Aberration measurement and correction on a large field of view in fluorescence microscopy.

The aberrations induced by the sample and/or by the sample holder limit the resolution of optical microscopes. Wavefront correction can be achieved using a deformable mirror with wavefront sensorless optimization algorithms but, despite the complexity of these systems, the level of correction is often limited to a small area in the field of view of the microscope. In this work, we present a plug and play module for aberration measurement and correction. The wavefront correction is performed through direct wavefront reconstruction using the spinning-pupil aberration measurement and controlling a deformable lens in closed loop. The lens corrects the aberrations in the center of the field of view, leaving residual aberrations at the margins, that are removed by anisoplanatic deconvolution. We present experimental results obtained in fluorescence microscopy, with a wide field and a light sheet fluorescence microscope. These results indicate that detection and correction over the full field of view can be achieved with a compact transmissive module placed in the detection path of the fluorescence microscope.

Ultrastructural analysis of climbing fiber-Purkinje cell synaptogenesis in the rat cerebellum.

Previous reports have described the transient expression of the neuropeptides calcitonin gene-related peptide and neuropeptide Y in selected subsets of rat olivocerebellar compartments during embryonic and postnatal development. Using these neuropeptides as endogenous markers for olivocerebellar fibers, the aim of this electron microscopic analysis was to reveal the synaptogenetic processes occurring between climbing fibers and their target Purkinje cells, from embryonic day 19 to postnatal day 16, the period during which Purkinje cells undergo intense emission and retraction of dendrites, and climbing fibers translocate their synapses along Purkinje cell membrane surfaces. The present findings provide the first direct evidence that climbing fiber synaptogenesis starts on embryonic day 19 and that these first synapses mainly involve the Purkinje cell embryonic dendrite rather than the Purkinje cell soma. At the same age, the presence of unlabeled synapses resembling calcitonin gene-related peptide-labeled synapses in the Purkinje cell plate makes it possible to conclude that climbing fibers form a major synaptic investment on embryonic Purkinje cells, a finding that strongly supports the hypothesis of an early differentiating role of climbing fibers on cerebellar development. Furthermore, during the period of intense dendritic remodeling of Purkinje cells, 'myelin figures' were often detected in Purkinje cell dendrites suggesting that they may at least in part represent real ultrastructural markers of membrane turnover that identifies the sites where Purkinje cell dendritic rearrangement is taking place. Finally the finding that the climbing fiber terminals apposed to degenerating dendrites did not generally show signs of degeneration leads us to suggests that climbing fiber translocation from a perisomatic to a dendritic location may be driven by the Purkinje cell dendritic remodeling.

Calcitonin gene-related peptide receptor expression in the neurons and glia of developing rat cerebellum: an autoradiographic and immunohistochemical analysis.

Quantitative autoradiography (using [125I]human alpha-calcitonin gene-related peptide as a ligand) and immunofluorescence (using monoclonal antibodies directed against a purified receptor) followed by confocal analysis were applied to analyse the distribution and cellular localization of the calcitonin gene-related peptide receptor in the rat cerebellum during development. From late embryonic days to the end of the second postnatal week, during the time window of calcitonin gene-related peptide expression in climbing fibers, high levels of calcitonin gene-related peptide binding sites were found in the white matter, where immunolabeling was present in oligodendrocytes. Lower levels were found in the cerebellar cortex, where receptor immunolabeling was found in Bergmann glia in a presumptive cell surface location and, during the second postnatal week, also in the cytoplasm of Purkinje cells. From the end of the second postnatal week to adulthood, when calcitonin gene-related peptide is no longer present in climbing fibers, the number of calcitonin gene-related peptide binding sites increased in the molecular layer, where not only Bergmann glia but also Purkinje cell distal dendritic branchlets were immunolabeled in a presumptive cell surface location. Concomitantly, the number of calcitonin gene-related peptide binding sites sharply decreased in the white matter. The developmental expression of the calcitonin gene-related peptide receptor and the previously described proliferating/differentiating effects of the peptide on glial cells suggest that calcitonin gene-related peptide and its receptor may promote a coordinated development of cerebellar glial cells, an effect driven mainly by the calcitonin gene-related peptide released by climbing fibers. As a result of glia-neuron interactions, an indirect effect on the differentiation of the cerebellar neuronal circuitry is also likely to occur.

