Differential accumulation of human ß-amyloid and tau from enriched extracts in neuronal and endothelial cells.

While Aß and Tau cellular distribution has been largely studied, the comparative internalization and subcellular accumulation of Tau and Aß isolated from human brain extracts in endothelial and neuronal cells has not yet been unveiled. We have previously demonstrated that controlled enrichment of Aß from human brain extracts constitutes a valuable tool to monitor cellular internalization in vitro and in vivo. Herein, we establish an alternative method to strongly enrich Aß and Tau aggregates from human AD brains, which has allowed us to study and compare the cellular internalization, distribution and toxicity of both proteins within brain barrier endothelial (bEnd.3) and neuronal (Neuro2A) cells. Our findings demonstrate the suitability of human enriched brain extracts to monitor the intracellular distribution of human Aß and Tau, which, once internalized, show dissimilar sorting to different organelles within the cell and differential toxicity, exhibiting higher toxic effects on neuronal cells than on endothelial cells. While tau is strongly concentrated preferentially in mitochondria, Aß is distributed predominantly within the endolysosomal system in endothelial cells, whereas the endoplasmic reticulum was its preferential location in neurons. Altogether, our findings display a picture of the interactions that human Aß and Tau might establish in these cells.

Neurogenesis in subclasses of vomeronasal sensory neurons in adult mice.

The vomeronasal sensory epithelium contains two distinct populations of vomeronasal sensory neurons. Apical neurons express G(i) (2) (α) -linked V1R vomeronasal receptors and project to the anterior portion of the accessory olfactory bulb, while basal neurons express G(o) (α) -linked V2R receptors and project to the posterior portion. Sensory neurons expressing V1R and V2R vomeronasal receptors are sensitive to different stimuli. Neurons in the vomeronasal system undergo continuous cell turnover during adulthood. To analyze over time neurogenesis of the different sensory cell populations, adult mice were injected with bromodeoxyuridine (BrdU) and sacrificed at postinjection days 1, 3, 5, 7, and 11. Newborn vomeronasal neurons were revealed by antibodies against BrdU while subclasses of vomeronasal neurons were identified using antibodies against G(o) (α) or G(i) (2) (α) proteins. To ascertain whether G proteins are early expressed during neurogenesis, multiple labeling experiments using PSA-NCAM and doublecortin were performed. Distribution of BrdU-labeled cells was analyzed in angular segments from the margin of the sensory epithelium. No sexual differences were found. Within survival groups, BrdU-G(o) (α) labeled cells were found more marginally when compared with BrdU-G(i) (2) (α) labeled cells. The number of BrdU-positive cells decreased from day 1 to day 3 to remain constant afterwards. The relative proportions of BrdU-G(i) (2) (α) and BrdU-G(o) (α) labeled cells remained similar and constant from postinjection day 1 onwards. This rate was also comparable with BrdU-positive cells starting day 3. These results indicate an early, constant, and similar rate of neurogenesis in the two major subclasses of vomeronasal neurons, which suggests that both cell populations maturate independently.

Somatostatin, tau, and beta-amyloid within the anterior olfactory nucleus in Alzheimer disease.

Impaired olfaction is an early symptom of Alzheimer disease (AD). This likely to reflect neurodegenerative processes taking place in basal telencephalic structures that mediate olfactory processing, including the anterior olfactory nucleus. Betaeta-amyloid (Abeta) accumulation in AD brain may relate to decline in somatostatin levels: somatostatin induces the expression of the Abeta-degrading enzyme neprilysin and somatostatin deficiency in AD may therefore reduce Abeta clearance. We have investigated the expression of somatostatin in the anterior olfactory nucleus of AD and control brain. We report that somatostatin levels were reduced by approximately 50% in AD brain. Furthermore, triple-immunofluorescence revealed co-localization of somatostatin expression with Abeta (65.43%) with Abeta and tau (19.75%) and with tau (2.47%). These data indicate that somatostatin decreases in AD and its expression may be linked with Abeta deposition.

Fate of marginal neuroblasts in the vomeronasal epithelium of adult mice.

