[Helping medical students to approach death]. / Aider les étudiants en médecine à approcher la mort.

A project recently launched by the Faculty of biology and medicine of Lausanne introduces the approach of facing death during both the dissection and the course of clinical activities. Existential questions relating to mortality are bound to arise sooner or later during the course of the study. For the sake of humanized clinical practice, these questions must be confronted. In response to a request by a student association, an accompanying curriculum with active student's contribution through encounters with death in anatomy and clinical situations was created in Lausanne. Students will benefit from this new program throughout their curriculum. This program is the first of its kind in Switzerland.

Excess of serotonin affects neocortical pyramidal neuron migration.

The serotonin transporter (SERT) is a key molecule involved in the homeostasis of extracellular levels of serotonin and is regulated developmentally. Genetic deletion of SERT in rodents increases extracellular levels of serotonin and affects cellular processes involved in neocortical circuit assembly such as barrel cortex wiring and cortical interneuron migration. Importantly, pharmacological blockade of SERT during brain development leads to phenotypes relevant to psychiatry in rodents and to an increased risk for autism spectrum disorders in humans. Furthermore, developmental adversity interacts with genetically-driven variations of serotonin function in humans and nonhuman primates to increase the risk for a variety of stress-related phenotypes. In this study, we investigate whether an excess of serotonin affects the migration of neocortical pyramidal neurons during development. Using in utero electroporation combined with time-lapse imaging to specifically monitor pyramidal neurons during late mouse embryogenesis, we show that an excess of serotonin reversibly affects the radial migration of pyramidal neurons. We further identify that the serotonin receptor 5-HT(6) is expressed in pyramidal neuron progenitors and that 5-HT(6) receptor activation replicates the effects of serotonin stimulation. Finally, we show that the positioning of superficial layer pyramidal neurons is altered in vivo in SERT knockout mice. Taken together, these results indicate that a developmental excess of serotonin decreases the migration speed of cortical pyramidal neurons, affecting a fundamental step in the assembly of neural circuits. These findings support the hypothesis that developmental dysregulation of serotonin homeostasis has detrimental effects on neocortical circuit formation and contributes to increased vulnerability to psychiatric disorders.

Transitory uptake of serotonin in the developing sensory pathways of the common marmoset.

Serotonin (5-HT) affects brain development during sensitive developmental periods. In rodents, transient sites of high affinity capture of 5-HT were demonstrated in the primary sensory neurons and in the sensory thalamocortical afferents. This uptake is required to adjust 5-HT receptor stimulation during the formation of sensory maps. To determine whether similar mechanisms exist in primates, we analyzed staged embryos and postnatal pups in the common marmoset (total gestation time, 142 days). Immunocytochemical analyses were performed using antisera to 5-HT, to the serotonin transporter (SERT), and to the vesicular monoamine transporter (VMAT2). 5-HT, SERT, and VMAT2 labeled the raphe neurons and their terminal network from embryonic day (E)70 to adulthood. In addition, from E70-130 VMAT2 and SERT were observed in all the sensory cranial nerves, the olfactory nerve, the gustatory, the trigeminal, the auditory fibers, in the retinal ganglion cells, and the optic tract up to the lateral geniculate nucleus and the superior colliculus. All the spinal sensory ganglia and their peripheral sensory branches were labeled. Accumulation of 5-HT was observed in all the sensory neurons expressing SERT and the corresponding axon tracts. Since these neurons were missing tryptophan hydroxylase (TPH), the synthesizing enzyme for 5-HT, they most likely accumulated 5-HT through the action of the amine transporters, as has been shown in rodents. No transient expression of 5-HT markers was detectable in the sensory thalamocortical axons at any of the ages examined. Thus, the existence of 5-HT uptake in nonserotoninergic neurons appears to be a conserved feature in primates, although the topographic extent of this transient expression is more restricted than that previously demonstrated in rodents.

Can intestinal innervation be preserved in pancreatoduodenectomy for cancer? Results of an anatomical study.

