Nanotechnology in reproduction: Vitamin E nanoemulsions for reducing oxidative stress in sperm cells.

Vitamin E is considered a powerful biological antioxidant; however, its characteristics such as high hydrophobicity and low stability limit its application. We propose to use nanotechnology as an innovative tool in spermatology, formulating nanoemulsions (NE) that accommodate vitamin E, protecting it from oxidation and promoting its release into the medium. The protective effect of the NE against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from eleven stags were thawed and extended to 400 × 106 sperm/ml in Bovine Gamete Medium (BGM). Once aliquoted, the samples were supplemented with the NE at different concentrations (0, 6 and 12 mM), with or without induced oxidative stress (100 µM Fe2+/ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. Motility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) were assessed. After 2 and 4 h of incubation, the NE were able to prevent the deleterious effects of oxidative stress, thus improving total and progression motility (P ˂0.05). Moreover, the highest concentration tested (12 mM) improved almost every sperm kinematic variable (P ˂0.05) and preserved sperm viability in samples subjected to oxidative stress. In addition, 12 mM of NE protected the acrosomes integrity, maintained and protected mitochondrial activity, prevented sperm lipoperoxidation and reduced ROS production (P ˂0.05) in samples subjected to oxidative stress. This work indicates for the first time that vitamin E formulated in NE could be a new approach against sperm oxidative damage. This could be highly relevant for sperm physiology preservation in the context of assisted reproduction techniques.

Ascorbyl-dipalmitate-stabilised nanoemulsions as a potential localised treatment of inflammatory bowel diseases.

Current efforts on inflammatory bowel diseases (IBD) treatment are focused on strategies for localised drug delivery at the intestinal mucosa. Despite the potential of curcumin (CC) for IBD treatment, its low solubility and stability limit its application. Thus, the design of nanocarriers that focus CC delivery at the intestinal epithelium is an area of interest. This work proposes α-tocopherol nanoemulsions (NE) stabilised by ascorbyl-2,6-dipalmitate (ADP) as intestinal CC-carriers. The antioxidant capacity of α-tocopherol and ADP could have a synergistic effect on IBD-affected tissues, characterised by an oxidative environment. We obtained nanoemulsions (NE-ADP) with size below 200 nm, negative surface charge, stable in gastrointestinal media and no toxic in the Caco-2 cell model. Intracellular retention of NE-ADP in Caco-2 cells was observed by confocal microscopy. The extremely low Papp values obtained for CC and α-tocopherol indicated the lack of transport across the Caco-2 monolayer. Control nanoemulsion stabilised by lecithin (NE-L) was greatly transported across the Caco-2 cells monolayer, confirming the relevance of ADP on the cellular retention of NE-ADP. The therapeutic potential of NE-ADP was shown by the significant decrease of intracellular ROS levels. Altogether, these results indicate the potential of NE-ADP as a novel approach for the treatment of IBD.

The role of the intestinal-protein corona on the mucodiffusion behaviour of new nanoemulsions stabilised by ascorbyl derivatives.

Nanoemulsions are vesicular systems with great potential for the delivery of drugs, which significantly depends on the appropriate selection of the components that constitute them. In this sense, the use of materials with adequate toxicity profiles for the oral route provides additional advantages in terms of safety concerns avoidance. This work describes the formulation of novel two-component nanoemulsions constituted by α-tocopherol and ascorbyl-palmitate derivatives. Among them, ascorbyl-dipalmitate allowed the formation of nanoemulsions with size values around 170 nm and negative charge; additionally, they showed strong antioxidant capacity. These nanoemulsions are proposed to the oral route, so their behaviour in intestinal conditions was evaluated by incubating the nanoemulsion in simulated intestinal fluid. This process led to the formation of an intestinal-protein corona (I-PC) at the colloidal surface that determined the interaction with the mucus barrier. The I-PC displaced the immobile-hindered particles towards a subdiffusive-diffusive population. These studies report for the first time the effect of the I-PC on the mucodiffusion behaviour of vesicular systems, a finding that may help to comprehend the performance of nanocarriers under intestinal conditions.

Vitamin transporters in mice brain with aging.

