protein immunoreactivity in the midbrain of a PKC-gamma mutant rat

A. Al-Kushi 1 , D. Russell 1 , P. G. Shiels 2 , R. Wayne Davies 1 and A. P. Payne 1 1 Institute of Biomedical and Life Sciences and 2 Department of Surgery, Glasgow Royal Infirmary, Glasgow University, UK

The AS/AGU rat is a spontaneous recessive mutation derived from Albino Swiss (AS) stock; it is characterised by movement disorders, a failure to release amines within the striatum and, eventually, a loss of midbrain aminergic neurons (Payne et al. J. Anat. 196, 629–633, 2000). The mutation is in the gene coding the gamma isoform of protein kinase C (Craig et al. Nature Neuroscience 4, 1161–1162, 2001).

Cellular inclusions containing misfolded proteins occur in many neurodegenerative disorders. For example in human Parkinson's disease, Lewy bodies contain ubiquitin, alpha-synuclein and parkin. However similar inclusions do not occur in common laboratory models of the disease, so it is not clear if these proteins are increased. A quantitative fluorescence immuno-cytochemical technique was therefore used to examine levels of these proteins in individual cell bodies or in the surrounding neuropil of matched sections of the substantia nigra pars compacta (SNc) pars reticulata (SNr) and pars lateralis (SNl), the dorsal raphe nucleus (DR), median raphe nucleus (MR) and ventral tegmental area (VTA) from AS/AGU (mutant) rats and the parent Albino Swiss (AS) stock at 6–18 months of age. The oculomotor and pontine nuclei served as controls.

Ubiquitin and parkin were significantly elevated in the cell bodies of SNc and DR nuclei of AS/AGU rats compared to AS controls, but no differences could be found in the SNr, SNl, MR, VTA, oculomotor or pontine nuclei. Ubiquitin and parkin increase with age within the SNc in both AS and AS/AGU animals, but are consistently higher in the latter. The presynaptic protein alpha-synuclein was elevated in the neuropil of the SNc area of AS/AGU rats, but was not detectable elsewhere.

The results suggest that the AS/AGU mutant has elevated levels of these proteins in affected midbrain regions, even though cellular inclusions themselves do not occur. It remains unclear whether these elevations are related to impaired transmitter release, or form part of a degenerative process within the neuron.

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Progressive neuronal atrophy in a mouse model of Huntington's disease

A. S. Raza and S. W. Davies Neurodegeneration Research Group, Department of Anatomy and Developmental Biology, University College London, UK

Genetic mouse models of the human neurodegenerative disease, Huntington's disease (HD), exhibit a series of characteristic pathological changes accompanied by progressive brain atrophy. Neuronal cell death, however, is not a major feature. We have investigated the morphological changes that occur in the cerebral cortex and striatum of the R6/2 exon 1 transgenic mouse model of HD. Using Golgi impregnation techniques together with both light and electron microscopic analysis, we have determined that there is a dramatic somal atrophy accompanied by dendritic pruning, shrinkage and spine loss. Sholl analysis suggests that there is a retraction/loss of distal dendritic elements with a preservation of proximal dendrites. Investigation of the temporal mechanisms of neuronal atrophy, suggest that HD transgenic mice fail to achieve the normal density of dendritic arborisation, before losing dendritic elements. The molecular mechanisms underlying this neuronal atrophy will be discussed.

The financial support of The Alzheimer's Research Trust (ART, UK) and The Huntington's Society of America (HDSA, USA) is gratefully acknowledged.

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Encephalopathy is associated with increased brain water content and microvessel changes in a rat model of sepsis

H. Brooks and D. C. Davies Department of Basic Medical Sciences, St George's University London, UK

Encephalopathy is a common complication of sepsis. To establish whether cerebral oedema develops in sepsis, the rat caecal ligation and puncture (CLP) model was used. Four treatment groups were employed: CLP, CLP + Steroid (methylprednisolone 30 mg/kg), Sham CLP and Unoperated rats. All animals were sacrificed approximately 24 h after surgery. Following a lethal dose of anaesthetic, the brains from 35 adult male Wistar rats were collected (CLP, n = 9; CLP + S, n = 8; Sham CLP, n = 8; Unoperated, n = 10). 1 mm3 blocks of frontal cortex were dissected and placed into gradient columns constructed from kerosene and bromobenzene and calibrated using potassium sulphate samples of known specific gravity. The percentage brain water content of each block was then calculated. In a separate experiment ultrastructural correlates of cerebral oedema were investigated. Following a lethal dose of anaesthetic 24 adult male Wistar rats (n = 6 for each treatment) were perfused and the brains processed for transmission electron microscopy. Images were captured and imported into NIH Image for morphometric analysis.

Gravimetry revealed a significant increase (1.12%, P > 0.05) in overall water content in the frontal cortex of CLP rats compared to all other groups. There were no significant differences between the brain water content of Sham CLP, CLP + S and Unoperated rats. Electron microscopy revealed that there was significantly more oedema surrounding cerebral microvessels in CLP rats (P < 0.001) compared to all other groups, but no significant difference between CLP + S, Sham or Unoperated rats. Microvessel endothelial cross-sectional cell area was significantly smaller in brains of CLP (P < 0.05) compared to Unoperated rats. Some microvessels in the brains of CLP and CLP + S rats appeared collapsed, but although there were numerical decreases in microvessel lumen cross-sectional area in CLP and CLP + S compared to Unoperated and Sham CLP rats, these only reached significance in the CLP + S rats. Therefore CLP-induced sepsis causes an increase in brain water content that was ameliorated by steroid treatment. The oedema localised around brain microvessels is likely to be associated with changes to astrocyte endfeet and microvessel endothelial cells. These changes are likely to play an important role in the encephalopathy associated with sepsis.

H. F. Brooks was supported by an A.S.G.B.I. PhD studentship.

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Molecular mechanisms of early axon scaffold formation in the chick embryonic brain

M. Ahsan, K. Riley and F. R. Schubert Institute of Biomedical and Biomolecular Science, University of Portsmouth, UK

The first neurons in the vertebrate brain form a stereotype array of longitudinal and commissural axon tracts, the early axon scaffold. This scaffold is thought to lay down the basic structure for the later, complex connections in the brain. Yet surprisingly little is known about the formation of these early pioneering neurons. Our lab is investigating the molecular control of fate determination and axon guidance during the formation of the medial longitudinal fascicle (MLF), the earliest longitudinal tract in the developing brain.

The MLF is initially formed from neurons located at the ventral midbrain–forebrain boundary. We have identified several homeobox genes that are expressed in distinct patterns in this area during the establishment of the MLF. In particular, Sax1 is active in MLF neurons, and ectopic expression of this transcription factor in the chick embryo results in an enlarged MLF. In addition, Sax1 suppresses the expression of other homeobox genes like Emx2. We are currently investigating the role of Emx2 and Uncx4.1in patterning the ventral midbrain. Our preliminary results suggest a network of reciprocally regulating transcription factors, resembling aspects of ventral patterning in the spinal cord.

Once the MLF neurons are formed, they project their axons caudally, in loose bundles on either side of the floor plate. To establish the tract, the MLF axons have to be prevented from growing in rostral direction. Interestingly, the repellent axon guidance molecule Sema3A is expressed in a small patch of cells adjoining the nucleus of the MLF rostrally. The MLF neurons themselves express the receptors Neuropilin1 and PlexinA4, suggesting that they are responsive to Sema3A signalling. We have used electroporation in the chick to test the function of Sema3A. We have found that ectopic expression of Sema3A, but not other Sema3 genes, can divert or even block the outgrowth of MLF axons.

Our results indicate that homeobox genes like Sax1 play a major role in fate determination of MLF neurons as part of patterning in the ventral midbrain, and furthermore that Sema3A signalling is involved in guiding MLF axons towards their correct path.

We are grateful for support from the BBSRC, The Royal Society, and The Nuffield Foundation.

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GABAergic control of retinal ganglion cell dendritic growth

F. Chabrol and E. Sernagor School of Neurology, Neurobiology and Psychiatry, University of Newcastle upon Tyne, UK

GABAergic activity is believed to play an important role in the development of neuronal networks by influencing events such as cell differentiation and migration, neurite growth, synapse formation and cell death. In immature neurons the activation of GABAA receptors often leads to membrane depolarisation (because of a high intracellular concentration of chloride) thereby inducing calcium transients that could be involved in dendritic proliferation. In this study we have investigated the effect of disrupting embryonic GABAergic activity on the development of ganglion cells dendritic arbours in the turtle retina. Using the slow release polymer Elvax we have chronically blocked GABAA receptors with bicuculline (a treatment known to cause GABAA responses to remain excitatory and to prolong the period of spontaneous bursting activity and wave-like activity in the developing retina) or inhibited the GABA-synthesizing enzyme glutamic acid decarboxylase with allylglycine (a treatment known to cause GABAA responses to remain excitatory). These treatments were performed in vivo, starting at embryonic stage 24 (10 days before hatching) until 5–6 weeks post hatching. Ganglion cells were labelled with horseradish peroxidase-conjugated biotin dextran (applied either by injection in the optic nerve or electroporated directly into the retina). Both treatments had profound effects on dendritic growth. Dendritic arbours of bicuculline-treated ganglion cells have significantly fewer branches than age-matched controls and they tend to spread further away from the cell body. On the other hand dendritic trees of allylglycine-treated cells were narrower, but with significantly more branches than age-matched controls. These results show that GABAergic activity is necessary for the normal development of retinal ganglion cells dendritic arbours. Moreover the contrasting effects of chronic exposure to bicuculline or allylglycine indicate that GABA is involved at different levels of dendritic development. GABAA receptor activity could be implicated in the process that stops maturational events such as dendritic growth and pruning. On the other hand different GABAergic pathways could be involved in earlier developmental stages, such as dendritic guidance and growth.

