Biomedical subjects

Piergiorgio Strata

Publications and source records attributed to Piergiorgio Strata.

At least 19 recordsLinked to original sources

PubMed2006

Composition of perineuronal nets in the adult rat cerebellum and the cellular origin of their components.

The decrease in plasticity that occurs in the central nervous system during postnatal development is accompanied by the appearance of perineuronal nets (PNNs) around the cell body and dendrites of many classes of neuron. These structures are composed of extracellular matrix molecules, such as chondroitin sulfate proteoglycans (CSPGs), hyaluronan (HA), tenascin-R, and link proteins. To elucidate the role played by neurons and glial cells in constructing PNNs, we studied the expression of PNN components in the adult rat cerebellum by immunohistochemistry and in situ hybridization. In the deep cerebellar nuclei, only large excitatory neurons were surrounded by nets, which contained the CSPGs aggrecan, neurocan, brevican, versican, and phosphacan, along with tenascin-R and HA. Whereas both net-bearing neurons and glial cells were the sources of CSPGs and tenascin-R, only the neurons expressed the mRNA for HA synthases (HASs), cartilage link protein, and link protein Bral2. In the cerebellar cortex, Golgi neurons possessed PNNs and also synthesized HASs, cartilage link protein, and Bral2 mRNAs. To see whether HA might link PNNs to the neuronal cell surface by binding to a receptor, we investigated the expression of the HA receptors CD44, RHAMM, and LYVE-1. No immunolabelling for HA receptors on the membrane of net-bearing neurons was found. We therefore propose that HASs, which can retain HA on the cell surface, may act as a link between PNNs and neurons. Thus, HAS and link proteins might be key molecules for PNN formation and stability.

Animals↗

PubMed2005

Axonal and synaptic remodeling in the mature cerebellar cortex.

By blocking electrical activity in the cerebellar cortex the Purkinje cell dendrites become a uniform territory with a high density of spines all bearing the glutamate receptor delta2 subunit (GluRdelta2) and being mainly innervated by parallel fibers. Such a subunit, which is constitutively targeted specifically to the parallel fiber synapses, appears in the spines contacted by the climbing fibers before they disconnect from the target. A similar pattern of hyperspiny transformation and innervation occurs a few days after a subtotal lesion of the inferior olive, the source of climbing fibers. During the climbing fiber reinnervation process which follows the removal of the electrical block or by collateral sprouting of surviving inferior olive neurons, the new active climbing fibers establish synaptic contacts with proximal dendritic spines that bear GluRdelta2s. After, they repress these subunits and displace the parallel fibers to the distal dendritic territory. These findings suggest the following operational principle in the axonal competition for a common target. The Purkinje cells have an intrinsic phenotypic profile which is compatible with the parallel fiber innervation, this mode being operational in targets innervated by a single neuronal population, like the neuromuscular system. An additional input, the climbing fibers, in order to achieve its own territory on the proximal dendrite needs the ability to displace the competitor. Such an inhibition is activity-dependent and the activity needs to be present in order to allow the climbing fiber to maintain its territory, even when the developmental period is over.

Age Factors↗

PubMed2005

Pharmacology of the metabotropic glutamate receptor mediated current at the climbing fiber to Purkinje cell synapse.

Different forms of synaptic plasticity in the cerebellum are mediated by metabotropic glutamate receptors (mGluRs). At parallel fiber (PF) to Purkinje cell (PC) synapses activation of mGluR gives rise to a well known slow synaptic current inhibited by antagonists of mGluR1. The distribution of mGluR types in the climbing fiber (CF) to PC synapses is not well known. However, a mGluR1alpha-mediated all-or-none postsynaptic current was also demonstrated at the CF-PC synapse (Dzubay and Otis, Neuron 36, 1159, 2002). Using whole cell patch-clamp recording from PCs in rat cerebellar slices with AMPA receptors blocked and glutamate uptake impaired we demonstrate a more complex pharmacology of a current obtained by single or train CF stimulation. The mGluR1 specific antagonist CPCCOEt in a group of cells suppressed this response while in a similar number of other cells it induced a potentiating effect. The antagonists of mGluR groups II and III (LY341495 and MSOP, respectively) predominantly suppressed the current. The ambiguous effect of CPCCOEt was checked by measuring the paired-pulse depression of the CF EPSC, which was not changed with the antagonist in normal as well as in low (0.5 mM) external Ca(2+) (used to prevent saturation of AMPARs), thus excluding a presynaptic effect. However, CPCCOEt induced a rise in the amplitude (by approximately 50%) as well as a prolongation (p<0.05) of the decay time of CF EPSCs at normal 2 mM Ca(2+), i.e. under conditions of AMPAR saturation, thus indicating an effect of postsynaptic origin. In 0.5 mM Ca(2+) the decay of CF EPSCs was longer but it was also significantly prolonged (p?0.01) by CPCCOEt. However, the CF EPSC amplitude was not significantly affected indicating an underlying Ca(2+)-dependent mechanism. Thus, the pharmacology of the PC mGluR-mediated response points to a dual postsynaptic role of mGluR1 giving rise to a slow postsynaptic current but also regulating other presumably mGluR-dependent currents via second messenger molecules and Ca(2+). The additional electrophysiological role of mGluR II & III types was also indicated. Such a complex regulatory mechanism may have an important role in the mGluR-dependent forms of homosynaptic plasticity and motor learning at the CF-PC synapse.