Its high metabolic rate and high polyunsaturated fatty acid content make the brain very sensitive to oxidative damage. In the brain, neuronal metabolism occurs at a very high rate and generates considerable amounts of reactive oxygen species and free radicals, which accumulate inside neurons, leading to altered cellular homeostasis and integrity and eventually irreversible damage and cell death. A misbalance in redox metabolism and the subsequent neurodegeneration increase throughout the course of normal aging, leading to several age-related changes in learning and memory as well as motor functions. The neuroprotective function of antioxidants is crucial to maintain good brain homeostasis and adequate neuronal functions. Vitamins E and C are two important antioxidants that are taken up by brain cells via the specific carriers αTTP and SVCT2, respectively. The aim of this study was to use immunohistochemistry to determine the distribution pattern of these vitamin transporters in the brain in a mouse model that shows fewer signs of brain aging and a higher resistance to oxidative damage. Both carriers were distributed widely throughout the entire brain in a pattern that remained similar in 4-, 12-, 18- and 24-month-old mice. In general, αTTP and SVCT2 were located in the same regions, but they seemed to have complementary distribution patterns. Double-labeled cell bodies were detected only in the inferior colliculus, entorhinal cortex, dorsal subiculum, and several cortical areas. In addition, the presence of αTTP and SVCT2 in neurons was analyzed using double immunohistochemistry for NeuN and the results showed that αTTP but not SVCT2 was present in Bergmann's glia. The presence of these transporters in brain regions implicated in learning, memory and motor control provides an anatomical basis that may explain the higher resistance of this animal model to brain oxidative stress, which is associated with better motor performance and learning abilities in old age.

PEGylated Nanoemulsions for Oral Delivery: Role of the Inner Core on the Final Fate of the Formulation.

Since the past decade, there has been growing interest to grant nanoparticles with diffusion properties across mucosae. In this sense, the nonionic block copolymer Pluronic F127 (PF127) has emerged as a promising coating agent to formulate mucus-penetrating particles. In the journey to find efficient coating agents, researchers have focused more on the effect of the coating agent architecture rather than on the role of the physicochemical properties of the nanoparticle used as the substrate. The current knowledge about mucodiffusive particles is in general based on model-like nanoparticles, such as polystyrene or poly(lactic-co-glycolic) acid nanoparticles, but there is a lack of information about the potential of PF127 on other colloidal systems. This work aims to shed some light on this issue by selecting three oils, palm (solid), coconut (semisolid), and wheat germ (liquid), with different physicochemical properties to formulate PF127-coated nanoemulsions. The obtained nanoemulsions were characterized, and their colloidal stability was tested. Their diffusion capacity was determined by particle tracking after challenging the nanoemulsions across an intestinal porcine mucus layer. In accordance with the evidence of model-like nanoparticles, our results state that PF127 allows mucodiffusion, but its effectiveness as a coating agent clearly depends on the physicochemical properties of the nanostructure core over which PF127 is placed. Among other physicochemical properties, the results certainly showed that the hydrophobic character of the nanostructure core emerges as a critical factor in the formulation of successful PF127 coatings.

Improving green enrichment of virgin olive oil by oregano. Effects on antioxidants.

This work is about improvement of a maceration method in order to achieve a green process for the enrichment of virgin olive oil (VOO) with natural antioxidants, specifically from oregano leaves. This goal was accomplished after evaluating different mechanical methods, i.e. magnetic stirring, sonication, vertical stirring and sonication in combination with vertical stirring, for promoting the extraction of the antioxidants from oregano. The results obtained indicated that the best extraction procedure was vertical stirring at 1000 r.p.m. for 3 h. Therefore, these conditions were selected to enrich VOO with phenolic acids (mainly rosmarinic acid) and endogenous antioxidants (o-coumaric and vanillic acids), and further determine their stability at room temperature or under temperature stress (50°C) during 45 days. Quantitative analysis of rosmarinic, o-coumaric and vanillic acids was carried out by an off-line, solid phase extraction, capillary zone, electrophoresis method combined with diode-array detector (SPE-CE-DAD).

Developmental study of vitamin C distribution in children's brainstems by immunohistochemistry.

