The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation.

The monoamine serotonin (5-HT), a well-known neurotransmitter, is also important in peripheral tissues. Several studies have suggested that 5-HT is involved in bone metabolism. Starting from our original observation of increased 5-HT(2B) receptor (5-HT(2B)R) expression during in vitro osteoblast differentiation, we investigated a putative bone phenotype in vivo in 5-HT(2B)R knockout mice. Of interest, 5-HT(2B)R mutant female mice displayed reduced bone density that was significant from age 4 months and had intensified by 12 and 18 months. This histomorphometrically confirmed osteopenia seems to be due to reduced bone formation because 1) the alkaline phosphatase-positive colony-forming unit capacity of bone marrow precursors was markedly reduced in the 5-HT(2B)R mutant mice from 4 to 12 months of age, 2) ex vivo primary osteoblasts from mutant mice exhibited reduced proliferation and delayed differentiation, and 3) calcium incorporation was markedly reduced in osteoblasts after 5-HT(2B)R depletion (produced genetically or by pharmacological inactivation). These findings support the hypothesis that the 5-HT(2B)R receptor facilitates osteoblast recruitment and proliferation and that its absence leads to osteopenia that worsens with age. We show here, for the first time, that the 5-HT(2B)R receptor is a physiological mediator of 5-HT in bone formation and, potentially, in the onset of osteoporosis in aging women.

Over-expression of TIMP-1 in osteoblasts increases the anabolic response to PTH.

Intermittent PTH treatment induces structural changes that affect cancellous bone mass and have led to its indication for the treatment of osteoporosis. PTH is also known to upregulate the expression of matrix metalloproteinases (MMP) in osteoblasts. We wanted to find out whether inhibiting osteoblastic MMPs can affect the anabolic action of PTH in vivo. We had shown previously that mice over-expressing TIMP-1 (tissue inhibitor of MMPs) specifically in osteoblasts display an increase in bone mineral density and bone mass combined with an overall decrease in bone turnover. In the present study, 10-week-old wild-type (WT) and transgenic (TG) mice were treated with PTH at 40 microg/kg/day for 1.5 months. DEXA analysis was performed before and after treatment, and histomorphometric and molecular analysis were carried out at the end of the experiment. Our findings indicate that the transgene boosted the anabolic action of PTH. The femurs of PTH-treated TG mice displayed a greater increase in bone mineral density and trabecular bone volume than treated WT mice. Interestingly, the positive effect of the transgene on the action of PTH resulted from both reduced bone resorption activity and an increase in the bone formation rate. Osteoclastic surfaces that were increased in PTH-treated WT mice remained unchanged in TG mice, suggesting a decrease in osteoclastic differentiation. Histomorphometric data also indicate that PTH administration increased osteoblast activity in TG mice and affected the number of osteoblasts in WT mice. In conclusion, we demonstrate that inhibiting osteoblastic MMPs can potentiate the anabolic effect of PTH by decreasing osteoclast activity and increasing osteoblast activity. Our data also suggest that osteoblastic MMPs have some role in mediating the anabolic effects of PTH in vivo and indicate that inhibitors of MMPs could constitute a new therapy for degenerative diseases.

How does the brain discriminate familiar and unfamiliar faces?: a PET study of face categorical perception.

