Synaptic elimination and protection after minimal injury depend on cell type and their prelesion structural dynamics in the adult cerebral cortex.

The axonal and synaptic mechanisms underlying dysfunction and repair of the injured CNS are poorly understood. Unresolved issues include to what degree, when, and how the surviving neurons degenerate and the extent of synaptic remodeling both along the severed axon and in the nearby area. One of the main reasons is the lack of tools to study the complex asynchronous and dynamic features of individual lesioned axon responses in the intact brain. To address these issues, we combined two-photon microscopy and laser microsurgery to image the real-time reorganization of cortical circuitry at synaptic resolution for periods of up to 1 year in the brain of living mice. Injured cortical axons were eliminated proximally through a two-phase retraction process, which continued for at least 3 months postlesion and was independent of the presence of scar tissue. Remarkably, axons which later attempt to regenerate in both the mature and juvenile brain retracted less, raising the possibility that targeting retraction may improve the chances of axon regrowth after axotomy. Comparing prelesion and postlesion dynamics on the same axons over several days and weeks revealed that, although synapse formation rates were unaffected, boutons on injured axons were either rapidly and persistently lost, or extremely resistant, depending on cell-type and their prelesion structural dynamics. Our data suggest a lasting deficiency in synaptic output on surviving injured cortical axons and a surprising difference in the vulnerability of synaptic boutons after axotomy, which depend on cell-type and their recent history.

In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits.

To what extent, how and when axons respond to injury in the highly interconnected grey matter is poorly understood. Here we use two-photon imaging and focused ion beam-scanning electron microscopy to explore, at synaptic resolution, the regrowth capacity of several neuronal populations in the intact brain. Time-lapse analysis of >100 individually ablated axons for periods of up to a year reveals a surprising inability to regenerate even in a glial scar-free environment. However, depending on cell type some axons spontaneously extend for distances unseen in the unlesioned adult cortex and at maximum speeds comparable to peripheral nerve regeneration. Regrowth follows a distinct pattern from developmental axon growth. Remarkably, although never reconnecting to the original targets, axons consistently form new boutons at comparable prelesion synaptic densities, implying the existence of intrinsic homeostatic programmes, which regulate synaptic numbers on regenerating axons. Our results may help guide future clinical investigations to promote functional axon regeneration.

Imaging of experience-dependent structural plasticity in the mouse neocortex in vivo.

The functionality of adult neocortical circuits can be altered by novel experiences or learning. This functional plasticity appears to rely on changes in the strength of neuronal connections that were established during development. Here we will describe some of our studies in which we have addressed whether structural changes, including the remodeling of axons and dendrites with synapse formation and elimination, could underlie experience-dependent plasticity in the adult neocortex. Using 2-photon laser-scanning microscopes and transgenic mice expressing GFP in a subset of pyramidal cells, we have observed that a small subset of dendritic spines continuously appear and disappear on a daily basis, whereas the majority of spines persists for months. Axonal boutons from different neuronal classes displayed similar behavior, although the extent of remodeling varied. Under baseline conditions, new spines in the barrel cortex were mostly transient and rarely survived for more than a week. However, when every other whisker was trimmed, the generation and loss of persistent spines was enhanced. Ultrastructural reconstruction of previously imaged spines and boutons showed that new spines slowly form synapses. New spines persisting for a few days always had synapses, whereas very young spines often lacked synapses. New synapses were predominantly found on large, multi-synapse boutons, suggesting that spine growth is followed by synapse formation, preferentially on existing boutons. Altogether our data indicate that novel sensory experience drives the stabilization of new spines on subclasses of cortical neurons and promotes the formation of new synapses. These synaptic changes likely underlie experience-dependent functional remodeling of specific neocortical circuits.

Bone metabolism in men: role of aromatase activity.