Vitamin C (Vit C) is an important antioxidant, exerts powerful neuroprotective brain effects and plays a role in neuronal development and maturation. Vit C is present in brain tissue at higher concentrations than in other organs, but its detailed distribution in brain is unknown. Immunohistochemical detection of this vitamin has been performed by using a highly specific antibody against Vit C. The aim of the present work was to analyze the distribution of Vit C in children's brainstems during postnatal development, comparing two groups of ages: younger and older than one year of life. In general, the same areas showing neurons with Vit C in young cases are also immunostained at older ages. The distribution of neurons containing Vit C was broader in the brainstems of older children, suggesting that brainstem neurons maintain or even increase their ability to retain Vit C along the life span. Immunohistochemical labeling revealed only cell bodies containing this vitamin, and no immunoreactive fibers were observed. The distribution pattern of Vit C in children's brainstems suggests a possible role of Vit C in brain homeostatic regulation. In addition, the constant presence of Vit C in neurons of locus coeruleus supports the important role of Vit C in noradrenaline synthesis, which seemed to be maintained along postnatal development.

Design of the interface of edible nanoemulsions to modulate the bioaccessibility of neuroprotective antioxidants.

Most frequently the use of bioactive molecules for the supplementation of food and beverages is hampered by stability limitations or inadequate intestinal absorption. This work evaluates in vitro the role that the interface of the nanoemulsion has on the physicochemical properties, the stability behavior and the enzymatic degradation after oral intake. For that purpose three soybean oil (SB) formulations were studied. These formulations were based on the emulsifier lecithin but modified with two non-ionic surfactants Pluronic(®) F68 (PF68) or Pluronic(®) F127 (PF127) yielding (i) SB-NE (only lecithin on the interface), (ii) SB-NE PF68 (lecithin plus PF68) and 9 (iii) SB-NE PF127 (lecithin plus PF127). All the formulations tested were low polydispersed and showed a size of about 200 nm and ζ-potential of -50 mV. The in vitro colloidal stability assay showed that lecithin itself was able to promote that formulations reach unaltered to the small intestine and facilitate the absorption of the antioxidant payload on a tunable fashion there (with in vitro bioaccessibility values from around 40% up to a 70%). PF68 was able to sterically stabilize the formulation against the aggregation induced by the pH and electrolytes of the simulated gastrointestinal track; however, this surfactant was easily displaced by the lipases of the simulated intestinal milieu being unable to modulate the digestion pattern of the oil droplets in the small intestine. Finally, PF127 displayed a strong steric potential that dramatically reduced the interaction of the oil droplets with lipases in vitro, which will compromise the capacity of the formulation to improve the bioaccessibility of the loaded antioxidant.

Distribution of peptidergic populations in the human dentate gyrus (somatostatin [SOM-28, SOM-12] and neuropeptide Y [NPY]) during postnatal development.

The postnatal development of the human hippocampal formation establishes the time and place at which we start autobiographical memories. However, data concerning the maturation of the neurochemical phenotypes characteristic of interneurons in the human hippocampus are scarce. We have studied the perinatal and postnatal changes of the dentate gyrus (DG) interneuron populations at three rostrocaudal levels. Immunohistochemically identified neurons and fibers for somatostatin (SOM-12 and SOM-28) and neuropeptide Y (NPY) and the co-localization of SOM-28 and NPY were analyzed. In total, 13 cases were investigated from late pregnancy (1 case), perinatal period (6 cases), first year (1 case), early infancy (3 cases), and late infancy (2 cases). Overall, the pattern of distribution of these peptides in the DG was similar to that of the adult. The distribution of cells was charted, and the cell density (number of positive cells/mm(2)) was calculated. The highest density corresponded to the polymorphic cell layer and was higher at pre- and perinatal periods. At increasing ages, neuron density modifications revealed a decrease from 5 postnatal months onward. In contrast, by late infancy, two immunoreactive bands for SOM-28 and NPY in the molecular layer were much better defined. Double-immunohistochemistry showed that NPY-positive neurons co-localized with SOM-28, whereas some fibers contained only one or other of the neuropeptides. Thus, this peptidergic population, presumably inhibitory, probably has a role in DG maturation and its subsequent functional activity in memory processing.

Developmental study of the distribution of hypoxia-induced factor-1 alpha and microtubule-associated protein 2 in children's brainstem: comparison between controls and cases with signs of perinatal hypoxia.