Where and how does the brain discriminate familiar and unfamiliar faces? This question has not been answered yet by neuroimaging studies partly because different tasks were performed on familiar and unfamiliar faces, or because familiar faces were associated with semantic and lexical information. Here eight subjects were trained during 3 days with a set of 30 faces. The familiarized faces were morphed with unfamiliar faces. Presented with continua of unfamiliar and familiar faces in a pilot experiment, a group of eight subjects presented a categorical perception of face familiarity: there was a sharp boundary in percentage of familiarity decisions between 40% and 60% faces. In the main experiment, subjects were scanned (PET) on the fourth day (after 3 days of training) in six conditions, all requiring a sex classification task. Completely novel faces (0%) were presented in Condition 1 and familiar faces (100%) in Condition 6, while faces of steps of 20% in the continuum of familiarity were presented in Conditions 2 to 5 (20% to 80%). A principal component analysis (PCA) indicated that most variations in neural responses were related to the dissociation between faces perceived as familiar (60% to 100%) and faces perceived as unfamiliar (0 to 40%). Subtraction analyses did not disclose any increase of activation for faces perceived as familiar while there were large relative increases for faces perceived as unfamiliar in several regions of the right occipito-temporal visual pathway. These changes were all categorical and were observed mainly in the right middle occipital gyrus, the right posterior fusiform gyrus, and the right inferotemporal cortex. These results show that (1) the discrimination between familiar and unfamiliar faces is related to relative increases in the right ventral pathway to unfamiliar/novel faces; (2) familiar and unfamiliar faces are discriminated in an all-or-none fashion rather than proportionally to their resemblance to stored representations; and (3) categorical perception of faces is associated with abrupt changes of brain activity in the regions that discriminate the two extremes of the multidimensional continuum.

A pet study of human skill learning: changes in brain activity related to learning an orientation discrimination task.

Using 15O-water 3D positron emission tomography we investigated the effect of training in orientation discrimination upon cerebral activity in healthy human adults. When subjects are trained in this discrimination task, they learn the visuo-motor stimulus-response association required by the task and they increase their perceptual abilities in orientation discrimination. The present study was designed to investigate the rCBF modifications related to both these learning processes induced by training in orientation discrimination. PET data were acquired on two separate days (before and after training). Comparing the activation pattern related to orientation discrimination before and after the training period we observed activity decreases located in the left cerebellar cortex, in the right precentral gyrus and bilaterally in the fusiform gyri. The only region showing an activity increase was located in the body of the right caudate nucleus. These findings confirm the role of the neostriatum in skill learning and highlight the importance of mechanisms resulting in cortical and cerebellar neuronal activity decreases in this type of learning.

Enteric neuroblasts require the phosphatidylinositol 3-kinase pathway for GDNF-stimulated proliferation.

The enteric nervous system (ENS) develops from neural crest cells that enter the gut, migrate, proliferate, and differentiate into neurons and glia. The growth factor glial-derived neurotrophic factor (GDNF) stimulates the proliferation and survival of enteric crest-derived cells. We investigated the intracellular signaling pathways activated by GDNF and their involvement in proliferation. We found that GDNF stimulates the phosphorylation of both the PI 3-kinase downstream substrate Akt and the MAP kinase substrate ERK in cultures of immunoaffinity-purified embryonic avian enteric crest-derived cells. The selective PI 3-kinase inhibitor LY-294002 blocked GDNF-stimulated Akt phosphorylation in purified crest cells, and reduced proliferation in cultures of dissociated quail gut. The ERK kinase (MEK) inhibitors PD 98059 and UO126 did not reduce GDNF-stimulated proliferation, although PD 98059 blocked GDNF-stimulated phosphorylation of ERK. We conclude that the PI 3-kinase pathway is necessary for the GDNF-stimulated proliferation of enteric neuroblasts.

Expression of the GDNF receptors ret and GFRalpha1 in the developing avian enteric nervous system.

The formation of the enteric nervous system (ENS) from neural crest-derived cell precursors requires the growth factor glial cell line-derived neurotrophic factor (GDNF) and the receptors Ret and GDNF family receptor alpha 1 (GFRalpha1). We investigated the location(s), the timing, and the extent to which these GDNF receptors appear in the population of crest-derived precursors that form the avian ENS using immunohistochemistry and in situ hybridization. Sections and whole mounts of embryonic chick gastrointestinal tract were costained with antibodies to the receptors and to HNK-1, a marker for crest-derived cells. Neural crest-derived precursors migrate through the primitive esophagus to colonize the gizzard where an extensive cellular network forms. Ret-immunoreactivity (ir) was found in a network of cells in the gizzard at embryonic day (E)3.5. As development proceeded, Ret-immunoreactive cells appeared at progressively more caudal positions and were present in the colon at E7.5. Costaining with Ret and HNK-1 was performed to determine the number of Ret-immunoreactive cells in the crest-derived population. Ret appeared in some HNK-1 cells in the esophagus and gizzard at embryonic day (E)3.5. During development, the number of crest cells with Ret increased in the ganglia of the gizzard and small intestine. GFRalpha1-ir was also found in HNK-1 cells in the esophagus at E3.5 but did not appear in the gizzard until E4.5. Surprisingly, the colonizing vanguard of crest-derived cells lacked both Ret- and GFRalpha-ir. Between E4.5 and E6.5, the fraction of HNK-1-positive cells expressing GFRalpha1 increased considerably in the foregut. Ret and GFRalpha1 were coexpressed in many cells at E6.5, and the number of such cells increased as development progressed. In the adult, GFRalpha1 and Ret were found in the neuropil of enteric ganglia. We conclude that the population of cells expressing the receptors increases during development and persists in the adult, findings that support a neurotrophic role for GDNF in the formation and maintenance of the avian ENS.