Twenty dissections were carried out, in all of which the splanchnic nerves, celiac plexuses, capital pancreatic plexus and superior mesenteric plexus were identified and traced. The capital pancreatic plexus was formed from two bundles, the first taking its origin from the right celiac plexus, the second from the superior mesenteric plexus. These two bundles joined together just behind the head of the pancreas. Two preganglionic bundles, a ganglion and two postganglionic bundles composed the superior mesenteric plexus. Postganglionic bundles received fibers from both right and left celiac plexuses. In small cancers a thin layer of nervous tissue around the superior mesenteric artery might be spared in order to avoid diarrhea from intestinal denervation. This study has provided anatomical evidence that a part of the mesenteric plexus, which receives fibers from both left and right celiac plexuses, maintains a sufficient intestinal innervation.

Gross anatomy in the surgical curriculum in Switzerland: improved cadaver preservation, anatomical models, and course development.

The invention of new techniques for surgery and interventional radiology demand improved training for ongoing specialists. The Anatomical Institutes in Switzerland support these requirements by establishing hands-on practical training courses by using new procedures for cadaver embalming and model construction. Improvements allow courses to provide students with more realistic simulations of both established and experimental surgical methods. Through these changes, the value of in-depth gross anatomy is enhanced as a topic of fundamental importance for the postgraduate medical and surgical curriculum. The web site http://www.unifr.ch/sgahe/snga.html contains information on courses using the Thiel embalming solution. Details about training courses in Switzerland using anatomical models are available at http://www.heartlab.org, http://www.vascular-international.org, and http://www.elastrat.com.

Contribution to the surgical anatomy of the ligaments of the rectum.

PURPOSE: Many authors have discussed the presence and the importance of the lateral ligaments of the rectum. Our contribution aims at clarifying some aspects of surgical anatomy that help in the preservation of the urogenital functions and may influence the surgical practice. METHODS: From 1994 to 1998 we examined 27 fresh cadavers and five embalmed pelves. We performed all dissections with a technique similar to that used for the surgical mobilization of the rectum. RESULTS: The lateral ligaments of the rectum are trapezoid structures originating from mesorectum and are anchored to the endopelvic fascia; as lateral extensions of the mesorectum, they must be included in the surgical specimen. According to our results, three main structures can be recognized laterally to the rectum: 1) the lateral ligament, which does not contain important structures; 2) the inferior hypogastric plexus and the urogenital bundle; and 3) the lateral neurovascular pedicle of the rectum that comprises the nervi recti and the middle rectal artery, both running under the lateral ligament, although at different angles. CONCLUSION: At the point of insertion into the endopelvic fascia, the lateral ligaments run close to the urogenital bundle. Nevertheless, the dissection at its attachment is safe if the urogenital bundle is kept under visual control.

Neonatal serotonin depletion affects developing and mature mouse cortical neurons.

The early expression of neurotransmitters and receptors in the developing brain has brought attention to their potential contribution in modulating neuronal developmental processes. Monoamines are among the first neurotransmitter systems to develop during embryogenesis. Depletion of neocortical serotonin or catecholamine afferents with selective neurotoxins resulted in a permanent alteration of the dendritic arborization of calretinin-containing interneurons, and a transient delay of parvalbumin and calbindin expression in a number of cortical neurones during the second postnatal week. The expression pattern of other developmentally regulated proteins, such as two subunits of the GABA(A) receptor, was not altered. Depletion of serotonin, and in part catecholamines, appeared to perturb several developmental processes of the cerebral cortex which would interfere with both its maturation and adult circuitry.

Dopamine affects parvalbumin expression during cortical development in vitro.