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Descending modulation of nociceptive activation of spinal cord neurons expressing NK1 or GABAB receptors during chronic pain

I. Tavares 1,3 , A. R. Castro 1,2,3,4 and D. Lima 2,3 1 Institute of Histology and Embryology and 2 Laboratory of Molecular Cell Biology of the Faculty of Medicine of Oporto, Portugal; 3 IBMC; and 4 University Fernando Pessoa, Oporto, Portugal

Chronic pain induces plastic changes at the central nervous system. The neurochemical alterations at the spinal dorsal horn have been established whereas the effects on supraspinal pain control centres were seldom evaluated. We studied the nociceptive activation of spinal dorsal horn neurons expressing NK1 or GABAB receptors, using a model of secondary hyperalgesia characterized by increases both in noxious-evoked c-fos expression and in behavioural responses to mechanical stimulation by von Frey filaments of the hindlimb skin close to an arthritic joint. An increase in nociceptive activation of spinal neurons expressing NK1 receptors paralleled with a decrease in nociceptive activation of GABAB-expressing neurons. The functional relevance of the NK1/GABAB imbalance was demonstrated by the reversal of both signs of secondary hyperalgesia after intrathecal delivery of substance P-saporine, a drug that destroys NK1-expressing neurons, and/or baclofen, the GABAB agonist. The participation of two pain control centres of the medulla oblongata was analysed by studying the effect of local lesions in the NK1/GABAB imbalance and in the behavioural signs of secondary hyperalgesia. Upon quinolinic acid lesions of the caudal ventrolateral medulla (VLMlat), an area involved in inhibition of acute pain, both the NK1/GABAB imbalance and the behavioural signs of secondary hyperalgesia were reversed, with no effects in noninflamed control animals. After lesioning the dorsal reticular nucleus (DRt), an area involved in facilitation of acute and sustained pain, the NK1/GABAB imbalance was attenuated due to a decrease in nociceptive activation of NK1 neurons and the secondary hyperalgesia was prevented, whereas in noninflamed animals only the nociceptive activation of GABAB-expressing neurons decreased. These data suggest that during chronic pain, the continuous arrival of peripheral nociceptive input to the spinal cord, namely substance P-mediated, induces neurochemical reorganization at the spinal dorsal horn and drastically affects the modulatory actions exerted by pain control medullary centres.

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Serotonergic innervation of the anterior, midline and mediodorsal thalamic nuclei in the macaque monkey

M. Del Álamo, B. Pagán and C. Cavada Department of Anatomy, Histology and Neurocience, Faculty of Medicine, Autonomous University of Madrid, Spain

Serotonin (5-hydroxytryptamine, 5HT) is a widespread brain neurotransmitter that is attributed a critical role in maintaining mood balance. We have examined the distribution of axons immunoreactive for 5HT in the thalamic nuclei most heavily connected with cortical limbic areas thought to play a role in mood regulation. Tissue from 7 adult male Macaca nemestrina was immunoreacted with either a polyclonal or a monoclonal antibody directed against 5HT. Axonal immunolabelling was charted using a computerized set-up run by the Neurolucida® application. The boundaries of the thalamic nuclei were drawn in adjacent sections processed for acetylcholinesterase histochemistry and then superimposed over the charts of immunostained axons.

Within the anterior group nuclei a moderate concentration of 5HT-immunoreactive (5HT-ir) axons, homogeneously distributed, was present in the anterodorsal, anteroventral and anteromedial nuclei. The most medial, densocellular portion of the anteromedial nucleus, however, showed a denser innervation which ranged from moderate to dense. The density of 5HT-ir axons was markedly lower in posterior levels of the anterior thalamic group where just a few labelled axons were observed.

The midline thalamic nuclei showed dense immunolabelling albeit heterogeneously distributed. The paraventricular, paratenial, central densocellular and central latocellular nuclei held the densest concentrations of 5HT-ir axons. Axonal immunolabeling decreased at intermediate and posterior levels of the midline nuclei, with the central superior, central intermediate, central inferior, reuniens and caudal paraventricular nuclei holding moderate densities of 5HT-ir axons.

In the mediodorsal nucleus weak, moderate and dense concentrations of 5HT-ir axons were present. Axonal immunostaining was densest in the medial and posterior sectors of the nucleus, moderate in the ventral sector and relatively weak in the lateral sector. Contrary to the anterior and midline nuclei no antero-posterior gradient in immunolabelling density was present in the mediodorsal nucleus.

We conclude that the thalamic nuclei, or their portions, connected with limbic cortices receive a rich and specific serotonergic innervation that is in a position to modulate the subcortico-thalamo-cortical and cortico-thalamo-cortical networks engaging the cortical areas playing a role in mood control.

Supported by BFI2002-00513 and SAF2005-05380.

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Adult organization and development of the ‘thalamic matrix’ system in the rat

M. J. Galazo, P. Rubio-Garrido, C. Porrero and F. Clascá Department of Anatomy and Neuroscience, School of Medicine, Autonomous University of Madrid, Spain

In addition to a highly topographic input to the middle cortical layers, the thalamus provides a more divergent/convergent input to cortical layer 1. The thalamocortical cells that originate this pathway have been collectively labelled as the thalamic matrix system. TCI axons spread tangentially in layer I, and are believed to be an important substrate for synchronous activity across extensive cortical territories. However even in well-studied model species such as rodents, anatomical data on the origin, extent, and arborization pattern of adult TCI axons are surprisingly scant, and little is also know about their development.

In adult rats we have systematically identified the location of TCI neurons in the thalamus by means of epipial deposits of retrograde tracers. We have also examined the tangential arborization of TCI axons in whole-hemisphere cortical flatmounts. Results show that several thousands of TCI axons innervate relatively small areas (2 mm2) of cortical surface. The neurons originating these axons are spread across several thalamic nuclei. Anterograde labeling of axons show that the axons from small groups of TCI cells diverge over large swaths of the cerebral hemisphere, and that their tangential arborization is far from diffuse.

In developing rats aged E16–P12 we have examined the timing of TCI axon extension, target invasion and layer-specific arborization. Our analysis focused on some nuclei (Ventromedial, Anteromedial, Posterior) that preferentially project to cortical layer 1. Developing axons were labelled with BDA microinjections or carbocyanine dye deposits. In addition TCI neurons somata were retrogradely labeled by epipial deposits of fluorescent tracers. Axons from the nuclei investigated first arrive into the subplate at E16–17 but do not invade layer I until P2–P3. Parallel retrograde labelling experiments reveal that the TCI neurons in all the nuclei that eventually inervate layer I, invade this layer simultaneously, suggesting that TCI axon ingrowth is regulated by local signalling in the cortical plate. Between P3–P12 the density of the subpial TCI plexus increases steadily, and reaches adult-like density by the end of the period analyzed (P12). In contrast to primates, carnivores, and marsupials, rodent thalamic axons arborize simultaneously in the middle and superficial cortical layers. Present results clarify the origin, magnitude and developmental sequence of the thalamic projection to layer I in rodents.

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Number of synaptic boutons on the axon initial segment of pyramidal neurons of rat infragranular neocortical layers

J. L. Mendizabal-Zubiaga, C. Reblet and J. L. Bueno-López Department of Neurosciences, School of Medicine and Dentistry, University of the Basque Country, Leioa, Spain

Herein we examine the axon initial segment (AIS) of typically and inversely oriented pyramidal neurons of the infra-granular cortical stratum (TP and IP neurons, respectively). TP neurons were retrogradely labelled after biotin dextranamine injections delivered into cerebral cortex. IP neurons were either Golgi-impregnated or filled with biocytin. AISs of all these neurons were subsequently reconstructed on drawings made from serial electron micrographs. Fully reconstructed AISs were 3 TP neurons with axon projection from area 18 to ipsi-lateral area 17, 2 TP neurons with projection from area 17 to contra-lateral area 17 and 11 IP neurons with unidentified axon projection. The cell bodies of 9 of the latter neurons lay in areas 17–18 and those of the rest in the precentral motor cortex. The results showed that the studied neurons could be subdivided into types and subtypes concomitant with their axo-dendritic traits. TP neurons had few synaptic boutons on the thickest, shortest and straight-most AISs of all neurons. Within this TP neuron type, ipsilateral-projection neurons had more synaptic boutons on their AISs (average, 21; range 20–22) than callosal-projection neurons (average, 15.5; range 15–16). In turn, synaptic boutons on IP-neuron AISs ranged between 11 and 37, the precise number varying with the AIS-origin site at the parent cell body. Thus the number of boutons on AISs sprouting from apical dendrites typically range between 11 and 18 (these AISs were the thinnest AISs of all neurons studied here), whereas 21–29 and 31–37 boutons were, respectively, featured by AISs coming out from cell-body flanks either basal or lateral. AIS thickness and length were found to increase from the first of these IP subtypes to the next, in order. The functional significance of these findings must be further investigated, though indeed it deals with unique axon potential modulation endorsed by the combination of cell type and radial and area location in the cortex.

This work was granted by MEC-BSA2001-1179, 9/UPV00212.327–15837/2004.