Effect of familiarity on the processing of human faces.

Most brain imaging studies on face perception have investigated the processing of unknown faces and addressed mainly the question of specific face processing in the human brain. The goal of this study was to highlight the effects of familiarity on the visual processing of faces. Using [15O]water 3D Positron Emission Tomography, regional cerebral blood flow distribution was measured in 11 human subjects performing an identical task (gender categorization) on both unknown and known faces. Subjects also performed two control tasks (a face recognition task and a visual pattern discrimination task). They were scanned after a training phase using videotapes during which they had been familiarized with and learned to recognize a set of faces. Two major results were obtained. On the one hand, we found bilateral activations of the fusiform gyri in the three face conditions, including the so-called fusiform-face area, a region in the right fusiform gyrus specifically devoted to face processing. This common activation suggests that different cognitive tasks performed on known and unknown faces require the involvement of this fusiform region. On the other hand, specific regional cerebral blood flow changes were related to the processing of known and unknown faces. The left amygdala, a structure involved in implicit learning of visual representations, was activated by the categorization task on unknown faces. The same task on known faces induced a relative decrease of activity in early visual areas. These differences between the two categorization tasks reveal that the human brain processes known and unknown faces differently.

Neuronal mechanisms of perceptual learning: changes in human brain activity with training in orientation discrimination.

Using 15O-water 3D positron emission tomography, regional cerebral blood flow was measured twice in six human subjects: before and after extensive training in orientation discrimination. In each session subjects performed two orientation discrimination tasks, during which they discriminated the orientation of a grating at either the trained or untrained reference orientation, and a control task, during which they detected a randomly textured pattern. By comparing the discrimination to the detection tasks, we observed a main effect of task bilaterally in the posterior occipital cortex, extending into the left posterior fusiform gyrus and the right inferior occipital gyrus, bilaterally in the intraparietal sulcus, as well as in the cerebellum, thalamus, and brainstem. When we compared the activation pattern before and after the training period, all the changes observed were activity decreases. The nonspecific changes, which were not related to the orientation used during the training, were situated in the cerebellum and bilaterally in the extrastriate visual cortex. The orientation-specific changes, on the other hand, were restricted to the striate and extrastriate visual cortex, more precisely the right calcarine sulcus, the left lingual gyrus, the left middle occipital, and the right inferior occipital gyrus. These findings confirm our hypothesis concerning the existence of learning related changes at early levels of visual processing in human adults and suggest that mechanisms resulting in neuronal activity decreases might be involved in the present kind of learning.

PET study of human voluntary saccadic eye movements in darkness: effect of task repetition on the activation pattern.