Sex steroid hormones play an important role in the maintenance of bone mass in males and in females. Even though androgens are the major sex steroids in men, direct and indirect evidence emerged suggesting that estrogens may also play a major role in male skeletal health. Since the testes account for only 15% of circulating estrogens in males, the remaining 85% comes from peripheral aromatization of androgen precursors in different tissues, including bone. Human models of aromatase deficiency clearly demonstrated the critical importance of the conversion of androgens into estrogens in regulating male skeletal homeostasis. Aromatase- deficient men showed tall stature due to continued longitudinal growth, unfused epiphyses, high bone turnover, and osteopenia. Interventional studies in adult men using aromatase inhibition confirmed that estrogens are important in controlling bone remodeling. Importantly either inherited (i.e. due to common polymorphisms at the human aromatase CYP19 gene) or acquired (i.e. by diseases or different compounds) variation in aromatase ability to convert androgen precursors into estrogen may also be relevant for skeletal homeostasis.

Serum OPG and RANKL levels before and after intravenous bisphosphonate treatment in Paget's disease of bone.

Paget's disease of bone (PDB) is a focal disorder of bone remodeling characterized by increased osteoclast-mediated bone resorption. Even though increasing evidence indicates enhanced nuclear factor-kB (NF-kB) signaling as a common mechanism involved in PDB and other related disorders, few studies investigated circulating osteoprotegerin (OPG) and receptor of activator of NF-kB-ligand (RANKL) levels in PDB patients. In this study we explored the relationships between OPG or RANKL levels and bone turnover markers in a group of patients with PDB, before and after intravenous bisphosphonate treatment (pamidronate 60 mg). Both OPG and RANKL were markedly elevated in PDB patients with respect to control groups (healthy or osteoporotic postmenopausal women and elderly men) and were positively associated with bone turnover markers. Higher levels of these cytokines were observed in polyostotic than monostotic PDB cases. The ratio between RANKL and OPG was more than 3-fold higher in PDB patients than in controls. Interestingly, in the group of patients treated with pamidronate, we found an increase in OPG levels that become statistically significant after 3 and 6 months from treatment. A trend toward a decrease in RANKL levels after treatment was also observed. The RANKL/OPG ratio was significantly reduced after 3 and 6 months of therapy. In contrast, in patients classified as non-responders, OPG and RANKL levels after pamidronate infusion did not significantly differ with respect to pre-treatment values. Thus, the positive effect of amino bisphosphonates in the treatment of PDB may be due to either direct or indirect suppression of RANKL-induced bone resorption through decreased RANKL and increased OPG production.

Effects of carvedilol on left ventricular diastolic function and chamber volumes in advanced heart failure.

AIM: Carvedilol treatment in chronic heart failure (CHF) demonstrated to reduce mortality and rehospitalisation, and improvement of cardiac systolic function with reduction of left ventricular volumes and remodelling. The effects of the drug on left ventricular (LV) filling are less studied. Systolic dysfunction is often associated to diastolic alteration, pseudonormal and restrictive filling pattern are related with poor prognosis. In this study we evaluated diastolic cardiac modifications during carvedilol therapy at early and long term in patients with advanced CHF and pseudonormal or restrictive filling pattern by echo Doppler method. METHODS: We studied 59 patients with severe but stable CHF (40 in class NYHA III and 19 in class NYHA IV) with both systolic and diastolic dysfunction due to idiopathic or ischemic cardiomyopathy. Group I (n=32) received preceding treatment plus carvedilol and Group II (n=27) continued standard therapy with ACE-inhibitors, diuretics, digossin and vasodilators. In all subjects were evaluated LV volumes mass and ejection fraction (EF). Therefore, we studied transmitral filling parameters: Early filling wave (E), atrial filling wave (A), E/A ratio, deceleration time of E (DT) and isovolumetric releasing time (IVRT) by echo Doppler method. RESULTS: After 4 months of therapy carvedilol group showed a significant increase of A wave (p<0.001) DT (p<0.0001) and IVRT (p<0.0001), and significant reduction of E/A ratio (p<0.0005) respect to Group II. Any significant changes were observed for volumes mass and EF. Transmitral Doppler measurements remained unchanged or further ameliorated at 12 months (A p<0.0005; DT p<0.00002; IVRT p<0.000004; E/A p<0.0008), we also observed E wave reduction (p<0.001) in Group I respect to controls. Besides, after 1 year of follow-up we observed a reduction of systolic volume (p<0.001) pulmonary pressure (p<0.0001) and consequent increase of EF (p<0.001) in group treated with beta-blockers. Multivariate analysis demonstrated that Doppler modification were minimally related to heart rate and blood pressure reduction. CONCLUSIONS: Carvedilol treatment improve diastolic function in CHF with severe diastolic impairment driving restrictive or pseudonormal filling pattern towards altered pattern at early time. These changes remained also after 1 year of therapy and appeared to precede increase of systolic function and improvement of emodynamic status.