Chemical stimuli are sensed through the olfactory and vomeronasal epithelia, and the sensory cells of both systems undergo neuronal turnover during adulthood. In the vomeronasal epithelium, stem cells adjacent to the basal lamina divide and migrate to replace two classes of sensory neurons: apical neurons that express G(i2alpha)-linked V1R vomeronasal receptors and project to the anterior accessory olfactory bulb, and basal neurons that express G(oalpha)-linked V2R receptors and project to the posterior accessory olfactory bulb. Most of the dividing cells are present in the margins of the epithelium and only migrate locally. Previous studies have suggested that these marginal cells may participate in growth, sensory cell replacement or become apoptotic before maturation; however, the exact fate of these cells have remained unclear. In this work we investigated the fate of these marginal cells by analyzing markers of neurogenesis (bromodeoxyuridine incorporation), apoptosis (caspase-3), and neuronal maturation (olfactory marker protein and Neurotrace Nissl stain). Our data reveal a pool of dividing cells in the epithelial margins that predominantly give rise to mature neurons and only rarely undergo apoptosis. Newly generated cells are several times more numerous than apoptotic cells. These marginal neuroblasts could therefore constitute a net neural addition zone during adulthood.

Subicular and CA1 hippocampal projections to the accessory olfactory bulb.

The hippocampal formation is anatomically and functionally related to the olfactory structures especially in rodents. The entorhinal cortex (EC) receives afferent projections from the main olfactory bulb; this constitutes an olfactory pathway to the hippocampus. In addition to the olfactory system, most mammals possess an accessory olfactory (or vomeronasal) system. The relationships between the hippocampal formation and the vomeronasal system are virtually unexplored. Recently, a centrifugal projection from CA1 to the accessory olfactory bulb has been identified using anterograde tracers. In the study reported herein, experiments using anterograde tracers confirm this projection, and injections of retrograde tracers show the distribution and morphology of a population of CA1 and ventral subicular neurons projecting to the accessory olfactory bulb of rats. These results extend previous descriptions of hippocampal projections to the accessory olfactory bulb by including the ventral subiculum and characterizing the morphology, neurochemistry (double labeling with somatostatin), and distribution of such neurons. These data suggest feedback hippocampal control of chemosensory stimuli in the accessory olfactory bulb. Whether this projection processes spatial information on conspecifics or is involved in learning and memory processes associated with chemical stimuli remains to be elucidated.

Vomeronasal inputs to the rodent ventral striatum.

Vertebrates sense chemical signals through the olfactory and vomeronasal systems. In squamate reptiles, which possess the largest vomeronasal system of all vertebrates, the accessory olfactory bulb projects to the nucleus sphericus, which in turn projects to a portion of the ventral striatum known as olfactostriatum. Characteristically, the olfactostriatum is innervated by neuropeptide Y, tyrosine hydroxylase and serotonin immunoreactive fibers. In this study, the possibility that a structure similar to the reptilian olfactostriatum might be present in the mammalian brain has been investigated. Injections of dextran-amines have been aimed at the posteromedial cortical amygdaloid nucleus (the putative mammalian homologue of the reptilian nucleus sphericus) of rats and mice. The resulting anterograde labeling includes the olfactory tubercle, the islands of Calleja and sparse terminal fields in the shell of the nucleus accumbens and ventral pallidum. This projection has been confirmed by injections of retrograde tracers into the ventral striato-pallidum that render retrograde labeling in the posteromedial cortical amygdaloid nucleus. The analysis of the distribution of neuropeptide Y, tyrosine hydroxylase, serotonin and substance P in the ventral striato-pallidum of rats, and the anterograde tracing of the vomeronasal amygdaloid input in the same material confirm that, similar to reptiles, the ventral striatum of mammals includes a specialized vomeronasal structure (olfactory tubercle and islands of Calleja) displaying dense neuropeptide Y-, tyrosine hydroxylase- and serotonin-immunoreactive innervations. The possibility that parts of the accumbens shell and/or ventral pallidum could be included in the mammalian olfactostriatum cannot be discarded.

Cell migration to the anterior and posterior divisions of the granule cell layer of the accessory olfactory bulb of adult opossums.

To test the hypothesis that differential addition of new cells occurs in the two functionally distinct divisions of the accessory olfactory bulb (AOB), the addition of bromodeoxyuridine-labeled cells was analyzed in the anterior and posterior subdivisions of the AOB as a function of post-injection survival time (0-11 days). One week postinjection, an increase of cells was detected in the granule layer but not in the glomerular or mitral/tufted cell layers. No evidence for differential addition of cells to the anterior and posterior divisions was observed.

Neural substrates for tongue-flicking behavior in snakes.