Animals↗

PubMed2005

International perspectives on engaging the public in neuroethics.

With an ever-increasing understanding of the brain mechanisms associated with core human attributes and values, there is an increasing public interest in the results of neuroscience research and the ways in which that new knowledge will be used. Here, we present perspectives on engaging the public on these issues on an international scale, the role of the media, and prospects for the new field of neuroethics as both a focus and a driver of these efforts.

Bioethical Issues↗

PubMed2005

Correlation between multiple climbing fibre regression and parallel fibre response development in the postnatal mouse cerebellum.

At the neuromuscular junction elimination of supernumerary synaptic connections during development is owing to a competitive process between single neuronal populations, whereas in the central nervous system the interaction of different types of input could affect the process. In the cerebellum, the regression from multiple- to mono-innervation of the Purkinje cells by climbing fibres is virtually completed during the first two weeks of postnatal development. While it is clear that parallel fibres are important in the control of the regression, there are conflicting results in relation to whether an early phase of regression is independent of parallel fibre effects. We studied the precise timing of climbing fibre synapse development and decline and the relationship with the functional maturation of parallel fibres. Until postnatal day (P) 6 or 7, the synaptic currents generated by different climbing fibres become progressively more uniform in amplitude. However, between P7 and 14, the amplitudes of the currents increasingly diverge until only one fibre remains connected. These data are taken as evidence that, in multiply innervated Purkinje cells, competition between different climbing fibres appears at P7 and continues during the second postnatal week. Morphological and electrophysiological data demonstrate that parallel fibres synapses appear at P7 and their development is significantly correlated with the time course of the climbing fibre regression. These results provide no evidence for climbing fibre regression independent of parallel fibres before P7 and also suggest a dominant role of the parallel fibre input in the later phase.

Age Factors↗

PubMed2005

Spontaneous electrical activity and dendritic spine size in mature cerebellar Purkinje cells.

Previous experiments have shown that in the mature cerebellum both blocking of spontaneous electrical activity and destruction of the climbing fibres by a lesion of the inferior olive have a similar profound effect on the spine distribution on the proximal dendrites of the Purkinje cells. Many new spines develop that are largely innervated by parallel fibers. Here we show that blocking electrical activity leads to a significant decrease in size of the spines on the branchlets. We have also compared the size of the spines of the proximal dendritic domain that appear during activity block and after an inferior olive lesion. In this region also, the spines in the absence of activity are significantly smaller. In the proximal dendritic domain, the new spines that develop in the absence of activity are innervated by parallel fibers and are not significantly different in size from those of the branchlets, although they are shorter. Thus, the spontaneous activity of the cerebellar cortex is necessary not only to maintain the physiological spine distribution profile in the Purkinje cell dendritic tree, but also acts as a signal that prevents spines from shrinking.

Action Potentials↗

PubMed2005

Purkinje cell spinogenesis during architectural rewiring in the mature cerebellum.