Estrogen receptor gene polymorphisms and the genetics of osteoporosis: a HuGE review.

Osteoporosis (OMIM166710) is a common skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue with increased susceptibility to fracture. Osteoporosis has a complex etiology and is considered a multifactorial polygenic disease in which genetic determinants are modulated by hormonal, environmental, and nutritional factors. Estrogens are known to play an important role in regulating bone homeostasis and preventing postmenopausal bone loss. They act through binding to two different estrogen receptors (ERs), ER alpha (OMIM133430) and ER beta (OMIM601663), which are members of the nuclear receptor superfamily of ligand-activated transcription factors. Different polymorphisms have been described in both the ER alpha and ER beta genes. Although a large number of association studies have been performed, the individual contribution of these polymorphisms to the pathogenesis of osteoporosis remains to be universally confirmed. Moreover, an important aim in future work will be to define their functional molecular consequences and their interaction with the environment in the causation of the osteoporotic phenotype. A further promising application of these polymorphisms comes from their pharmacogenomic implications, with the possibility of providing better guidance for therapeutic regimens, such as estrogen replacement therapy and selective ER modulators. At the moment, no recommendations for population-based screening can be made.

Professional sport activity and micronutrients: effects on bone mass.

Osteoporosis is the most prevalent metabolic bone disease among developed countries. Although bone mass and density are certainly determined by various concurrent factors such as genetics, hormones, life-style and the environment, and although the genetic program has a critical role in growth and in bone peak development, for their realization an adequate nutritional intake of nutrients and regular exercise are always necessary and may represent a way to prevent osteoporosis and fractures. Exercise and especially high-impact sport activity during growth and adolescence increases bone mineral density (BMD) in weight-loaded skeletal regions. Aerobics, weight bearing and resistance exercises may also be effective in increasing BMD in post-menopausal women. Even though most of the research on nutritional components has focused almost exclusively on calcium and vitamin D, there is now considerable interest in the effects of a variety of other nutrients on bone status.

AMPA-receptor activation regulates the diffusion of a membrane marker in parallel with dendritic spine motility in the mouse hippocampus.

Dendritic spines are the site of most excitatory connections in the hippocampus. We have investigated the diffusibility of a membrane-bound green fluorescent protein (mGFP) within the inner leaflet of the plasma membrane using Fluorescence Recovery After Photobleaching. In dendritic spines the diffusion of mGFP was significantly retarded relative to the dendritic shaft. In parallel, we have assessed the motility of dendritic spines, and found an inverse correlation between spine motility and the rate of diffusion of mGFP. We then tested the influence of glutamate receptor activation or blockade, and the involvement of the actin cytoskeleton (using latrunculin A) on spine motility and mGFP diffusion. These results show that glutamate receptors regulate the mobility of molecules in the inner leaflet of the plasma membrane through an action upon the actin cytoskeleton, suggesting a novel mechanism for the regulation of postsynaptic receptor density and composition.