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Temporal and prefrontal reorganization of declarative memory functions in patients with left mesial temporal sclerosis

F. Maestú, L. Arraez, P. Campo, I. García-Morales, S. Moratti, A. Gil-Nagel and T. Ortiz MEG Center Dr Pérez-Modrego and Department of Human Anatomy and Embryology II, Faculty of Medicine. Complutense University of Madrid; and Department of Neurology, Ruber International Hospital, Spain

Functional neuroimaging and animal studies have demonstrated a declarative memory pathway between the medial temporal lobe and the orbitofrontal regions. In order to test how a lesion could affects the reorganization of the pathway we studied Medial Temporal Lobe (MTL) sclerosis patients. These patients usually show declarative memory impairment. However, it is not well known how they reorganize their memory functions in the cortex, in order to remember some information. Here we present a study in which 8 left MTL epilepsy patients and 12 healthy age-matched control subjects have to memorize list of words and then asked to remember as many words as they could from each list. To test the reorganization of the memory functions we analyse the brain magnetic activity, by means of magnetoencephalography (MEG), from those words that were subsequently recalled. The comparison between patients and control subjects showed an increased activity in the left (between 1 and 300 ms; 700–800 ms time window) and right (between 1 and 100 ms; 600–900 ms) temporal lobe and in the left dorsolateral (between 300 and 500 ms time window) and ventral prefrontal cortex (between 300 and 900 ms time window) for patients than control subjects. Finally, control subjects showed higher number of activity sources over the left MTL than patients (between 400 and 500 ms). These results indicates how memory functions are reorganized in patients with left MTL. The increased activity in the prefrontal cortex in the patients group, during the encoding stage, may be related to a greater involvement of control and strategic processing that the patients have to apply in order to compensate for their declarative memory dysfunction. The bilateral activation of the neocortical regions of the temporal lobe during word processing seems to be another kind of compensation for their failure to activate the left MTL.

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3D Reconstruction of Déjérine's brain atlas: the thalamic syndrome revisited

R. Makhoul 1 , J. B. Thiebaut 2 , J. F. Uhl 1 , O. Plaisant 1 and V. Delmas 1 1 Department of Anatomy, René Descartes University, Faculty of Medicine Paris V; and 2 Unité de traitement de la douleur – Fondation Rothschild, Paris, France

In 1895 and 1901, Jules Déjérine published the ‘Anatomie des Centres Nerveux’, based upon serial slices of the brain with projection pathways studies from human degenerative lesions. This was the starting point of the anatomoclinical method, which established the description of the thalamic syndrome on the 8th of June 1906 at the Society of Neurology in Paris.

Our objectives were (1) to build a 3D model of Déjérine's brain atlas, and to match the degenerative lesions described for the thalamic syndrome; and (2) to use this 3D model to describe the thalamic syndromes assessed by MRI.

Using the original slices of Déjérine's atlas, a 3D reconstruction of the main brain structures, in particular the basal nuclei, was performed. Reconstruction software (Surfdriver) was used to delineate brain structures and build the 3D model on a PC Pentium 4 (2 GHz; 512 MB RAM) under Windows XP operating system. The resulting interactive 3D model was put into Talairach's 3D reference space, based on the CA-CP line. MRI studies from patients presenting with thalamic syndrome were correlated with the new 3D model.

Using our method, it was possible to reconstruct Déjérine's atlas in 3D, and to coregister this volume in Talairach space. Registration of the thalamic lesions described by Déjérine in this new space gives a modern spatial description of the thalamic syndrome. Patient lesions imaged by MRI were colocalised in this reconstructed 3D space and correlated with clinical signs, which allowed us to reproduce in 3D the anatomoclinical method used by Déjérine 100 years ago.

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GABAAR lateral mobility and localization

M. Perán 1 , H. Boulaiz 2 , J. A. Marchal 3 , C. Melguizo 1 , F. Hita 3 , C. Vélez 3 , J. C. Prados 2 , F. Rodríguez-Serrano 3 , A. Martinez-Amat 3 , O. Caba 2 and A. Aranega 2 1 Department of Neuroscience and Health Sciences, University of Almería; 2 Department of Human Anatomy and Embryology, University of Granada; and 3 Department of Health Sciences, University of Jaén, Spain

The major inhibitory neurotransmitter in the vertebrate brain is gamma-aminobutyric acid (GABA). From the therapeutic point of view its relevance is determined by the fact that its function is regulated by pharmaceutically significant drugs.

The clustering of GABA receptors type A (GABAAR) at discrete and functionally significant domains on the nerve surface is an important determinant in the integration of synaptic inputs. To discern the role that specific GABAAR subunits play in determining the receptor's cell surface topography and mobility, recombinant GABAAR, comprising different alpha1 subunit chimeras were transiently expressed in cerebellar granule cells. This was done in order to elucidate which regions of the protein are important in mobility/anchoring of receptors. The cellular localization and lateral mobility of the recombinant expressed GABAAR were determined by immunocytochemistry and Fluorescence Photobleach Recovery (FPR).

Some important conclusions were drawn as a result of the expression of these constructs in non-neuronal and neuronal cells. Receptor clustering does not imply receptor immobility. The M3/M4 intracellular domain of the α1 subunit restricts the mobility of the receptor complexes but is not necessary for the aggregation between receptors. Finally, the inclusion of at least one α1 subunit, with an intact intracellular loop, in the receptor complex seems to be necessary for immobilisation of the GABAA receptors.

We hope that these results will help to gain a better understanding of the complexity neuronal synaptic plasticity.

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Long range projections of the antero-lateral subependymal zone and adjacent white matter neurons of the rabbit

C. Reblet, I. Blanco., A. Alejo, T. Fuentes, P. Pró-Sistiaga, J. L. Mendizabal-Zubiaga and J. L. Bueno-LópezL Department of Neurosciences, School of Medicine and Dentistry, University of the Basque Country, Leioa, Spain

In the proliferative antero-lateral telencephalic subependymal zone (al-SEZ) and adjascent WM of pre- and postnatal rabbits there are cells that express calbindin and calrretinin (Reblet et al. 2005). We have seen Nissl and immuno-stained MP2 neurons in these localizations in adult rabbits (personal observations). The aim of this study was to ascertain if these neurons have long-range axonal projections. Rabbits (age 2 months) received pressure or iontophortical injections of 0.2–0.4 µL of 1% CTb in several cortical areas: primary somatosensory (SI), visual (VI), somatosensory-visual (S-V), anterior limbic (24b) and precentral medial (PrCM) and lateral (PrCL). Two additional animals were injected in the insular claustrum.

The cortical injections yielded retrograde labelling of neurons in the white matter (WM) between the al-SEZ and the insular claustrum, especially in the 24b and PrcM injections. In the claustral injections few labelled neurons appeared in the WM. In addition some neurons were retrograde labelled in the al-SEZ after the injections in the 24b and PrcM areas. These labelled neurons were in continuity with the labelled neurons in the WM. Some of these cells in the WM resembled migratory neuroblasts. We also found, after the injections in the 24b and PrCM areas, labelled axons that proceeded towards the caudate in which they formed extensive terminal fields. Interestingly some of these axons leaved terminal fields in the al-SEZ, indicating a reciprocal connection. The results show for the first time that some neurons in the antero-lateral SVZ and contiguous WM have long-range projections especially to the limbic cortex. In addition the al-SVZ of the adult rabbit could be a proliferative zone because the existence of some retrograde labelled neurons in the WM with neuroblast appearance.

This work was granted by MEC-BSA2001-1179, 9/UPV00212.327-15837/2004

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Comparative analysis of selected linear measurements of human and baboon brains

J. Hassanali 1 and G. Pokhariyal 2 1 Department of Human Anatomy and 2 Department of Mathematics, University of Nairobi, Kenya

Morphometric parameters of brains of various mammals and primates show differences which are likely to be associated with cognitive and other functions necessary for survival as well as evolution. Seven human and 8 baboon formalin fixed brains were used to show comparative aspects. The whole brains were weighed and 3 of each, human and baboon were separated into components, cerebrum, cerebellum and brainstem and their weights recorded. The linear measurements of the cerebrum were occipito-frontal (O-F), fronto-temporal (F-T) temporo-occipital (T-O), height of temporal lobe (HTL), inter: frontal pole (F-F), occipital pole (O-O), parietal (P-P), temporal pole (T-T), and occipital lobe (OL-OL) from superior, lateral and inferior aspects. The proportions of brain weight relative to body weight and those of the brain components were higher in the human. The length, height and width in human were higher than in baboon. F – F was 6 times O-O in humans while other parameters were nearly double in humans as compared to baboons. The ratio of P-P to F-F is nearly 20 times in baboon as compared to 6 times in humans. The overall increase in human brain in length, width and size of lobes could be related to increased body size, functional complexity, upright posture and evolution. The lateral expansion in the size of frontal, temporal and occipital lobes may be due to complex circuitry associated with cognitive functions and life style.

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Adaptability, flexibility and versatility in stem cell populations

D. Brynmor Thomas Bute Medical School, University of St Andrews, Scotland, UK

The term ‘stem cell plasticity’ is frequently used to designate the versatility of self-maintaining cell populations. It does not however, differentiate between their versatility, flexibility and adaptability. The adaptability of a stem cell population can be defined as the ability to adjust its output of precursors to a single maturation compartment. It can usefully be distinguished from flexibility, the ability of a multipotent stem cell population to regulate the distribution of such adjustments between two or more maturation compartments and versatility, the ability of a stem cell population to contribute to the production of previously unexpected progeny. The concept of stem cell versatility has been generated during the past decade by the demonstration of donor specific markers and the concurrent expression of cell specific markers in transplanted bone marrow-derived cells, which appear to have been assimilated into several populations of host cells derived from each of the three germ layers. The initial contention that versatility can be attributed to the trans-differentiation of transplanted cells has subsequently been endorsed. The use of host specific markers, in addition to donor specific markers and cell specific markers, has however, revealed the formation of hepatocytes, skeletal muscle fibres and neurons, in which heterokaria that have derived markers from both the donated cells and the cells of the host reflect the fusion of donor cells with host cells. It has thus become evident that while versatility may depend upon trans-differentiation, apparent versatility may result from cell fusion. These alternatives may both be important during development and regeneration as well as in cell replacement therapy. Thus in some instances the replacement of damaged host cells by donor cells, with or without trans-differentiation, may be the only available option but in others the modification of viable but imperfect host cells by fusion with donor cells may be infinitely preferable to replacement. Both alternatives await further evaluation and the potential dangers of aneuploid heterokaria await assessment. Meanwhile it remains important to recognize the possibility that in some instances apparent versatility may be due to the heterogeneity of donor cell populations.