This study was undertaken to determine how dopamine influences cortical development. It focused on morphogenesis of GABAergic neurons that contained the calcium-binding protein parvalbumin (PV). Organotypic slices of frontoparietal cortex were taken from neonatal rats, cultured with or without dopamine, harvested daily (4-30 d), and immunostained for parvalbumin. Expression of parvalbumin occurred in the same regional and laminar sequence as in vivo. Expression in cingulate and entorhinal preceded that in lateral frontoparietal cortices. Laminar expression progressed from layer V to VI and finally II-IV. Somal labeling preceded fiber labeling by 2 d. Dopamine accelerated PV expression. In treated slices, a dense band of PV-immunoreactive neurons appeared in layer V at 7 d in vitro (DIV), and in all layers of frontoparietal cortex at 14 DIV, whereas in control slices such labeling did not appear until 14 and 21 DIV, respectively. The laminar distribution and dendritic branching of PV-immunoreactive neurons were quantified. More labeled neurons were in the superficial layers, and their dendritic arborizations were significantly increased by dopamine. Treatment with a D1 receptor agonist had little effect, whereas a D2 agonist mimicked dopamine's effects. Likewise, the D2 but not the D1 antagonist blocked dopamine-induced changes, indicating that they were mediated primarily by D2 receptors. Parvalbumin expression was accelerated by dopaminergic reinnervation of cortical slices that were cocultured with mesencephalic slices. Coapplication of the glutamate NMDA receptor antagonist MK801 or AP5 blocked dopamine-induced increases in dendritic branching, suggesting that changes were mediated partly by interaction with glutamate to alter cortical excitability.

Medium-sized neurofilament protein related to maturation of a subset of cortical neurons.

Neurofilaments are typical structures of the neuronal cytoskeleton and participate in the formation and stabilization of the axonal and dendritic architecture. In this study, we have characterized a murine monoclonal antibody, FNP7, that is directed against the medium-sized neurofilament subunit NF-M. This antibody identifies a subset of neurons in the cerebral cortex of various species including human and in organotypic cultures of rat cortex. In the neocortex of all species examined, the antibody labels pyramidal cells in layers III, V, and VI, with a distinctive laminar distribution between architectonic boundaries. In comparison with other antibodies directed against NF-M, the FNP7 antibody identifies on blots two forms of NF-M that appear relatively late during development, at the time when dynamic growth of processes changes to the stabilization of the formed processes. Dephosphorylation with alkaline phosphatase unmasks the site, making it detectable for the FNP7 antibody. The late appearance suggests that the site is present during early development in phosphorylated form and with increasing maturation becomes dephosphorylated, mainly in dendrites. This event may relate to changes in cytoskeleton stability in a late phase of dendritic maturation. Furthermore, mainly corticofugal projections and only few callosal axons are stained, suggesting a differential phosphorylation in a subset of axons. The antibody provides a useful marker to study subsets of pyramidal cells in vivo, in vitro, and under experimental conditions.

Increased exploratory activity and altered response to LSD in mice lacking the 5-HT(5A) receptor.

In order to determine the distribution and function of the 5-HT5A serotonin receptor subtype, we generated knockout mice lacking the 5-HT5A gene. Comparative autoradiography studies of brains of wild-type (wt) and 5-HT5A knockout (5A-KO) mice revealed the existence of binding sites with high affinity for [125I]LSD that correspond to 5-HT5A receptors and that are concentrated in the olfactory bulb, neocortex, and medial habenula. When exposed to novel environments, the 5A-KO mice displayed increased exploratory activity but no change in anxiety-related behaviors. In addition, the stimulatory effect of LSD on exploratory activity was attenuated in 5A-KO mice. These results suggest that 5-HT5A receptors modulate the activity of neural circuits involved specifically in exploratory behavior and suggest that some of the psychotropic effects of LSD may be mediated by 5-HT5A receptors.

Laminar development of the mouse barrel cortex: effects of neurotoxins against monoamines.