Spines can grow and retract within hours of activity perturbation. We investigated the time course of spine formation in a model of plasticity involving changes in brain architecture where spines of a dendritic domain become innervated by a different neuronal population. Following a lesion of rat olivocerebellar axons, by severing the inferior cerebellar peduncle, new spines grow on the deafferented proximal dendrite of the Purkinje cells (PCs) and these new spines become innervated by parallel fibres (PFs) that normally contact only the distal dendrites. The varicosities of climbing fibre (CF) terminal arbors disappear within 3 days of the lesion. Spine density in the proximal dendritic domain begins to rise within 3 days and continues to increase towards a plateau at 6-8 days. In 'slow Wallerian degeneration' mice, in which axonal degeneration is delayed, climbing fibre varicosities virtually disappear at 14 rather than 3 days. Spine density in the proximal dendritic domain is similar to control Purkinje cells up to 14 days and increases significantly 18 days postlesion. The delayed spinogenesis in the latter mutant is the result of a persistence of the climbing fibre presynaptic structure in the absence of activity. Therefore, climbing fibre activity itself is not directly responsible for the suppression of spine formation, but suppression mechanisms tend to become weaker as long as the structural dismantling of the presynaptic varicosities proceeds. Thus, spinogenesis is guided by two different mechanisms; a rapid one related to changes in homotypic remodeling and a slower one, which requires the removal of a competitive afferent.

Animals↗

PubMed2005

The cerebellum: synaptic changes and fear conditioning.

In addition to coordinating movement, the cerebellum participates in motor learning, emotional behavior, and fear memory. Fear learning is reflected in a change of autonomic and somatic responses, such as heart rate and freezing, elicited by a neutral stimulus that has been previously paired with a painful one. Manipulation of the vermis affects these responses, and its reversible inactivation during the consolidation period impairs fear memory. The neural correlate of cerebellar involvement in fear consolidation is provided by a behaviorally induced long-term increase of synaptic efficacy between parallel fibers and a Purkinje cell. Similar synaptic changes after fear conditioning are well documented in the amygdala and hippocampus, providing a link between emotional experiences and changes in neural activity. In addition, in hotfoot mice, with a primary deficiency of parallel fiber to Purkinje cell synapse, short- and long-term fear memories are affected. All these data support the idea that the cerebellum participates in fear learning. The functional interconnection of the vermis with hypothalamus, amygdala, and hippocampus suggests a more complex role of the cerebellum as part of an integrated network regulating emotional behavior.

Animals↗

PubMed2005

Spontaneous electrical activity and structural plasticity in the mature cerebellar cortex.

The Purkinje cell of the cerebellar cortex presents two distinct dendritic domains: a distal one, with spiny branchlets and a high density of spines innervated by many parallel fibers, and a proximal one, with a few clusters of spines innervated by a single climbing fiber terminal arbor. In adult rats, after 7 days of blocked electrical activity by the administration of TTX into the cerebellar parenchyma, the proximal dendritic domain of the Purkinje cell shows a remarkable growth of new spines that are innervated by parallel fibers. At the same time, the climbing fiber terminal arbor tends to become atrophic. In contrast, in the branchlets, spine density remains unmodified. These changes are reversible when TTX is removed. TTX treatment also leads to a decrease in spine size both in the branchlets and in the new spines of the proximal dendritic compartment. Spontaneous electrical activity should therefore be regarded not simply as noise, but as a significant signal for maintaining the typical profile of afferent innervation of the Purkinje cell and for preventing spines from shrinking.

Animals↗

PubMed2005

Short-term facilitation and depression in the cerebellum: some observations on wild-type and mutant rodents deficient in the extracellular matrix molecule tenascin C.

Short-term plasticity was studied on synapses to Purkinje cells (PC): paired-pulse facilitation in parallel fibers (PF) and paired-pulse depression in climbing fibers (CF). Both phenomena relate to synaptic strength. These forms of short-term plasticity were tested on cerebellar slices in rat by early postnatal synchronous stimulation of olivary neurons (i.e., CFs) with harmaline and by inhibition of a metabotropic glutamate receptor (mGluR) as well as in mice that were deficient in the extracellular matrix glycoprotein tenascin-C. Harmaline stimulation delayed the developmental competition between CF inputs and maintained multiple innervation. Paired-pulse depression of the CF-PC synapse after harmaline treatment was more expressed. However, paired-pulse facilitation in PF-PC synapses remained unchanged. Electrophysiological responses of postsynaptic mGluR1 in CF-PC synapses could be obtained only with AMPA receptors blocked and glutamate uptake impaired. The mGluR1-specific antagonist CPCCOEt suppressed the CF-mGluR EPSC in some PCs and potentiated it in other PCs. CF paired-pulse depression was not changed with CPCCOEt, thus excluding a presynaptic effect. The postsynaptic effect was underlined by CPCCOEt-induced rise in amplitude of EPSC and by a prolongation of its decay time. Tenascins are extracellular matrix glycoproteins that may restrict the regenerative capacity of the nervous tissue. Testing short-term presynaptic plasticity in tenascin-C-deficient mice showed that CF paired-pulse depression was less expressed while PF paired-pulse facilitation was augmented except in a group of cells where there was even depression. The results underline differences in forms of short-term plasticity with regard to susceptibility to diverse modulatory factors.