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Study of juxtaoral organ in rat embryos

J. R. Mérida Velasco, J. F. Rodríguez Vázquez, C. de la Cuadra Blanco, J. J. Pozo Kreilinger S. Verdugo López, I. Sánchez Montesinos and J. A. Mérida Velasco Institute of Morphological Sciences and Department of Anatomy and Embryology II, Complutense University of Madrid; and Department of Anatomy, University of Granada, Spain

The juxtaoral organ was first described by Chievitz (1885) in human embryos near the parotid duct. This structure has been given various names, including the Chievitz organ. The objective of this study was to analyse the origin, position and relations of the juxtaoral organ in rat embryos.

A total of 30 rat embryos (Wistar) were used from 14 to 19 d gestation. The juxtaoral organ was studied using light microscopy. The 14 d gestation rat embryos presented a thickening of the epithelium of the transversal groove of the primitive mouth, close to the buccal nerve. We consider this to correspond to the future region of the juxtaoral organ. Rat embryos of 15 days gestation presented an invaginated epithelium. The buccal nerve was observed to inervate the juxtaroal organ at 19 d gestation.

The function of the juxtaroal organ is controversial. Some authors suggest that it may play a role in the mechanical activity of the region.

The authors thank Ms. P. Chimeno, Ms. A. Garc’a and Mr J. de la Calle for their technical participation. The study was supported by a grant from the Instituto de Salud Carlos III, PI03/0275.

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Differential expression of cytokeratins 7 and 18 in villous and extravillous trophoblast cells: a confocal laser scanning microscopy study

J. Ahenkorah, B. A. Hottor, N. Davis, S. Byrne, J. Waugh, P. Bosio and C. D. Ockleford Department of Infection, Immunity and Inflammation, University of Leicester, UK

Cytokeratins (CKs) are a subfamily of proteins that form intermediate filaments and constitute the major cytoskeletal system of epithelial cells. CKs are subdivided into Type I (Acidic) including CK9- CK20 and Type II (Neutral-Basic) consisting of CK1-CK8. During pregnancy, trophoblast differentiates into either villous trophoblast or extravillous trophoblast (EVTs). EVT's remodel spiral arterioles. Trophoblasts express greater antipan-cytokeratin immunoreactivity in EVTs than in villous trophoblast. Here we investigate expression of cytokeratins 7 and 18 using indirect immunofluorescence confocal laser scanning microscopy (CLSM) of freshly delivered cryo-fixed term-placental basal plate tissues. For both CKs, EVTs were more immunofluorescent than villous trophoblast. Non-epithelial tissue levels of fluorescence were close to background. For CK7, there was a > 10 fold difference [Wilcoxon Sign Rank Test Z = – 6.501, P = 0.000] in the mean area percent of a banded zone of the most intensely fluorescent pixels, when equal size areas containing villous trophoblast and EVTs were compared. A similar result was obtained for CK18 [Wilcoxon Sign Rank Test Z = – 8.339, P = 0.000]. Similar results are indicated using CK19 and CK5. Summarising, cytokeratins 7 and 18 are up-regulated in extavillous trophoblast following differentiation. Since these proteins are representatives of the two subfamilies, and taken together with the pancytokeratin data it appears that this may be a generalised phenomenon in the CK gene family. The up-regulation of CKs in EVTs may favour their migration into the basal plate to remodel the spiral arterioles in normal placentation.

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Anatomical study of Reichert's cartilage in human fetuses

J. F. Rodríguez-Vázquez, J. R. Mérida-Velasco, S. Verdugo-López, C. De La Cuadra-Blanco, H. Gonzalez and J. V. Sanz Casado Department of Anatomy and Embryology II, Complutense University of Madrid, Spain

The developmental pattern of the cartilage of the second branchial arch has been recently described by Rodríguez-Vázquez et al. 2006. The cartilage has 2 segments: the cranial or styloid segment which has 2 ends (the superior end joined to the otic capsule and the inferior end, which is angulated); and the caudal or hyoid segment, which terminates caudally in the hyoid cartilaginous structure between the body and the greater horn. This morphological arrangement is totally new in contrast with the classical conception of a continuous Reichert's cartilage (Hamilton & Mossman, 1975; Corliss, 1979; Sperber, 1989; O’Rahilly & Müller, 1996; Sadler, 1996; Larsen, 2003).

The objectives of our investigation were to study the anatomical relationships of the cranial segment and their possible clinical implications. Forty human fetuses between 43 and 240 mm (weeks 10–24 of development) were studied with a light microscope and microdisections.

The cranial segment was located in the lateropharyngeal region and had important vasculonervous relationships with the elements of the retrostyloid space:

Relationship with the external carotid artery: the artery surrounded by the external carotid nerves passed through the hiatus or space defined by Reichert's cartilage and stylohyoid muscle. This took place where Reichert's cartilage changes direction and begins to angulate.

Relationship with the glossopharyngeal nerve: this crossed medial to the inferior end of cranial segment. The more angulated this segment the closer the nerve crossed to its end.

Relationship with the pharynx: when angulation of the inferior end of the cranial segment persisted, depending on the degree of angulation, the end of the cartilage may be observed very close to the pharyngeal wall.

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Nano-gold immuno-electron microscopic localization of caveolin-1 in human placenta

S. Byrne, J. Ahenkorah, C. Lockwood and C. D. Ockleford Laboratory for Developmental Cell Sciences, Department of Infection, Immunity and Inflammation, University of Leicester and Warwick Medical Schools, Leicester; and Centre for Medical Education, Lancaster University, UK

We have localized the placental endothelial marker caveolin-1 at the ultrastructural level using indirect immuno-labelling with particles of colloidal gold 10 nm in diameter. The particulate label has been quantified per unit area to assess the distribution of the target protein within placental chorionic villi and basal plate in term placentae. Very low levels of labelling were detectable over embedding medium that was free of tissue, erythrocytes, serum-filled cavities and extracellular matrix. The mesodermal compartment of the tissue was more heavily labelled than the ectodermally derived chorionic villus trophoblast. Within the mesodermally derived cells, the distribution of anticaveolin-1 labelling within endothelial cells was maximal. Intracellularly there was a clear tendency for the labelling pattern to be marginated in the endothelial cells. Our approach to quantitating this was to measure particles per unit area in apical, basal and central (‘ABC’) strips in order to assess whether it was polarised or not. In both villous and basal plate lining endothelium labelling of this fixed term tissue B > A > C. Caveolin-1 distribution is considered in the light of possible transport vesicle and signaling platform rôles relevant to the materno–fetal interaction.

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Changes in muscle cells and caveolin-3 immunofluorescence labelling during skeletal muscle cell differentiation

K. Smith and C. D. Ockleford Department of Infection, Immunity and Inflammation, University of Leicester, UK

As myoblasts differentiate, several changes occur such as elongation, fusion into multinucleated fibres, alignment of organelles, and changes in membrane protein composition. In fully differentiated skeletal muscle fißres immunolabelling of caveolin-3 was observed using confocal microscopy (CLSM) as a regular pattern of bright nodes, 1.48 µm apart in the long axis. The aim of this study was to observe the changes that occur in myoblasts as they differentiate, and to observe the development of any pattern of caveolin-3 immunolabelling.

A mouse muscle myoblast cell line, C2C12, was cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 2 mm l-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% fetal bovine serum (FBS). To trigger differentiation this culture medium was replaced with one containing 10% horse serum in place of FBS. Cultures (approximately 80% confluent) were allowed to differentiate for 0–21 days, and then fixed in 2.5% formaldehyde solution. An indirect immunofluorescence method was used to label caveolin-3 in coverslip-attached cells. Cultures were observed using CLSM and DIC optics.

As the days of differentiation increased, changes were observed. Before differentiation little caveolin-3 immunofluorescence was observed. Most cells had only one nucleus. From day 3 multinucleated syncytia began to appear, and caveolin-3 labelling increased. This was largely perinuclear. Cells elongated and became better aligned as time increased. Subsequently caveolin-3 labelling was marginalised towards the membranes, and the regular pattern of fluorescent nodes began to be observed. By day 9 many long, multinucleated fibres were seen, in a regular arrangement with bright caveolin-3 labelling. However, some mono-nucleated cells were still seen at this and later time points.

This method is useful to monitor morphological and local compositional changes that occur in skeletal muscle myoblasts as they form myotubes. Immunolabelling of muscle proteins allows their expression to be observed during differentiation. These images show that not all myoblasts in this culture differentiate at the same rate. Some cells still divide and grow while others differentiate.

Funding was provided by the ASGBI.

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Selective vulnerability to ischaemic-hypoxic injury of the subnucleus gelatinosus of the human nucleus tractus solitarii

A. Porzionato, V. Macchi, G. Sarasin, A. Parenti and R. De Caro Department of Human Anatomy and Physiology, University of Padova, Italy

The nucleus tractus solitarii (NTS) is an integration centre which has been divided in subnuclei with specific anatomical and functional characteristics. In preceding studies selective lesions have been described in the dorsal portion of the NTS in adults who died after acute heart failure. In the present study we examined NTS in 32 adults and 8 infants, who died shortly after an ischaemic-hypoxic event. Medullary sections were subjected to histological stainings (haematoxylin-eosin, Nissl, Klüver-Barrera), immunohistochemistry by anti-Fos, and TUNEL method.