The rodent somatosensory cortex is characterized by a unique cellular organization in the field of representation of the whiskers, called the barrelfield, which develops in layer IV during the 1st postnatal week in parallel with the establishment of the thalamo-cortical connections. This area is transiently densely innervated by serotonergic afferents during this period. Serotonin depletion delays the formation of barrels in the rat somatosensory cortex. However, no information is available to date on the time-course of the laminar differentiation of the cortex after monoaminergic depletion and the relative contribution of different monoaminergic inputs to this process. To address these issues, newborn mice were treated with selective neurotoxins (6-hydroxydopamine or 5,7-dihydroxytryptamine) at birth to destroy the catecholaminergic and monoaminergic cortical innervation, respectively. The parietal cortex of these animals was examined in Nissl-stained coronal sections prepared on different days of postnatal development (between P2 and P30). Compared with the controls, delayed growth and differentiation of the cortical layers II-IV were observed in the treated animals, most prominently between P2 and P16. From the 3rd postnatal week, no cytoarchitectonic difference could be detected. Although neonatal depletion of the cortical monoaminergic innervation does not affect the laminar organization of the adult mouse barrelfield, it significantly delays the time-course of development of several cortical layers. This delay generates a mismatch in the degree of maturation between cortical neurons and their afferents at a time when neuronal interactions are critical for the establishment of local circuitry.

Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 microm diode laser beam.

The safety of microdrilling the zona pellucida of mouse oocytes with a 1.48 microm diode laser has been investigated by determining the ability of mouse oocytes to fertilize in vitro and develop in vivo. Mice born after transfer of control and zona pellucida-microdrilled embryos into foster mothers were submitted to anatomical and immunohistochemical investigations, and their aptitude to breed was assessed in two subsequent generations. Decoronization of the oocytes with hyaluronidase induced a reduction of the fertilization and implantation rates, which was attributed to a zona hardening phenomenon. After laser zona pellucida microdrilling, these rates were restored to those obtained with embryos derived from untreated oocyte-cumulus complexes. Pups derived from zona pellucida microdrilled embryos were comparable with those obtained from control embryos, confirming the lack of deleterious effects of the laser treatment. In conclusion, the 1.48 microm diode laser allows safe microdrilling of the zona pellucida of mouse oocytes after decoronization with hyaluronidase. Based on the health of the F2 generation and the lack of neuroanatomical and neurochemical differences, we concluded that this technology may be investigated in the human, particularly when the zona pellucida represents the main impediment for fertilization or embryo hatching.

Developmental profile of GABAA-receptors in the marmoset monkey: expression of distinct subtypes in pre- and postnatal brain.

Gamma aminobutyric acid (GABA)A-receptors are expressed in fetal mammalian brain before the onset of synaptic inhibition, suggesting their involvement in brain development. In this study, we have analyzed the maturation of the GABAA-receptor in the marmoset monkey forebrain to determine whether distinct receptor subtypes are expressed at particular stages of pre- and postnatal ontogeny. The distribution of the subunits alpha 1, alpha 2, and beta 2,3 was investigated immunohistochemically between embryonic day 100 (6 weeks before birth) and adulthood. Prenatally, the alpha 2- and beta 2,3-subunit-immunoreactivity (-IR) was prominent throughout the forebrain, whereas the alpha 1-subunit-IR appeared in selected regions shortly before birth. The alpha 2-subunit-IR disappeared gradually to become restricted to a few regions in adult forebrain. By contrast, the alpha 1-subunit-IR increased dramatically after birth and replaced the alpha 2-subunit in the basal forebrain, pallidum, thalamus, and most of the cerebral cortex. Staining for the beta 2,3-subunits was ubiquitous at every age examined, indicating their association with either the alpha 1- or the alpha 2-subunit in distinct receptor subtypes. In neocortex, the alpha 1 -subunit-IR was first located selectively to layers IV and VI of primary somatosensory and visual areas. Postnatally, it increased throughout the cortex, with the adult pattern being established only during the second year. The switch in expression of the alpha 1- and alpha 2- subunits indicates that the subunit composition of major GABAA-receptor subtypes changes during ontogeny. This change coincides with synaptogenesis, suggesting that the emergence of alpha 1- GABAA-receptors parallels the formation of inhibitory circuits. A similar pattern has been reported in rat, indicating that the developmental regulation of GABAA-receptors is conserved across species, possibly including man. However, the marmoset brain is more mature than the rat brain at the onset of alpha 1-subunit expression, suggesting that alpha 1-GABAA-receptors are largely dispensable in utero, but may be required for information processing after birth.