Perinatal asphyxia and hypoxia are common causes of morbidity in neonates. Prenatal birth associated with hypoxemia often results in several disorders because of the lack of oxygen in the brain. Survival rates from perinatal hypoxia have improved, but appropriate treatments for recovery are still limited, with great impact on patients, their families, society in general and health systems. The aim of this work is to contribute to a better understanding of the cellular mechanisms underlying the brainstem responses to hypoxia. For this purpose, distributions of two proteins, hypoxia-inducible factor-1 alpha (HIF-1α) and microtubule-associated protein 2 (MAP-2) were analyzed in brainstems of 11 children, four of them showing neuropathological evidence of brain hypoxia. They were included in control or hypoxic groups, and then in several subgroups according to their age. Immunohistochemical labeling for these proteins revealed only cell bodies containing HIF-1α, and both cell bodies and fibers positive for MAP-2 in the children's brainstems. The distribution of HIF-1α was more restricted than that of MAP-2, and it can be suggested that the expression of HIF-1α increased with age. The distribution pattern of MAP-2 in the medulla oblongata could be more due to age-related changes than to a response to hypoxic damage, whereas in the pons several regions, such as the nucleus ambiguus or the solitary nucleus, showed different immunolabeling patterns in controls and hypoxic cases. The distribution patterns of these two proteins suggest that some brainstem regions, such as the reticular formation or the central gray, could be less affected by conditions of hypoxia.

Mapping of tyrosine hydroxylase in the diencephalon of alpaca (Lama pacos) and co-distribution with somatostatin-28 (1-12).

Based on previous work describing the distribution of somatostatin-28 (1-12) in the male alpaca (Lama pacos) diencephalon, and owing to the well known interactions between this peptide and the catecholaminergic system, the aims of this work are (1) to describe the distribution of putative catecholaminergic cell groups in the alpaca diencephalon and (2) to study the possible morphological basis of the interactions between these substances in the diencephalon of the alpaca by using double immunohistochemistry methods. Thus, the distribution of catecholaminergic cell groups in the alpaca diencephalon agrees with that previously described in the diencephalon of other mammalian species of the same order: the A11, A12, A13, A14 and A15d cell groups have been identified; however, we have observed an additional hitherto undescribed cell group containing tyrosine hydroxylase in the medial habenula. In addition, double-labelling procedures did not reveal neurons containing tyrosine hydroxylase and somatostatin, suggesting that the hypothalamic interactions between catecholamines and somatostatin at intra-cellular level must be carried out by a somatostatin molecule other than fragment (1-12). Otherwise, the overlapping distribution patterns of these substances would suggest some interconnections between groups of chemospecific neurons. These results could be the starting point for future studies on hypothalamic functions in alpacas, for example those concerning reproductive control, since other physiological studies have suggested that this species could have different regulatory mechanisms from other mammalian species. Our results support the Manger hypothesis that the same nuclear complement of neural systems exists in the brain of species of the same order.

Mapping of tyrosine hydroxylase in the alpaca (Lama pacos) brainstem and colocalization with CGRP.

The distribution of tyrosine hydroxylase (TH) in the brainstem of alpaca (Lama pacos) has been analysed using immunohistochemical methods. The following catecholaminergic cell nuclei have been detected: A1, C1, A2, C2 and area postrema in the medulla oblongata; A5, A6d, A7sc and A7d in the pons; as have several mesencephalic groups: A8, A9l, A9m, A9v, A9pc, A10, A10c, A10d and A10dc. This nuclear parcellation differs from that found in rodents, but agrees with the results reported in other members of the Artiodactyla order, such as giraffe or pig, and with the catecholaminergic distribution detected in species of other mammalian orders. Thus, these findings support the hypothesis that the animals included in the same order show the same nuclear complement in the neuromodulatory systems. In addition, it seems that other species share the same catecholaminergic groups as the alpaca, suggesting that a specific nuclear disposition was important and worth maintaining throughout evolution. Moreover, the distribution of TH has been compared with that of CGRP by double immunohistochemistry. Double-labelled neurons were very isolated and observed only in a few catecholaminergic groups: A1 and C2 in the medulla oblongata, A6d, A7sc and A7d in the pons, and A9l in the mesencephalon. However, interaction between TH and CGRP may be possible in more brainstem regions, particularly the area postrema. This interaction may prove important in the regulation of the specific cardiovascular control of alpacas given their morphological characteristics.

Convergence of unimodal and polymodal sensory input to the entorhinal cortex in the fascicularis monkey.