In 21 cases (17 adult and 4 infant cases), 2 symmetrical hypereosinophilic areas were found at the level of the dorsal portion of the NTS, corresponding to the subnucleus gelatinosus (SG). In these areas neurons were shrunken, staining darkly with cresyl violet, and had markedly eosinophilic cytoplasm. The dendrites were clearly recognisable due to intense eosinophilia and, in some sections, presented a curved course at the periphery of the involved neurons. In these cases strong Fos-like immunoreactivity was detected at the level of the subnucleus gelatinosus. In the other cases the dorsal portion of the NTS did not show hypoxic-ischaemic lesions and, particularly, the subnucleus gelatinosus showed pale-staining neurons and was not clearly defined with respect to the surrounding tissue. In 10 (7 adults and 2 infants) of these cases Fos-like immunoreactivity was present at the level of a round area, in the context of the dorsal NTS, corresponding to the subnucleus gelatinosus. In all cases both nuclear and cytoplasmic neuronal positivity was present. In both adults and infants a specific subnuclear distribution of apoptotic neurons in the NTS was not found – SG showing no higher percentage of apoptotic neurons with respect to the other subnuclei.

Findings at the level of the SG were ascribed to hypoxic-ischaemic injury developing before death. The cases showing only Fos expression were due to the rapidity of death, which permitted only immediate-early gene expression without the onset of morphologically evident hypoxic-ischaemic lesions. The selective vulnerability of the subnucleus gelatinosus to hypoxic-ischaemic injury may be explained with reference to vascularization of the medullary tegmentum and structural-functional characteristics of the subnucleus itself.

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Topographical and laminar distribution of cortical input to the monkey entorhinal cortex

R. Insausti, A. Mohedano-Moriano, P. Pro Sistiaga, M. M. Arroyo, E. Artacho Pérula, A. M. Insausti, P. Marcos Rabal and A. Martínez Marcos Human Neuroanatomy Laboratory, Department of Health Sciences and CRIB, School of Medicine, University of Castilla-La Mancha, Albacete, Spain

The hippocampal formation is a key structure in memory formation and consolidation. The hippocampus receives direct information from different cortical and subcortical sources. Cortical information is mostly funneled through the entorhinal cortex (EC) to the hippocampus in a bi-directional circuit that eventually returns to the neocortex. Retrograde tracing studies showed that neocortical afferents to the EC originates almost exclusively in polymodal association cortical areas. However the use of retrograde studies leaves unanswered the question of the laminar and topographical distribution of cortical terminal fields as well as the input from both visual and auditory unimodal association areas. In addition, the convergence of cortical inputs, and the relationship between unimodal and polymodal inputs in the EC remain largely undisclosed. We examined material from 60 Macaca fascicularis monkeys in which cortical deposits of either 3H labeled aminoacids or Biotinylated Dextran Amine (BDA) as anterograde tracers were made into different cortical areas including portions of visual and auditory unimodal association cortices in the temporal lobe, and in the polymodal association cortex at the upper bank of the superior temporal sulcus.

Results showed that cortical afferents to the entorhinal cortex present a heterogeneous topographical distribution, ranging from a majority of the EC extent (afferents from medial area 13 and areas TF and TH), to focal termination, either rostrally (lateral orbitofrontal cortex, insular cortex, anterior cingulate cortex, perirhinal cortex, area TE), intermediate (upper bank of the superior temporal sulcus, unimodal auditory association cortex) or caudally (parietal and retrosplenial cortices). Many of those inputs overlapped, particularly at the rostro-lateral portion of the EC (subfields ELR and ELC). Laminar distribution followed either a superficial (layers I–III) or deep patterns (V–VI) although dense spots of projections spanned all layers.

The present study suggests that the distribution of cortical afferents to the EC may be critical for the elaboration and integration of complex stimuli for the transference of both EC input to the dentate gyrus and CA3-CA1 fields of the hippocampus as well as the hippocampal output to the EC prior to its cortical output. This local interaction would support a controlled processing of declarative memory formation and consolidation.

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A multiarchitectonic approach for the definition of functionally distinct areas and domains in the monkey frontal lobe

A. Belmalih, E. Borra, M. Contini, M. Gerbella, S. Rozzi and G. Luppino Department of Neuroscience, University of Parma, Italy

Pioneering anatomical studies of the last century clearly showed that the cerebral cortex can be subdivided into regions of specific structure, giving rise to a new branch of neuroanatomy, i.e. ‘architectonics’. Since then architectonics has been often accused of giving uncertain results due to its subjective nature and its possible contribution to understand brain functions has been seriously questioned and discarded for several years. From the late eighties, this issue became topical in functional studies of the the primate agranular frontal cortex (AFG) showing that: (a) the macaque brain contains an increasing number of different motor areas; (b) human motor cortex shares several general organizational principles with monkey motor cortex.

Over the last 20 years, we embarked on a series of cytoarchitectonic studies in which different areas of the macaque AFG were defined on the basis of multiple criteria. In this way, we found that the location and extent of the identified areas becomes much more constant among different individuals. This approach revealed to be valid in providing a reliable anatomical framework for the definition of functionally distinct areas and for predicting the existence of new functional areas, not previously identified.

A new impulse to architectonics was recently given by the introduction of immunoarchitectonic approaches. In particular we found that, in the macaque AFG, differences in regional and laminar distributions of pyramidal or nonpyramidal neurons, positive for SMI-32 or calcium-binding proteins immunochemistry, respectively, very well characterize individual areas and different domains of the frontal cortex. By using this approach we recently identified 2 distinct areas in the inferior arcuate sulcus, which appear to be related to higher order aspects of skeletomotor and eye movement control.

In conclusion, after more than one century, architectonics is still a modern discipline, whose power in understanding neural functions has been often underestimated and by far goes beyond the original aim of generating cortical maps.

Work supported by EU and MIUR

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Organization of primate prefrontal association cortex

H. Barbas Department of Health Sciences, and Program in Neuroscience, Boston University, USA

The prefrontal cortex in primates guides behaviour by selecting relevant information on the sensory and spatial aspects of the environment and mnemonic information acquired through experience. Moreover decisions and actions in behavior are intricately linked to emotional context. These complex functions are likely mediated by specialized prefrontal pathways that target excitatory and inhibitory systems in other cortical and subcortical structures. In a nonhuman primate, the rhesus monkey, lateral prefrontal cortices are robustly linked with occipital, temporal, parietal, and premotor cortices, implicated in sensory perception, cognition, working memory, and motor action. Medial and orbitofrontal cortices, on the other hand, are richly connected with the entorhinal and perirhinal cortices, the hippocampal formation, and the amygdala, associated with long-term memory and emotional memory. Caudal orbitofrontal areas, in particular, receive input from cortices associated with each sensory modality, and target dual systems in the amygdala, poised to either drive or dampen autonomic activity, which may depend on emotional context. Medial prefrontal cortices are preferentially connected with hypothalamic, brainstem, and spinal autonomic structures specialized for emotional expression. The different sectors of the prefrontal cortex communicate with each other, inextricably linking pathways associated with thoughts and emotions that guide actions. The different sectors of the prefrontal cortex are linked through laminar-specific pathways that suggest the sequence of recruitment of cortical and subcortical structures in cognitive-emotional processes. Moreover, prefrontal cortical pathways synapse with both excitatory and inhibitory neurons in sensory association cortices, in thalamic nuclei and in the amygdala, providing the structural basis for the selection of relevant signals and suppression of irrelevant signals for the task at hand. Disconnection of the intricate pathways that link distinct prefrontal cortices with other structures disrupts normal behavior, as seen in several psychiatric diseases affecting preferentially distinct prefrontal cortices.

Supported by NIH grants from NIMH and NINDS.

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Specializations of the human neocortex

J. De Felipe and R. Benavides-Piccione Cajal Institute (CSIC), Madrid, Spain

One of the fundamental questions in neuroscience is what is special about the neocortex of humans and how does it differ from that of other species? At present it is generally thought that during evolution, the neocortex expanded in larger brains due to the addition of microcircuits with the same basic structure, based around pyramidal cells and their input-output connections. However, it is clear that distinct cortical areas show important differences within both the same and different species. For example, the number of neurons contained in a discrete vertical cylinder of cortical tissue varies across species. Additionally it has been shown that the neuropil in different cortical areas of the human, rat and mouse has a characteristic layer specific synaptology. Moreover there appear to be special types of interneurons, double bouquet cells and tyrosine hydroxylase neurons that are particularly abundant in the human neocortex. These differences clearly highlight the variability in the design of microcircuits between cortical areas and species and probably indicate the evolutionary adaptation of excitatory and inhibitory circuits to particular functions. Based on our studies and those of a number of other laboratories, we support the idea that the human neocortex shows unique specialization that is likely to be crucial for human cortical function.

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Issues about integration of basic science in innovative curricula

K. J. A. H. Prince, J. Drukker, C. P. M. Van der Vleuten and A. J. J. A. Scherpbier Department of Educational Research and Development, Maastricht University, the Netherlands

Several developments in society in general and medicine in particular, such as the knowledge explosion, decreasing time for medical education and new insights from psychology and education, have led to changes in medical curricula. This has caused concerns about students’ levels of basic science knowledge and discussions about what is core knowledge.

Empirical evidence shows that innovative educational methods do not necessarily lead to less knowledge of anatomy in comparison to traditional medical training. Research into the perceived level of undergraduate students’ anatomy knowledge showed that perceived insufficiency is common among all students, regardless of the type of curriculum. Moreover on an anatomy test students from a problem-based curriculum obtained scores that were similar to those of other students. Further research showed that there is a discrepancy between students’ levels of anatomy knowledge and standards set by experts. Significant differences between various groups of experts indicate that there is no consensus among staff about what medical students need to know.

Clear guidelines as to the required anatomy knowledge should be developed and agreed on by anatomists and clinicians, with other colleagues and students contributing to the process.