Identification of distinct GABAA-receptor subtypes in cholinergic and parvalbumin-positive neurons of the rat and marmoset medial septum-diagonal band complex.

GABAA-receptor heterogeneity is based on a multiplicity of subunits (alpha 1-6, beta 1-4, gamma 1-4, delta, rho 1-2) encoded by distinct genes. Flexibility in GABAergic signal transduction and allosteric modulation is expected to arise from the differential assembly of subunits into receptor subtypes. The aim of the present study was to investigate the potential diversity of receptor subtypes expressed by defined neuron populations, as identified by their neurotransmitter phenotype. To this end we have determined immunohistochemically the subunit repertoire of cholinergic and GABAergic neurons in the basal forebrain of rat and marmoset monkey, focusing on the medial septum-diagonal band complex. Co-localization of the GABAA-receptor subunits alpha 1, alpha 3, beta 2, beta 3, and gamma 2 with markers of cholinergic and GABAergic neurons (choline acetyltransferase and parvalbumin, respectively) was assessed by double- and triple immunofluorescence staining. The results reveal that cholinergic neurons in the rat basal forebrain are typically characterized by the subunit combination alpha 3/beta 3/gamma 2, whereas most of the parvalbumin-positive GABAergic neurons express either the subunit combination alpha 1/beta 2/gamma 2 or the combination alpha 1/alpha 3/beta 2/gamma 2. A similar pattern was observed in marmoset monkey, with GABAA-receptors containing the alpha 1-subunit being associated with parvalbumin-positive cells, but never with cholinergic neurons. Thus, the expression of distinct subunit repertoires by cholinergic and GABAergic neurons points to a functional specialization which is conserved across species. These subunit combinations are likely to correspond to different receptor subtypes, and may reflect the engagement of cholinergic and GABAergic neurons in distinct neuronal circuits in the basal forebrain.

Distribution of GABA-containing neurons in human frontal cortex: a quantitative immunocytochemical study.

Fresh biopsy specimens of human cerebral cortex were collected from patients suffering from deep-seated tumors requiring resection. GABAergic neurons were revealed in 50-microns-thick sections, for pre-embedding, and 1-micron-thick sections, for post-embedding GABA immunocytochemistry. In both thick and thin sections, the reaction product was found in neuronal cell bodies and in small profiles in the neuropil. In both preparations, GABA-containing somata were distributed evenly throughout the depth of the cortex. As best appreciated in the thicker sections, GABA-immunoreactive neurons belonged to a variety of morphological cell types with multipolar, bitufted or bipolar, and horizontal dendritic arbors. In the semi-thin sections sampled in the frontal cortex, the proportion of these neurons could be accurately evaluated and was found to be 21.2% +/- 4.8% of all cortical neurons. The average size of GABA-immunoreactive neurons was, in each layer, smaller than that of immunonegative neurons. The average soma size of both neuronal populations, immunoreactive and immunonegative for GABA, increased with depth. The comparison between the rat, cat, macaque monkey, and human GABAergic interneurons revealed similarities among primate brains, contrasting with the parameters (morphology, size, density) measured in rodents. These data are pertinent to the involvement of the GABAergic neurons in the shaping of receptive-field properties of cortical neurons in healthy brains and in pathologies involving the impairment of inhibitory neurotransmission.

Monoaminergic afferents to cortex modulate structural plasticity in the barrelfield of the mouse.