Using H2(15)O 3D Positron Emission Tomography (PET), regional cerebral blood flow (rCBF) was measured in six human subjects under two different conditions: at rest and while performing self-paced horizontal saccadic eye movements in darkness. These two conditions were repeated four times each. First, the comparison between the four saccadic and four resting conditions was investigated in a group and a single subject analysis. Saccades elicited bilateral rCBF increases in the medial part of the superior frontal gyrus (supplementary eye field), precentral gyrus (frontal eye field), superior parietal lobule, anterior medial part of the occipital lobe involving striate and extrastriate cortex (lingual gyrus and cuneus), and in the right inferior parietal lobule. At the subcortical level, activations were found in the left putamen. These results mainly replicate previous PET findings on saccadic control. Second, the interaction between the experimental conditions and their repetition was examined. When activations throughout repetition of the same saccadic task are compared, the supplementary eye fields show a progressive increase of activation. On the contrary, the activation in the cerebellum, left superior parietal lobule and left occipital cortex progressively decreases during the scanning session. Given the existence of such an interaction, the pattern of activations must be interpreted as a function of task repetition. This may be a factor explaining some apparent mismatch between different studies.

Variation in the inflammatory properties of basic calcium phosphate crystals according to crystal type.

OBJECTIVE: To determine the inflammatory potential of basic calcium phosphate (BCP) crystals, which have been identified in human joints. METHODS: Hydroxyapatite, carbonate apatite, whitlockite, and octacalcium phosphate crystals were injected in rat air pouches. Volume and cellularity of the exudate were measured. Physicochemical properties of the injected BCP crystals were determined, and correlations with the magnitude of induced inflammatory responses were sought. RESULTS: Significant differences were observed among the volumes and white blood cell (WBC) counts of the pouch exudates, based on the various crystal types used to induce inflammation. A strong correlation was demonstrated between the specific surface (SS) area of the injected crystals and the area under the curve for induced WBC count versus time (R2 = 0.88, P = 0.05). This correlation was observed for SS area values below 50 m2/gm, but when SS area increased further, this parameter plateaued. Another parameter of inflammatory response was obtained by dividing the area under the curve figuring WBC counts versus time by the corresponding SS area for each crystal type. This parameter increased linearly with the Ca:P ratio (R2 = 0.97, P = 0.0003). CONCLUSION: The inflammatory potential of BCP crystals appeared to vary according to crystal features. SS area and the Ca:P ratio (which correlates with crystal solubility) influenced inflammatory properties. These results could explain the variable clinical consequences of BCP deposits, and must be taken into account in the choice of apatite ceramics for use as biomaterials.

Inhibition and prevention of monosodium urate monohydrate crystal-induced acute inflammation in vivo by transforming growth factor beta1.

OBJECTIVE: We investigated the effects of transforming growth factor beta 1 (TGF beta 1) on monosodium urate monohydrate (MSU) crystal-induced acute inflammation in vivo. METHODS: One hour after MSU crystal-induced acute inflammation was produced in the rat subcutaneous air pouch model, the effects of recombinant human TGF beta 1 (rHuTGF beta 1; 10-100 pg/animal) and ultrapure TGF beta 1 (UPTGF beta 1; 100 and 500 pg/animal) were assessed, based on absolute and differential white blood cell counts in the exudate. The effects of 10 pg of rHuTGF beta 1 preincubated with a specific anti-TGF beta antibody, and the effects of coinjection of crystals and rHuTGF beta 1, were also studied. RESULTS: UPTGF beta 1 and rHuTGF beta 1 markedly reduced MSU crystal-induced inflammation. Recombinant human TGF beta 1 also reduced inflammation when administered concomitantly with MSU crystals. Moreover, rHuTGF beta 1 and UPTGF beta 1, injected 1 hour after MSU crystal injection, reduced the inflammatory response in a dose-dependent manner. Injection of rHuTGF beta 1 (100 pg/animal) resulted in a > 90% reduction in the maximal white blood cell count, achieved 6 hours after crystal injection. Preincubation of rHuTGF beta 1 with a specific anti-TGF beta 1 antibody significantly (P < 0.01) reversed the inhibitory effect of rHuTGF beta 1 on the inflammatory response. Consistent with the regulation of inflammatory cell recruitment into the joint, the percentage of monocytes markedly decreased (P < 0.01) following local injection with rHuTGF beta 1 6 hours after MSU crystal injection. CONCLUSION: Exogenous TGF beta 1 prevents and inhibits MSU crystal-induced acute inflammation in vivo. Its role in the self-limitation of gouty attacks deserves consideration, among the various other factors involved.