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Adding ‘common sense’ to ‘the need to know’ in anatomy teaching

S. McHanwell, M. Atkinson, C. Davies, R. Dyball, J. Morris, C. Ockleford, I. Parkin, S. Whiten and J. Wilton c/o School of Dental Sciences, Newcastle University, UK

It can be argued that past basic sciences curricula for students of medicine and dentistry were frequently overloaded with facts as a consequence of their being designed to provide knowledge on the basis of ‘just in case’. The need to provide students with a far wider range of skills to enable them to be safe and effective professionals has led to a drastic reduction in the basic science content towards a ‘need to know’ basis. It is recognised that this approach can lead to deficiencies in fundamental knowledge and serious uncorrected misconceptions. Common sense dictates that there must be a necessary minimum of factual knowledge for effective medical practice so professional bodies such as the General Medical Council (http://www.gmc-uk.org) and General Dental Council (http://www.gdc-uk.org) have attempted to define the necessary minimum standards. However, these statements are often too general and lacking in detail to constitute a blueprint for a curriculum.

The Anatomical Society of Great Britain and Ireland thus attempted to define the level of detail necessary for medical students. It can be accessed on http://www.anatsoc.org.uk/listed under ‘Education’ and ‘Core curriculum’ and topics include Language, Vertebral column, Upper limb, Lower limb, Head and neck, Neuroanatomy, Thorax, Abdomen and Pelvis. Similar work is in progress for a dental curriculum.

Space precludes the inclusion of all the material encompassed on the website but for the vertebral column an understanding would be expected of normal posture (curvatures); intervertebral joints and the anatomy underlying common pathology (back pain, disc lesions, spinal cord and nerve injury, whiplash injuries); the anatomy underlying lumbar puncture and interpretation of vertebral images.

We now propose the development of curricula based on a combination of the need to know together with sufficient background knowledge to give students the awareness of what they do not know. This talk will focus on some of the theoretical and practical issues that have to be confronted when designing courses and suggest ways in which they might be improved in future.

The authors are members of the Education Committee of the Anatomical Society of Great Britain and Ireland.

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Anatomy, a basic subject in biomedical education and science: a German perspective

Eveline Baumgart-Vogt Institute for Anatomy and Cell Biology, University of Giessen, Germany

Anatomy is one of the major basic subjects in medicine and in biomedical sciences. An educational background and training in anatomy is necessary for each beginner, entering professions in those subjects. Therefore a good quality standard is necessary for instructors and teachers to achieve optimal results in their educational programmes for the students. Additional efforts of the teaching personal are necessary to achieve optimal standards in scientific performance. In many cases personal scientific achievements in research give the major impact on a decision whether or not a candidate is selected for a higher university position. Therefore in this talk the major issues concerning anatomical education and science in Germany will be discussed and details of an anatomical education combined with a scientific career in Germany presented. The anatomy curricula of different Universities in Germany will be introduced and future perspectives for a common European Anatomy Curriculum will be discussed.

The author is President-elect of the Anatomische Gesellschaft.

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Supporting modern postgraduate surgical training programmes in the UK through greater use of cadaveric material

R. Rainsbury Raven Department of Education. The Royal College of Surgeons of England, London, UK

A new Intercollegiate Surgical Curriculum (ISC) has established a new modular framework for postgraduate surgical training. The curriculum has defined the surgical standards that are required before a trainee can progress from module to module. The core conditions within each specialty module have been defined, and the required operative and other skills needed for competent management of these conditions have been agreed.

Full implementation of the new ISC will commence in Autumn 2007. The Raven Department of Education at the English College views implementation as a major opportunity to extend the use of cadaveric material to support the new training programmes. To this end, a Core Specialty Skills Project has been launched to build on the Department's experience of practical skills courses based on cadaveric material, by improving access to appropriate facilities in Regional Departments of Anatomy. The aims of this project are to support each module within the 9 surgical specialties by producing a range of educational materials for participants and faculty, in association with the major specialty stakeholders. Use of cadaveric material, supported by step-by-step dissection guides and integrated assessment, will be a crucial component of this programme.

Nine University Departments of Anatomy have been visited in England, Wales and Northern Ireland to establish the current facilities available, and the type and supply of cadaveric material. Levels of staffing, current involvement in postgraduate education and the degree of interest in the extended use of services were investigated. The results of this perambulation will be presented to open up a debate, and to explore the level of interest in supporting the greater use of cadaveric material in this context.

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How anatomy departments could participate in Continuing Professional Development (CPD) for surgeons

J. R. Sanudo 1 , M. T. Vazquez 1 , E. Maranillo 1 , R. M. Mirapeix 2 , B. Mompeo 3 , J. Reig 2 and L. Arraez 4 1 Department of Anatomy and Embryology I, Medical School, Complutense University of Madrid; 2 Unit of Anatomy and Embryology, Medical School, Autonomous Unversity of Barcelona; 3 Department of Anatomy and Embryology, Medical School, Las Palmas University; and 4 Department of Anatomy and Embryology II, Medical School, Complutense University of Madrid, Spain

The Medical Professional Colleges currently advise the introduction of periodical assessment with the aim of improving medical and surgical competence. In other words they want to promote CPD.

An institution like the Royal College of Surgeons of England, committed to enabling surgeons to achieve and maintain the highest standards of surgical practice and patient care does not exist in the present Spain, but equivalents existed during the 18th and 19th centuries in Cadiz, Barcelona and Madrid. Nowadays if Spanish surgeons want to do CPD courses using cadaveric material they must contact Anatomy Departments.

Two types of attitude could be observed from the Anatomy Departments: those that simply offer their facilities to surgeons or commercial companies, and those that become involved in the project.

Courses using cadaveric material should be the result of a balance between surgical and anatomical sensibilities. They should not be made exclusively with the aim of performing surgical procedure (implanting a prosthesis, performing an arthroscopy, etc.) because in this way a lot of the potential of the human cadaver is lost (review of the whole regional anatomy) and furthermore it ‘betrays’ in some way the donor's will. Therefore we recommend courses that combine a deep review of the regional anatomy in correlation with the performance of the most relevant or attractive surgical techniques.

For getting quality a good standard is necessary: clear objectives for each session must be recorded in a dissection guide, they must be more practical than theoretical, they must guarantee individual work with good supervision and to have all equipment and facilities, and finally there must be evaluation of outcomes (knowledge and satisfaction).

The results of questionnaires answered by 344 Spanish medical doctors and surgeons about the relevance of anatomy in daily clinical practice will be presented.

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Influence of body composition on bronchopulmonary function in healthy young men

R. Martín, R. Rodriguez and L. Gómez Pellico Department of Anatomy and Embryology. Alcala University, Madrid, Spain

In recent years several studies have tried to clarify the influence of body compartments on bronchopulmonary function, in order to see if any parameter of body composition can be used as an individual risk factor of bronchopulmonary diseases, and in order to see if changes in body composition can contribute to improve the bronchopulmonary function in patients with chronic bronchopulmonary diseases.

We studied a group of 20 healthy non smoking young men with the purpose of determining the influence of different body compartments on bronchopulmonary function. We carried out for each one forced spirometry in order to obtain parameters of bronchopulmonary function, and dual energy X-ray absorptiometry to obtain total and regional values of the different body compartments. Using the SPSS 12.0 program, we examined the statistics of every parameter, and analysed the correlation between them.

We found positive and statistically significant correlations between total and regional values of fat free mass and FVC and FEV1, the 2 main parameters of spirometry. The bone mass showed an influence specially on FVC. The fat mass compartment had no statistically significant correlation with the parameters of bronchopulmonary function.

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Evaluation of the variability found in orbital fossae of human skulls

M. D. Cabañas Armesilla, M. I. Maestre López and M. G. Fernández García Department of Anatomy and Embryology II, Complutense University of Madrid, Spain

The detailed study of the orbital fossa is of great importance in the scientific world for anthropometric purposes, as well as anthropological forensics and ophthalmology. We have analyzed the population sample of skulls in the Department of Anatomy and Human Embryology II and in the Museum of Villa of the Medicine.

Our objectives were: (1) Morphometric study of the orbital fossae and their possible malformations in the sample; (2) Analysis of skulls to review the craneometric variables normally used; (3) Discriminant analysis, defining criteria to analyze and to identify individuals; and (4) To determine the mathematical relation existing between the anatomy of the orbital fossae and nasal morphology, and the morphology of the hard palate.

The sample consisted of 34 skulls, all of adult individuals, the sex and age were unknown in each case. This study followed the protocol described by Martin-Saller (1957) and a new protocol designed by the investigating group. The determination of age was established based on the closing of the exocranial sutures, described by Meindl and Lovejoy 1985. For the diagnosis of sex, the qualitative criteria by Boyd and Trevor (in Brigg, 1989) were used. Program SAS 9.1.3 has been used.

This study is an integrated preliminary analysis of all the available material. We emphasize that to date work has concentrated on taking measurements and defining correlations in the right side between variables: verticality of the foramen with the variable vertical length, and on the left side the variable perimeter with length. The rest of the variables do not contribute information given the small sample studied, since the data are calculated in independent variables. Between the vertical measures in respect of the peripheral the best metric correlation existed between rim perimeter and the maximum horizontal length on the right side. Any malformations found were detailed.

We conclude that: (1) There were better measures of correlation on the right side between verticality of the optical hole and the variable vertical length; (2) The measures of best correlation on the left side were variable perimeter with length.

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Age of commencement of union of the epiphyses at the knee: an Irish population study

J. E. O’Connor 1 , L. Spence 2 , C. Bogue 2 and J. Last 1 1 University College Dublin School of Medicine and Medical Science; and 2 Department of Radiology, Cork University Hospital, Ireland

Characteristic changes during epiphyseal union provide a skeletal age, which when compared with age-based standards provide an estimation of chronological age. Currently, there is no data on epiphyseal union for the purposes of age estimation specific to an Irish population. This information is necessary as environmental variation influences skeletal maturation (Lampl 1996). This cross-sectional study aims to investigate the relationship between stage of epiphyseal union at the knee joint and chronological age in a modern Irish population. Anteroposterior and lateral knee radiographs of 148 males and 86 females, aged 9–19 years were staged according degree of union (O’Connor 2005). Fusion was scored as Stage 0, nonunion; Stage 1, beginning union; Stage 2, active union; Stage 3, recent union or Stage 4, complete union.