Electrolytic lesions of the follicles of a set of mystacial vibrissae, and their innervation, of the mouse placed during the early postnatal period result in a modification in appearance of the corresponding and of adjacent barrels in the somatosensory cortex of the adult animal. These changes can be evoked during the first 6 days of postnatal life--the so-called critical period. The pattern of these modifications varies with the age of the animal at which the lesion was placed. In order to evaluate the contribution of the monoaminergic cortical input to this type of plasticity, the noradrenergic and/or serotonergic afferents to the cerebral cortex of newborn mice were destroyed by systemic administration of various selective neurotoxic drugs (6-hydroxydopamine, 5,7-dihydroxytryptamine, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine). The animals were then subjected, on postnatal day 3 (P3; P0 = day of birth), to a lesion of the follicles of the large, caudal mystacial vibrissae of row C. Control animals were injected with vehicle solution only but had the same follicles lesioned. Compared with animals with intact monoaminergic afferents, those treated with neurotoxins showed a different changed barrel pattern, i.e. one that corresponded to a pattern normally obtained after a lesion placed at an earlier stage of development, i.e. at P2 or P1. Thus, monoaminergic depletion of the cortex results in a retardation of the maturation of the parietal cortex as defined by its plastic response to peripheral nerve injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology and distribution of neuropeptide-containing neurons in human cerebral cortex.

Biopsies of human cerebral cortex were fixed by immersion and immunostained for the detection of neuropeptides in neuronal cell bodies and axons. Four neuropeptides (neuropeptide Y, somatostatin, , substance P and cholecystokinin) were visualized in a series of adjacent sections. All populations of immunoreactive neurons had a morphology characteristic of interneurons, with variations in dendritic arborizations and laminar distribution. The cholecystokinin-immunoreactive neurons were most numerous in the supragranular layers, whereas neurons containing the other three peptides occurred mainly in infragranular layers, or even in neurons populating the subcortical white matter. Quantitatively, each population of neuropeptide-containing neurons accounted for 1.4-2.5% of the total neuronal population. The distribution of these neurons varied slightly between cytoarchitectonic divisions, with substance P- and somatostatin-immunoreactive neurons dominating in the temporal lobe and cholecystokinin-immunoreactive neurons in the frontal lobe. Neuropeptide Y-immunoreactive neurons dominated in the gray matter of the frontal half of the hemisphere and in the subcortical white matter of the caudal half of the hemisphere. Furthermore, co-existence of neuropeptide Y or substance P immunoreactivity within somatostatin-immunoreactive neurons could be demonstrated using double labeling immunofluorescence techniques. The axonal plexuses immunoreactive for neuropeptide Y, somatostatin, or substance P were distributed in all layers, with a strong predominance of horizontally oriented fibers in layer I, a moderate plexus of randomly oriented fibers in the supra- and infragranular layers, and a slightly weaker innervation of layer IV. Immunoreactive axons formed, in addition, complex terminal arbors, mostly in older subjects, suggesting that they resulted from an as yet undefined aging process. The present study underlines several aspects of the organization of the neuropeptide-containing neurons of the human cerebral cortex, which are of particular interest in the light of the involvement of these neurons in several neurodegenerative diseases.

The selective innervation by serotoninergic axons of calbindin-containing interneurons in the neocortex and hippocampus of the marmoset.

The serotoninergic input to the mammalian cerebral cortex originates in the median and the dorsal raphe nuclei. Median raphe neurons have been previously shown to give rise to beaded varicose axons which form dense pericellular arrays (baskets) surrounding the soma and the proximal dendrites of certain cortical neurons. In the present study, we have searched for specific markers characterizing the neurons of the marmoset neocortex and hippocampus surrounded by these thick varicose serotonin-containing fibers. The non-pyramidal nature of these neurons, suggested by their dendritic arborization, was correlated, in immunocytochemical experiments with double-labelling to demonstrate their surrounding serotonin-containing basket and their content of glutamic acid decarboxylase (GAD) or of the calcium-binding protein calbindin. Another calcium-binding protein common in numerous non-pyramidal cortical neurons, parvalbumin, was never found in neurons surrounded by serotonin-containing baskets. This organization was found in all areas of the neocortex and of the hippocampus where serotonin-containing baskets were present. One of the serotoninergic cortical inputs which originates from the brainstem tegmentum, traditionally described as "diffuse," proves to be highly selective in that a subset of its axons terminates preferentially on a subpopulation of inhibitory interneurons of the cerebral cortex. It may be emphasized that this subset of cortical interneurons has now been shown to be characterized not only by its axonal and dendritic arborization and its neurotransmitter, but also by a specific type of input which can modulate cortical function in a unique manner.