The hippocampal formation is a key structure in memory formation and consolidation. The hippocampus receives information from different cortical and subcortical sources. Cortical information is mostly funneled to the hippocampus through the entorhinal cortex (EC) in a bi-directional way that ultimately ends in the cortex. Retrograde tracing studies in the nonhuman primate indicate that more than two-thirds of the cortical afferents to the EC come from polymodal sensory association areas. Although some evidence for the projection from visual unimodal cortex to the EC exists, inputs from other visual and auditory unimodal association areas, and the possibility of their convergence with polymodal input in the EC remains largely undisclosed. We studied 10 Macaca fascicularis monkeys in which cortical deposits of the anterograde tracer biotinylated dextran-amine were made into different portions of visual and auditory unimodal association cortices in the temporal lobe, and in polymodal association cortex at the upper bank of the superior temporal sulcus. Visual and auditory unimodal as well as polymodal cortical areas projected to the EC. Both visual unimodal and polymodal association cortices presented dense projections, while those from unimodal auditory association cortex were more patchy and less dense. In all instances, the projection distributed in both the superficial and deep layers of the EC. However, while polymodal cortex projected to all layers (including layer I), visual unimodal cortex did not project to layer I, and auditory unimodal cortex projected less densely, scattered through all layers. Topographically, convergence from the three cortical areas studied can be observed in the lateral rostral and lateral caudal subfields. The present study suggests that unimodal and polymodal association cortical inputs converge in the lateral EC, thereby providing the possibility for the integration of complex stimuli for internal representations in declarative memory elaboration.

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

Hippocampal formation plays a prominent role in episodic memory formation and consolidation. It is likely that episodic memory representations are constructed from cortical information that is mostly funnelled through the entorhinal cortex to the hippocampus. The entorhinal cortex returns processed information to the neocortex. Retrograde tracing studies have shown that neocortical afferents to the entorhinal cortex originate almost exclusively in polymodal association cortical areas. However, the use of retrograde studies does not address the question of the laminar and topographical distribution of cortical projections within the entorhinal cortex. We examined material from 60 Macaca fascicularis monkeys in which cortical deposits of either (3)H-amino acids or biotinylated dextran-amine as anterograde tracers were made into different cortical areas (the frontal, cingulate, temporal and parietal cortices). The various cortical inputs to the entorhinal cortex present a heterogeneous topographical distribution. Some projections terminate throughout the entorhinal cortex (afferents from medial area 13 and posterior parahippocampal cortex), while others have more limited termination, with emphasis either rostrally (lateral orbitofrontal cortex, agranular insular cortex, anterior cingulate cortex, perirhinal cortex, unimodal visual association cortex), intermediate (upper bank of the superior temporal sulcus, unimodal auditory association cortex) or caudally (parietal and retrosplenial cortices). Many of these inputs overlap, particularly within the rostrolateral portion of the entorhinal cortex. Some projections were directed mainly to superficial layers (I-III) while others were heavier to deep layers (V-VI) although areas of dense projections typically spanned all layers. A primary report will provide a detailed analysis of the regional and laminar organization of these projections. Here we provide a general overview of these projections in relation to the known neuroanatomy of the entorhinal cortex.

Reciprocal connections between olfactory structures and the cortex of the rostral superior temporal sulcus in the Macaca fascicularis monkey.

Convergence of sensory modalities in the nonhuman primate cerebral cortex is still poorly understood. We present an anatomical tracing study in which polysensory association cortex located at the fundus and upper bank of the rostral superior temporal sulcus presents reciprocal connections with primary olfactory structures. At the same time, projections from this polysensory area reach multiple primary olfactory centres. Retrograde (Fast Blue) and anterograde (biotinylated dextran-amine and 3H-amino acids) tracers were injected into primary olfactory structures and rostral superior temporal sulcus. Retrograde tracers restricted to the anterior olfactory nucleus resulted in labelled neurons in the rostral portion of the upper bank and fundus of superior temporal sulcus. Injections of biotinylated dextran-amine at the fundus and upper bank of the superior temporal sulcus confirmed this projection by labelling axons in the dorsal and lateral portions of the anterior olfactory nucleus, as well as piriform, periamygdaloid and entorhinal cortices. Retrograde tracer injections at the rostral superior temporal sulcus resulted in neuronal labelling in the anterior olfactory nucleus, piriform, periamygdaloid and entorhinal cortices, thus providing confirmation of the reciprocity between primary olfactory structures and the cortex at the rostral superior temporal sulcus. The reciprocal connections between the rostral part of superior temporal sulcus and primary olfactory structures represent a convergence for olfactory and other sensory modalities at the cortex of the rostral temporal lobe.

Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors.

The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the alpha4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the alpha4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125I alpha-conotoxin MII. These receptors are comprised of the alpha4, beta2 and alpha6 nAChR subunits and non-alpha4, beta2, and alpha6 nAChR subunits. Non-alpha4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and alpha4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and non-habituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that alpha4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs.