A significant difference was noted between the mean age of union for males and females for each of Stages 1 and 2 for the femur and Stages 0, 1, 2 and 3 for the tibia and the fibula. The youngest subject demonstrating beginning union of the distal femoral epiphysis was a 12.0-year-old male (mean 14.0 years) and a 10.2-year-old female (mean 12.5 years). Beginning union of the proximal tibial epiphysis was recorded in a male aged 12.0 years (mean 14.1 years) and a female aged 10.2 years (mean 12.5 years). The youngest individuals displaying beginning union of the proximal fibular epiphysis were aged 12.9 (mean 14.9 years) and 11.6 (13.3 years) years, respectively, for males and females. Johnston's (1961) examination of an American Indian population is the only study to provide data on commencement of union. In this cohort union of the three physes at the knee begins at 18–18.5 years in males and 16–17 years in females.

Irish children are comparable to those from Johnston's study with females commencing union approximately 2 years earlier than males. This data however, reveals that there are instances of union commencing at earlier ages in this Irish population. This has not previously been documented and larger samples are required to investigate if this is due to a secular or population variation in maturation or due to a methodology which clearly identifies beginning union.

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Emotional reactions to human dissection and perception of death in anatomy students

L. Arraez-Aybar 1 , G. Castaño-Collado 2 and M. I. Casado-Morales 3 1 Department of Human Anatomy and Embryology II, Faculty of Medicine; 2 Department of Differential Psychology and Work Psychology; and 3 Department of Basic Psychology (Cognitive Processes). Faculty of Psychology, Complutense University of Madrid, Spain

There is a lack of papers about the emotional reactions of Human Anatomy students before dissection, and about the evolution of these emotional reactions across different sessions (Tschernig et al. Anat. Record 261, 11–13, 2000). Previous studies supported the thesis that the novelty or uncertainty of the situation plays a fundamental role in the anxiety reaction of students to dissection (Arraez et al. Anat. Record 279B, 16–23, 2004).

This research analysed the emotional reactions of students to the dissection of human cadavers at 4 points: before and after the first and the last session of the practical program. We also studied the student's perception about death. We found differences in emotional reaction across these different points and some relationships between the emotional reactions of students in the dissection room and their perception of death.

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New teaching technologies applied to a continuous assessment method

D. Escolar Castellón, M. A. Escolar Castellón, J. Blasco Oquendo, C. Martínez Ciriano and A. Cavero Cavero Department of Human Anatomy and Histology, University of Zaragoza, Spain

Teaching of anatomy within the Medicine Degree should not be a problem because the students who are admitted have obtained high marks in the University Entrance Examination (7.9 in Zaragoza). This leads us to propose the working hypothesis that the percentage of students passing any Medicine Degree subject should be over 95%. However, given this situation a 5% failure rate per year will gradually be accumulated, constituting a significant number of students who are likely to drop out.

Our objectives were to develop a learning method in which the failure rate will be lower than 5%, and a method for early detection of possible failure candidates, in order to avoid dropout.

Human Anatomy I and II Medical student results were compared with those of Ocular Anatomy and Histology students who studied Optics. The method was based on the day-to-day work of the student in the dissection room. The student was given a work schedule with the lecture, alongside distance learning (digital teaching ring, WebCT) and a system of continuous assessment (assessment every 5 topics). Because learning is a conductive procedure, the aim was to provide the student with a pleasant experience of the subject.

As a result the pass rate was above 96%. Half of the chronic repeaters eventually passed their exams. It was observed that students who failed the first three assessments did not succeed in passing the subject. We consider that this method is a good way to stimulate the student and prevent dropout. However we have not succeeded in reversing trends for problem students.

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Results of applying a cooperative learning method and study using cases to anatomy students in the first year of physiotherapy training

M. A. Fdez-Villacañas Marín, M. Moreno Cascales and G. Doménech Ratto Department of Human Anatomy and Psychobiology, University of Murcia, Spain

We applied the method of cooperative learning in the practical groups for Anatomy in the curriculum of the Physiotherapy degree. It allowed us to distribute tasks, in rotation, between the students. Based on the use of a study guide to aid the students, different roles are designed. One task consisted of directing the practical based on the designation of a person in charge; a person to take charge of text and atlas guides; a person to fill in the bland sheets to identify different structures; and a person in charge of the group portfolio. The role of the professor is to introduce the practical; to establish a mechanism of systematic work; to verify the fulfillment of objectives and to answer questions.

The same group of students had to write a paper (course work) that they have to choose between 3 cases (shoulder girdle; knee or respiratory mechanics). The work was based on the study of the Method of Cases that culminated with the accomplishment of a Conceptual Map. The paper, which must be given at the end of course, scores up to 0.5 of the final mark, has to be presented in public. This activity is made by students in their practical groups and on the day for practicals assigned the group. For this reason the subjects were distributed so that every day each of the proposed subjects is guaranteed to appear.

A survey was conducted in which the students evaluated the 2 activities. The answers could range between nothing (1) and high value (5). In relation to the practicals, 61% of the students thought that they had acquired enough or much knowledge. 50% thought that they had been stimulated to participate. 66% thought that the method was in the interest of the students and motivated them to learn between enough and a lot.

In relation to the course work; we present the answers scored 4 and 5. For acquisition of knowledge, 38%. For degree of satisfaction: 42%. 33% thought that this activity encouraged investigation and critical spirit.

Supported by a grant from ICE (Murcia) Project EDUCA05 P106.

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Anatomy and e-learning

O. González-Sequeros 1 , C. Bernal-Mañas 1 and J. Egea 2 1 Department of Anatomy, Faculty of Medicine; and 2 Section for Information Technology and Applied Communication, University of Murcia, Spain

The reform of the structure and organization of Spanish Universities within the European Area of Higher Education are changing the teaching and learning process in Schools and Faculties of Health Sciences, based on the achievement of skills and competencies essential for beginning independent unsupervised clinical practice. In dental schools, by the time the dentist obtains the professional degree he or she must have acquired not only a broad academic and dental education necessary for responding the normal range of circumstances in dental general practice, but to achieve competences in: (1) continuing professional development; (2) working together with other health care professionals; and (3) using contemporary information technology (as stated in the General Assembly of the Association for Dental Education in Europe, Cardiff 2004).

We have designed a virtual subject ‘Applied Odontologic Anatomy’ (AOA) in order to develop these 3 general competences and a specifically anatomical one (number 4). Students must be competent to identify different aspects of maxillary and mandibular alveolar apophysis morphology and to connect it with oral diseases.

In the virtual campus of Murcia Universtiy ‘SUMA’ students have the contents of AOA separated into 6 units, each one with different tasks that students must complete following these premises: (1) working individually or in a team, as indicated in each unit; (2) looking for the answers using internet resources; and (3) managing the tools of the virtual campus, like tutorials, fora or chatrooms to be in contact and work with the teacher and other students.

We present the results of AOA evaluation using a quantitative questionnaire of the Lickert type with scale points 1–5, in order to assess if students have acquired the competences above mentioned and discuss how to improve it.

Acknowledgements: Institute for Educational Sciences of Universitiy of Murcia, for its economical support to develop this project.

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Learning and teaching human anatomy through virtual reality: functional and clinical study of the knee joint

J. A. Marchal 1 , F. Hita 1 , M. Perán 2 , A. Martínez-Amat 1 , F. Rodríguez-Serrano 1 , H. Boulaiz 3 , J. C. Prados 3 , C. Vélez 1 , C. Melguizo 2 , O. Caba 3 , C. J. Ogáyar 4 , J. R. Jiménez 4 , R. J. Segura 4 and A. Aránega 3 1 Department of Health Sciences, University of Jaén; 2 Department of Neuroscience and Health Sciences, University of Almer’a; 3 Department of Human Anatomy and Embryology, University of Granada; and 4 Department of Informatics, University of Jaén, Spain

Virtual reality (VR) is the simulation of a real or imagined environment that can be experienced visually in 3 dimensions and that may additionally provide an interactive experience visually in full real-time motion. Moreover VR provides the impression of being mentally immersed or present in the simulation or virtual world. Virtual anatomy is the life-like appearance of visible anatomy, a good example is the evolution of the Visible Human or the Anatomic VisualizeR. Nowadays an increasingly demand of 3-dimensional tools for learning and training medical sciences have emerged. For that reason the main objective of this work was to develop a VR system for the anatomical study of the knee to show the main elements of this joint as well as some of its pathologies.

Firstly we selected human bone material to scan by using a Polhemus Fast-Scan 3D handy scanner. Next the scanned models were simplified and purged in order to achieve an interactive visualization of them. In a second phase, a prototype of a VR system has been derived by integrating the bone models using the VRML 2.0 language. Furthermore a 3D system for visualizing nuclear magnetic resonance (NMR) images of the knee was implemented. This system is able to visualize selected slices from the set of images by using an interactive knife. The implementation of this system was also carried out by using the VRML language. All the previously referred elements have been integrated using different frames in a web environment in order to take advantage of the hypermedia features of this kind of environments. Additionally, the prototype has bonds towards functional, exploratory and clinical audio-visual contents of the knee joint.

In short, we have developed a prototype of virtual anatomy that it tries to give a complete and integrated vision of the knee joint with the purpose of allowing the teaching and autonomous learning of the medical sciences students in this concrete anatomy field.

Grant support: This study was supported by the Universidad de Jaén through the ‘Proyectos de Innovación Docente’ (project no. PID69B).