GAT-1 developmental expression in the rat cerebellar cortex: basket and pinceau formation.

In view of the key role exerted by neurotransmitter transporters in the synaptic transmission, the expression of GABA transporter GAT-1 was analysed during cerebellar development, when relevant processes of synapse maturation take place. GAT-1-immunoreactive (IR) structures started to be detected on PD 8-9, at the low molecular and Purkinje cell layer, coincident with the onset of functional inhibitory synapses on Purkinje neurons. By PD 18, GAT-1-IR structures completely ensheathed the Purkinje cell somata thus outlining the characteristic perisomatic formation, whereas GAT-1 wrapping on the axon initial segment started to be detected only at PD 15, and the mature form of the pinceau was fully developed from PD 23 on. These results, when compared with the functional maturation of the GABAergic input to Purkinje cells, indicate that GAT-1 may play a significant role in the differentiation of basket interneuron-Purkinje cell circuit.

GAT-1 developmental expression in the rat cerebellar cortex: basket and pinceau formation.

In view of the key role exerted by neurotransmitter transporters in the synaptic transmission, the expression of GABA transporter GAT-1 was analysed during cerebellar development, when relevant processes of synapse maturation take place. GAT-1-immunoreactive (IR) structures started to be detected on PD 8-9, at the low molecular and Purkinje cell layer, coincident with the onset of functional inhibitory synapses on Purkinje neurons. By PD 18, GAT-1-IR structures completely ensheathed the Purkinje cell somata thus outlining the characteristic perisomatic formation, whereas GAT-1 wrapping on the axon initial segment started to be detected only at PD 15, and the mature form of the pinceau was fully developed from PD 23 on. These results, when compared with the functional maturation of the GABAergic input to Purkinje cells, indicate that GAT-1 may play a significant role in the differentiation of basket interneuron-Purkinje cell circuit.

Monoclonal antibodies reveal expression of the CGRP receptor in Purkinje cells, interneurons and astrocytes of rat cerebellar cortex.

The molecular layer of adult rat cerebellum displays high levels of calcitonin gene-related peptide (CGRP) receptors, but the cellular location of the receptor remains unidentified. In an attempt to reveal the expression sites of these receptors, monoclonal antibodies raised against purified CGRP receptors from porcine cerebellar membranes were used in double-immunofluorescence experiments combined with confocal microscopy. PEP-19, a marker that is highly enriched in Purkinje cells (Pc), revealed that CGRP receptors are located in Pc cytoplasm and dendrites, where they label small puncta sometimes arranged in a row along the course of the dendrite itself. CGRP receptors were also located in inhibitory interneurons. Furthermore, as shown by double-labeling experiments with GFAP, CGRP receptor-IR labeled Golgi epithelial cells and their radial fibers (Bergmann fibers), as well as astrocytic processes encircling Pc somata. The simultaneous presence of CGRP receptors in Purkinje cells and in the glial cells that heavily enshroud Purkinje cells allows us to hypothesize that these receptors may be involved in neuron-glia interactions influencing neuronal activity.

Somatotopic nucleocortical projections to the multiple somatosensory cerebellar maps.