Postnatal development of calcium-binding proteins immunoreactivity (parvalbumin, calbindin, calretinin) in the human entorhinal cortex.

The entorhinal cortex is an essential component in the organization of the human hippocampal formation related to cortical activity. It transfers, neocortical information (ultimately distributed to the dentate gyrus and hippocampus) and receives most of the hippocampal output directed to neocortex. At birth, the human entorhinal cortex presents similar layer organization as in adults, although layer II (cell islands) and upper layer III have a protracted maturation. The presence of interneurons expressing calcium-binding proteins (parvalbumin, calbindin-D28K (calbindin) and calretinin) is well documented in the adult human entorhinal cortex. In many of them the calcium binding is co-localized with GABA. Parvalbumin-immunoreactive cells and fibers were virtually absent at birth, their presence increasing gradually in deep layer III, mostly in the lateral and caudal portions of the entorhinal cortex from the 5th month onwards. Calbindin immunoreactive cells and fibers were present at birth, mainly in layers II and upper III; mostly at rostral and lateral portions of the entorhinal cortex, increasing in number and extending to deep layers from the 5th month onwards. Calretinin immunoreactivity was present at birth, homogeneously distributed over layers I, II and upper V, throughout the entorhinal cortex. A substantial increase in the number of calretinin neurons in layer V was observed at the 5th month. The postnatal development of parvalbumin, calbindin and calretinin may have an important role in the functional maturation of the entorhinal cortex through the control of hippocampal, cortical and subcortical information.

Comparative aspects of the olfactory portion of the entorhinal cortex and its projection to the hippocampus in rodents, nonhuman primates, and the human brain.

The entorhinal cortex is a component of the hippocampal formation characterized by its projection to the dentate gyrus. Cortical input is relayed to the hippocampus through the entorhinal cortex. The entorhinal cortex can be recognized best in mammals, and it reaches its maximal importance in both human and nonhuman primates. In primates, but not in rodents, the olfactory projection from the olfactory bulb is the only direct sensory projection reaching the entorhinal cortex, where it defines a rostromedial subfield (olfactory subfield of the entorhinal cortex, EO). In this communication, we consider some comparative aspects of this projection in rodents, nonhuman primates, and human brains, in relation to the forward projection to the hippocampus. We base our analysis on the following three considerations: (1) Topology: Topologically, the entorhinal cortex lies medial to the rhinal fissure, well developed in all mammals except in humans. EO maintains the same topological relationship to the rhinal fissure in the three mammalian orders. (2) Cytoarchitecture: The rostromedial portion shows poorer lamination compared with caudomedial portions of the entorhinal cortex in the three mammalian orders examined. (3) Connectivity: In rodents, the olfactory projection covers the whole extent of the entorhinal cortex, whereas it decreases substantially in macaques, where it roughly covers 15% of the entorhinal cortex. The human EO as defined by topological and cytoarchitectonic criteria may be even less than in macaques. Interestingly, good evidence exists that all of these mammalian orders maintain the same topographical scheme of projection to the rostral end of the hippocampus.

Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell populations in the central nervous system.

The KAL gene is responsible for the X-chromosome linked form of Kallmann's syndrome in humans. Upon transfection of CHO cells with a human KAL cDNA, the corresponding encoded protein, KALc, was produced. This protein is N-glycosylated, secreted in the cell culture medium, and is localized at the cell surface. Several lines of evidence indicate that heparan-sulfate chains of proteoglycan(s) are involved in the binding of KALc to the cell membrane. Polyclonal and monoclonal antibodies to the purified KALc were generated. They allowed us to detect and characterize the protein encoded by the KAL gene in the chicken central nervous system at late stages of embryonic development. This protein is synthesized by definite neuronal cell populations including Purkinje cells in the cerebellum, mitral cells in the olfactory bulbs and several subpopulations in the optic tectum and the striatum. The protein, with an approximate molecular mass of 100 kDa, was named anosmin-1 in reference to the deficiency of the sense of smell which characterizes the human disease. Anosmin-1 is likely to be an extracellular matrix component. Since heparin treatment of cell membrane fractions from cerebellum and tectum resulted in the release of the protein, we suggest that one or several heparan-sulfate proteoglycans are involved in the binding of anosmin-1 to the membranes in vivo.