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The design of virtual practicals in anatomy learning: one experience

M. C. Mias 1 , R. Sola 1 , E. Brescó 2 and J. Bitterhoff 2 1 Department of Human Anatomy, Faculty of Medicine; and 2 Department of Virtual Learning, University of Lleida, Spain

The use of information and communication technologies could help anatomical learning and also could facilitate adaptation of anatomical programs to the ECTS. For this reason the authors have used virtual practicals to teach one anatomical subject during this academic course, but they think it is necessary to define the characteristics that must be included in the design of an anatomical virtual practical.

The objective of this work was to justify the importance of good design of virtual practicals and to define their characteristics. To design these practicals the anatomical group has needed the help of the Virtual Learning Team of our University and they have used a virtual platform named Sakai in order to create a virtual class room where the students and the teachers could work.

The practicals have been designed with this structure: theoretical explanation about the practical; instructions; interactive tools which make the problems; description in text of the result; verification of results; and sending by e-mail to the teacher. The authors present their experience using an example.

The design of virtual practicals must permit students to reach the objectives and the competencies of the subject using media tools. For this reason it is necessary to work with a team involving informatics, teachers, and psychologists of learning, to set up a virtual platform and to create a virtual classroom.

The virtual practicals also need a structure that permits students to obtain theoretical information about the content of the practical, to solve it with interactive tools and to send it to the teacher in order to control the work of the student. So the design of this type of practicals permit the student to be self-taught in anatomical subjects and permit the teacher to control the progress of the students in this subject.

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Coping methods in the dissection room of first-year medical students in human anatomy practicals

M. Miguel, N. Porta, J. C. Ortiz, A. Martínez, J. M. de Anta and V. Götzens Section of Embryology and Human Anatomy, Department of Pathology and Clinical Therapeutics, Faculty of Medicine (Bellvitge Campus), University of Barcelona, Spain

The aim of this study was to study the coping methods used by the first-year medical students at the University of Barcelona (Campus de Bellvitge) in preparation for the stress that the dissecting room is to these students. The students answered 2 questionnaires prepared and administered to students after the first dissection, and at the end of the course.

We found that the students used methods such as being and joking with friends, expanding their study of anatomy, and seeking advice to their friends and teachers, to avoid an adverse reaction to the dissecting room. There were significant differences between men and women and between students.

We concluded that the dissection room produced in first-year medical students a series of adverse reactions that required mechanisms of adaptation in order to cope with them. Teachers of anatomy have to instruct the subject but they also have to help to provide support and adaptation for students throughout the course.

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Anatomical terminology in surgery

J. A. Pereira 1 , A. Merí 1 , E. Pleguezuelos 1 , A. Molina-Ros 1 and A. Sitges-Serra 2 1 Department of Experimental Health Sciences, Pompeu Fabra University; and 2 Department of Surgery, Hospital del Mar, Barcelona, Spain

Knowledge of Human Anatomy is essential for surgeons. It allows understanding the pathophysiology of a large number of disease processes, interpreting radiological imaging and to feel assured in the operating field and in choosing the appropriate surgical access.

Historically, thorough knowledge of Anatomy has been closely related to advances in surgical technique and has been furthered by surgeons themselves in search of continuous technical improvement. Currently however, the value of Anatomy has been underappreciated in many fields of Medicine.

Anatomists are partially responsible for this worrying scenario since they have moved away from clinical medicine and have procrastinated in conservative attitudes when teaching morphology. Still, their teaching methodology is almost exclusively based on memorizing and accumulating anatomical terms without taking into account the practical importance of anatomical issues, not encouraging reasoning, using confusing names and abusing eponyms.

At present ignorance of accurate anatomical terminology is quite prevalent and this is often reflected in surgical forums in which new anatomical designations have born which are incorrect from a scientific terminology point of view.

This paper analyses the use of some anatomical terms commonly used in current surgical debates and its consistency with the new international terminology. For this purpose, a questionnaire regarding surgical anatomy terminology was presented to 40 surgeons of 4 hospitals to assess their opinion concerning the relevance of anatomical knowledge and asking them to answer 6 MCQs on surgical anatomy and terminology.

Our results showed a lack of knowledge of basic aspects of terminology and essential surgical anatomy. Only 36% of surgeons answered correctly 3 or more questions (Average mark = 3.5, scale 0–10 points). In our opinion anatomists should develop strategies to improve effective teaching of Anatomy in the clinical setting, especially for surgeons in their training period.

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Establishment of a programme of anatomy for physiotherapy based on competencies

M. A. Fdez-Villacañas Marín, M. Moreno Cascales and G. Doménech Ratto Department of Human Anatomy and Psychobiology, University of Murcia, Spain

In order to establish a model of competency based learning we have designed the curricular competencies, generic and specific, in anatomy within the requirements of a physiotherapy degree.

The relationship between discipline specific and generic competencies were analyzed. This was part of the change from a traditional credit system to ECTS, and included the work undertaken by a student globally (theoretical and practical study, theoretical and practical independent study, tutored sessions, group work, examination time). We propose to introduce anatomical terminology, relating to communication in English. Methods of working, such as criteria for evaluation and bibliography, were included. After establishing thematic blocks and components of the competencies in the discipline, the planned activities were listed and evaluated. Student surveys were developed for evaluation of activities, the design and development of the programme, and the acquisition of competencies.

Supported by a grant from ICE (Murcia) Project EDUCA05 P106.

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Interactive anatomy of the locomotor system: a new multimedia application for anatomy teaching

B. Torres and Q. Morales Department of Human Anatomy and Embryology, School of Medicine, University of Barcelona, Spain

With our universities entering the European Higher Education Area, new challenges and needs arise. The education paradigm has changed, and now the student is expected to walk the path of learning, based on his own experience and previous knowledge. The teacher will accompany and guide him. This new approach to teaching brings the necessity of generating new ways and strategies to teach and learn.

The application Interactive Anatomy of the Locomotor System has been created with this new paradigm, and can be used in multiple contexts: (1) As a complementary teaching material in presential education; (2) As a teaching material in a virtual classroom; (3) As an autonomous learning tool; and (4) As a reference tool for professionals. The application is based on completely interactive screens containing text and images. Just by hovering the mouse or clicking text and icons, users can access certain image details, construct a topographic complex region layer by layer or visualize text information.

At the end of every chapter there are different hotspot screens, and there are also 3 multiple choice tests for the user to rate his own knowledge whenever he wants.

Screens can be grouped in 3 categories: (1) Descriptive screens: a text describes a structure (or its details), always showing an image of it; (2) Image-only screens: a previously explained structure is presented with hotspots (visible or hidden at user's request). Users can hover the mouse and the name of the detail will show. These screens are intended for autoevaluation purposes; and (3) Layer screens: users can show or hide the different layers of a region just by clicking its names.

This project has been funded by the University of Barcelona. PID projects 2003 and 2004 (2003PID-UB/01, 2004PID-UB/002 and 2005PID-UB/04).

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Educational strategies for the teaching of gross anatomy in the medical curriculum – student attitudes, learning outcomes and teaching methods in medical education

B. Moxham Cardiff School of Biosciences, Cardiff University, UK

Recent developments in medical curricula have led to marked changes in the teaching of Gross Anatomy. This has resulted from the belief that anatomy is largely ‘content-driven’ and not ‘skills-based’. This presentation describes and evaluates, primarily from the perspective of medical students, different methods of teaching anatomy and includes the ‘teaching’ of such skills as: team skills, relating their dissecting room experience to the study of pathology and to the clinic, relating their experience to medical humanities issues such as their responses to death. The assessment of student attitudes was conducted by employing Thurstone and Chave attitude analyses and also a matrix questionnaire that evaluated different methods of teaching anatomy in relation to an array of potential course aims/learning outcomes. Comparisons were made with the attitudes of professional anatomists working in Europe.

The findings show that: (1) Professional anatomists and medical students differ little in their evaluation of the importance of anatomy and of the relationships between teaching methods and course aims/learning outcomes; (2) Medical students believe that anatomy is very important to clinical medicine (before entering their medical course, after completing their anatomy courses, and towards the end of their medical training); (3) Medical students would prefer that anatomy is taught practically (via dissection, use of prosection, with living and radiological anatomy) than theoretically (via didactic teaching, models, CAL).

Because of anatomy's perceived clinical importance, because of the preference for practical teaching and learning, and because both professional anatomists and medical students do not believe that anatomy contributes greatly to other basic sciences, anatomy ought to be a ‘stand-alone’ component in a medical curriculum.

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Educational strategies applied in teaching anatomy

R. Vázquez, J. M. Riesco, J. A. Juanes, E. Blanco, M. Rubio and J. Carretero Department of Human Anatomy and Histology, Faculty of Medicine, University of Salamanca, Spain

Until recent years there was no doubt that a combination of lectures and dissection were the 2 pillars for teaching anatomy, and the human cadaver the best book for learning it.

The topics and the depth of knowledge to teach have changed through different periods of time, from Galen to now. Many differently oriented textbooks have been published reflecting the historical evolution of anatomy. Nowadays it seems more or less accepted that anatomy should be clinically oriented, or it would not be useful for medical students.

A reduction in the time assigned to teach anatomy and new pedagogic approaches where the student takes the main role in the teaching-learning process (self-learning) have resulted in a Copernican revolution in the anatomical world. Anatomy has been an independent discipline for many years but must become integrated with other basic sciences and/or clinical disciplines and to be taught by many different methods, for example Problem Based Learning (PBL).

On the other hand, new technologies as well as the internet have replaced in most Anatomy Departments a number of lecture presentations by unsupervised teaching. At the same time the time dedicated to cadaveric dissection has also been reduced – if it has not been completely removed from the anatomical laboratories – on the argument that it is sufficient to use clinical images for teaching medical students.

This presentation attempts an overview of the different strategies and methods that through time have been developed for teaching anatomy: lectures vs. self learning, different orientation of textbooks, dissection vs. clinical images, anatomical models, computer programs, etc.