The cerebellum is organized in a series of parasagittal compartments: in C1-C3 and C2 compartments Purkinje cells receive climbing fibre afferents from the rostral part of the accessory olives, and project their axon to the nucleus interpositus anterior and posterior, respectively. Within these compartments electrophysiological studies have shown that the cutaneous input carried by climbing fibre afferents is topographically organized so as to design a map of peripheral body districts. The body map is replicated over the anterior lobe-pars intermedia and the paramedian lobule, and anatomical studies have indicated that the replication is partly due to the axonal branching of olivocerebellar neurons. The aim of this study was to analyse the presence of a somatotopic organization and of a branching pattern in the nucleocortical projections, in relation to the replicated body maps within C1-C3 and C2 compartments. By using double retrograde neuronal tracing we explored, in the cat, the topographic distribution of single- and double-labelled cells in the interposed nuclear subdivisions, after tracer injections into forelimb or hindlimb regions of the anterior lobe-pars intermedia, paramedian lobule and hemisphere (medial crus II). Most of the nucleocortical neurons were found in ipsilateral nucleus interpositus posterior, with smaller numbers in the ipsilateral nucleus interpositus anterior. Nucleocortical neurons projecting to forelimb- or hindlimb-related areas are completely segregated, the forelimb neurons being located laterally and the hindlimb neurons medially in the nucleus interpositus posterior. Within their respective domains both the forelimb and hindlimb populations projecting to the anterior lobe-pars intermedia are partly segregated from those projecting to the paramedian lobule, in that the two populations are slightly shifted along the dorsoventral axis of the nucleus. Although mostly different, some of the cells are common to the two forelimb populations, since they send axonal branches to the homologous areas of the anterior lobe and paramedian lobule. Contralateral fastigial or interposed nucleocortical projections are restricted to the anterior lobe-pars intermedia, and their neurons of origin are different from those that project to the ipsilateral cerebellar cortex: i.e. they are not a bilateral, but a separate contralateral component.

Neuropeptide Y (NPY) expression is up-regulated in the rat inferior olive during development.

The aim of this study was to analyse the developmental expression of the neuropeptide Y (NPY) in the rat inferior olivary (IO) complex by immunoperoxidase and immunofluorescence techniques. The spatial distribution of NPY-immunoreactivity (IR) did not vary during development, whereas NPY-IR intensity levels varied significantly. The peak of NPY-IR expression occurred during the second postnatal week, but differed in intensity in individual IO subnuclei, the highest levels being present in the dorsal fold of the dorsal accessory olive and in the ventro-lateral outgrowth. In the adult, NPY-IR could only be rescued in colchicine pretreated animals, but its distribution overlapped the one found during development. These findings show that NPY-IR is transiently up- regulated, during development, in specific compartments of the IO complex, and that the peptide is rescued in the same specific olivocerebellar compartments in the adult. These observations are here taken to support the hypothesis that NPY may exert different trophic-differentiating and/or neuromodulatory roles during development, when its expression is transiently up-regulated, or at adult stages, when it can be rescued, according to the different biological contexts.

Neuronal and glial localization of the GABA transporter GAT-1 in the cerebellar cortex.

THE distribution and neuronal or glial localization of GAT-1, a high affinity GABA transporter, in the cerebellar cortex was analysed by means of double-label immunofluorescence experiments with GAT-1 combined with calbindin D, synaptophysin, or GFAP antibodies. In the Purkinje cell (Pc) layer, prominent synaptic GAT-1-immunoreactivity (IR) was found in the axon terminals of basket cells surrounding the Pc axon hillock. GAT-1-IR was also found in neuronal and glial processes ensheathing Pc somata and dendrites. Numerous immunoreactive fibres and puncta originating from basket and stellate cells, or from Golgi cells, were also detected in the molecular or granular layer, respectively. These observations suggest that GAT-1 is involved in the termination of the action of GABA at the inhibitory synapses of all cerebellar interneurones, primarily of basket cell terminals at the Pc axon hillock. GAT-1 in the astroglial processes presumably plays the additional role of regulating the extracellular concentrations of GABA.

Developmentally regulated expression of alpha- and beta-calcitonin gene-related peptide mRNA and calcitonin gene-related peptide immunoreactivity in the rat inferior olive.