Snakes deliver odorants to the vomeronasal organ by means of tongue-flicks. The rate and pattern of tongue-flick behavior are altered depending on the chemical context. Accordingly, olfactory and vomeronasal information should reach motor centers that control the tongue musculature, namely, the hypoglossal nucleus (XIIN); however, virtually nothing is known about the circuits involved. In the present work, dextran amines were injected into the tongue of garter snakes (Thamnophis sirtalis) to identify the motoneurons of the XIIN. Tracers were then delivered into the XIIN to identify possible afferents of chemical information. Large injections into the XIIN yielded retrograde labeling in two chemosensory areas: the medial amygdala (MA) and the lateral posterior hypothalamic nucleus (LHN). Smaller injections only yielded labeled neurons in the LHN. In fact, the MA, which receives afferents from the accessory olfactory bulb, the rostroventral lateral cortex, and the nucleus sphericus, projects to the LHN. Injections into the MA did not show terminal labeling in the XIIN but in an area lateral to it. However, injections into the LHN gave rise not only to labeled fibers in the XIIN but also to retrograde labeling in the MA, thus confirming the chemosensory input to LHN. Injecting different fluorescent tracers into the tongue and into the LHN corroborated the projection from the LHN to the XIIN. The present report investigates further connections of the olfactory and vomeronasal systems and describes the afferent connections to XIIN in a nonmammalian vertebrate. The circuit for tongue-flicking behavior described herein should be evaluated using functional studies.

Neurogenesis in the vomeronasal epithelium of adult rats: evidence for different mechanisms for growth and neuronal turnover.

The pattern of cell migration during neuronal turnover in the vomeronasal sensory epithelium (VN-SE) is controversial. In mice, proliferating cells were detected at the edges and were described as migrating to the center of the VN-SE. In rats, in addition to proliferating cells at the margins of the epithelium, dividing cells are also present along the entire basal lamina of the VN-SE. In marsupials, dividing cells have also been observed in the margins and in the center of the VN-SE, the latter of which migrate vertically and become neurons. To investigate whether the process of neuronal turnover in placental mammals consists of horizontal and/or vertical migration, and whether or not this process is common to mammals, adult rats were injected with bromodeoxyuridine (BrdU) and allowed to survive for different periods of time. The distribution of BrdU-labeled cells in the horizontal and vertical dimension of the VN-SE was analyzed as a function of time. Both horizontal and vertical migrations of BrdU-labeled cells were detected. Since cells in the center of the VN-SE migrate vertically, and, as demonstrated by coexpression of markers of neuronal maturity and BrdU, become mature one day after undergoing mitosis, it is very likely that these cells participate in neuronal turnover. Conversely, because cells in the margins of the VN-SE stop migrating horizontally on day 14 before they have reached the center of the VN-SE, and since the VN-SE continues to grow during adulthood, it is likely that most of these latter cells constitute pools for growth.

Cell turnover in the vomeronasal epithelium: evidence for differential migration and maturation of subclasses of vomeronasal neurons in the adult opossum.

Previous investigations of cell turnover in the mammalian vomeronasal sensory epithelium (VN-SE) raised two issues. First, if, in addition to the already demonstrated vertical migration, horizontal migration from the edges of the VN-SE participates in neuronal replacement. Second, whether or not migration and maturation is differential in upper and lower populations of vomeronasal neurons, since these two cell populations are chemically, physiologically, functionally, and perhaps evolutionarily different. By injecting bromodeoxyuridine (BrdU) into adult opossum (Monodelphis domestica) and permitting different survival times, the pattern of distribution of BrdU-labeled cells was analyzed. No evidence of horizontal migration in neuronal replacement was found. To investigate vertical migration and maturation of subclasses of vomeronasal neurons, double immunohistochemistry of BrdU and markers of the lower (G(oalpha) protein) and upper [G(i2alpha) protein and olfactory marker protein (OMP)] cell populations were performed. Three days after administration of BrdU, some mature neurons were observed in both lower and upper layers of the VN-SE, as demonstrated by coexpression of BrdU with G(oalpha) protein and OMP, respectively. The data on vertical distribution, however, indicate that most of the daughter cells enter the G(oalpha)-protein-expressing zone of the VN-SE by day 5, whereas most daughter cells do not reach the G(i2alpha)-protein-expressing zone until day 7, suggesting that these two populations mature at slightly different rates. These results are the first evidence of differential neurogenesis of subclasses of vomeronasal neurons.