Immunohistochemical methods have revealed the transient neonatal expression of calcitonin gene-related peptide (CGRP) in olivocerebellar compartments, and it has been hypothesized that this peptide plays a role in the development of olivocerebellar connectivity. Furthermore, the distribution of the CGRP binding sites in the cerebellar cortex also favors this hypothesis. In this study, the pattern of postnatal expression of alpha- and beta-CGRP mRNAs in the inferior olive (IO) complex was analyzed using in situ hybridization histochemistry with RNA probes complementary to specific sequences of alpha- and beta-CGRP mRNAs, and the results were compared with the pattern of CGRP immunoreactivity. High levels of alpha-CGRP mRNA expression were found in specific subnuclei of the IO complex, i.e., the medial part of the dorsal fold of the dorsal accessory olive, the beta nucleus, the dorsal cap, the caudal third of the medial accessory olive, and the rostral part of the dorso-medial cell column; in the same subnuclei beta-CGRP mRNA was detected. The olivary distribution of the two CGRP mRNA coincided with that of CGRP immunoreactivity. The expressions of alpha-CGRP mRNA and CGRP immunoreactivity were restricted to the first 2 postnatal weeks, the peak being reached at the end of the first week; beta-CGRP mRNA was transiently expressed in the same olivary compartments, but only from postnatal day 6 to 9. In general, the alpha-CGRP signal was also more intense than the beta-CGRP signal. The present findings indicate that the alpha- and beta-CGRP mRNA expression in the olivary complex is under developmental control and restricted to specific olivocerebellar compartments. The data provide a basis for the transient expression of a CGRP olivocerebellar compartment and further support the hypothesis of a role for CGRP in the complex postnatal cerebellar phenomena of connectivity reshaping and synapse stabilization.

CGRP as a marker of the climbing fibers during the development of the cerebellum in the rat.

Olivary fibers express CGRP-LI until the nest phase of their development and never in the next postnatal steps, that is, the "capuchon" stage. Together with other results presented in this volume regarding the postnatal development of CGRP binding sites in the cerebellar cortex, these immunohistologic findings suggest a role for CGRP in reshaping connectivity and in synapse stabilization of cerebellar circuitry during postnatal development.

Modulation of cerebellar CGRP binding sites induced by climbing fibre activation.

The neuropeptide CGRP is transiently expressed at neonatal stages in the rat olivocerebellar system, while high affinity binding sites for the peptide are permanently expressed in the molecular layer of the adult cerebellum. In this study, an increase in the density of cerebellar high affinity binding sites for calcitonin gene-related peptide (CGRP) is induced by harmaline, given at a dose appropriate to induce tremor. The present results, taken together with the demonstration that harmaline is a potent stimulator of the olivocerebellar system, suggest that the expression of cerebellar CGRP receptors can be modulated by the level of activity in the cerebellar afferents.

CGRP expression in the rat olivocerebellar system during postnatal development.

CGRP-like immunoreactivity was studied in the cerebellar cortex and precerebellar nuclei of neonatal rats. From postnatal day (PD) 0 to PD 13-15, CGRP immunoreactivity was transiently found in fiber-like structures around Purkinje cells, defining parasagittal bands. Following the same time course, transient labeling was also found in the inferior olive neurons which, in the adult, distribute their axons to the olivocerebellar compartments corresponding to the immunoreactive bands. It is concluded that CGRP is transiently expressed in specific climbing fiber compartments.

The cerebrospinal fluid transferrin/tau proteins. A study by two-dimensional polyacrylamide gel electrophoresis (2D) and agarose isoelectrofocusing (IEF) followed by double-antibody peroxidase labeling and avidin-biotin amplification.

The cerebrospinal fluid (CSF) transferrin/Tau proteins were studied by two-dimensional polyacrylamide gel electrophoresis (2D) followed by immunoblotting and by agarose isoelectrofocusing (IEF), and subsequent double immunofixation, peroxidase staining and Avidin-Biotin Complex (ABC) amplification. The pattern of the Tau protein was similar but not equal to that of the transferrin (Tf). When a genetic variant of Tf was present in the serum, the same variant was also observed in the corresponding CSF Tf and in the Tau fraction. After neuraminidase treatment, both serum and CSF Tf moved to the Tau position on IEF and 2D. On 2D, no desialized precursors of the Tau proteins were detected, whereas the Tf precursors were always detected. No synthesis of the Tau globulin in the brain can, therefore, be inferred. In CSF not treated with neuraminidase, Tf is the only sialoglycoprotein clearly desialized, showing that the Tau fraction cannot be generated by neuraminidase action at CSF level. In fact, the treatment of serum and CSF proteins with neuraminidase produced a clear shift in the isoelectric mobility of all sialoglycoproteins. We clearly demonstrate that the Tau globulin is the result of neuraminidase activity not located in the CSF compartment. We suggest that Tf could be desialized by the action of neuraminidase at the brain level and then be "washed" into the CSF. Brain utilization of Tf, meeting the brain iron requirement, seems likely.