Animals↗

PubMed2005

MAPK activation in cerebellar basket cell terminals after harmaline treatment.

The mitogen-activated protein kinases (MAPKs) are a family of signal transduction mediators that regulate a number of cellular activities, including cell growth and proliferation, differentiation and survival, via phosphorylation (activation) of protein kinases. MAPKs are also recruited during synaptic plasticity and remodeling. In the present study we used Western blotting and immunohistochemistry to examine the effects of harmaline administration on the phosphorylation state of three MAPKs: the extracellular signal-regulated kinase (ERK1/2), c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 MAPK. Harmaline is a tremorigenic drug known to induce enhanced and rhythmic firing of the inferior olive. In rats, synchronous activity of the inferior olive cells induced by harmaline administered for four days from postnatal day 9 to 12 resulted in prolonged maintenance of polyinnervation of Purkinje cells by climbing fibers (axons of olivary cells). Immunohistochemistry showed small but sustained cytoplasmic positivity to phospho-ERK in Purkinje cells and a strong signal for phospho-ERK in the "pinceaux," terminals of the interneuronal basket cells onto Purkinje cells. A similar pattern was observed for JNK/SAPK, while no changes in p38 were noticed. Thus, it was revealed that the activation of two members of the MAPK family in these inhibitory presynaptic terminals is also one consequence of synchronous olivary input to Purkinje cells known to affect developmental plasticity.

Animals↗

PubMed2004

Long-term synaptic changes induced in the cerebellar cortex by fear conditioning.

To better understand learning mechanisms, one needs to study synaptic plasticity induced by behavioral training. Recently, it has been demonstrated that the cerebellum is involved in the consolidation of fear memory. Nevertheless, how the cerebellum contributes to emotional behavior is far from known. In cerebellar slices at 10 min and 24 hr following fear conditioning, we found a long-lasting potentiation of the synapse between parallel fibers and Purkinje cells in vermal lobules V-VI, but not in the climbing fiber synapses. The mechanism is postsynaptic, due to an increased AMPA response. In addition, in hotfoot mice with a primary deficiency of the parallel fiber to Purkinje cell synapse, cued (but not contextual) fear conditioning is affected. We propose that this synapse plays an important role in the learned fear and that its long-term potentiation may represent a contribution to the neural substrate of fear memory.

Animals↗

PubMed2004

Reparative mechanisms in the cerebellar cortex.

In the adult brain, different neuronal populations display different degrees of plasticity. Here, we describe the highly different plastic properties of inferior olivary neurones and Purkinje cells. Olivary neurones show a basal expression of growth-associated proteins, such as GAP-43 and Krox24/EGR-1, and remarkable remodelling capabilities of their terminal arbour. They also regenerate their transected neurites into growth-permissive territories and may reinnervate the lost target. Sprouting and regrowing olivary axons are able to follow specific positional information cues to establish new connections according to the original projection map. In addition, they set a strong cell body reaction to injury, which in specific olivary subsets is regulated by inhibitory target-derived cues. In contrast, Purkinje cells do not have a constitutive level of growth-associated genes, and show little cell body reaction, no axonal regeneration after axotomy, and weak sprouting capabilities. Block of myelin-derived signals allows terminal arbour remodelling, but not regeneration, while selective over-expression of GAP-43 induces axonal sprouting along the axonal surface and at the level of the lesion. We suggest that the high constitutive intrinsic plasticity of the inferior olive neurones allows their terminal arbour to sustain the activity-dependent ongoing competition with the parallel fibres in order to maintain the post-synaptic territory, and possibly underlies mechanisms of learning and memory. Such a plasticity is used also as a reparative mechanism following axotomy. In contrast, in Purkinje cells, poor intrinsic regenerative capabilities and myelin-derived signals stabilise the mature connectivity and prevent axonal regeneration after lesion.

Animals↗

PubMed2003

Glutamate receptor delta2 subunit in activity-dependent heterologous synaptic competition.

In the adult cerebellum, the glutamate receptor delta2 subunit (GluRdelta2) is selectively targeted to the spines of the distal Purkinje cell dendrites, the spiny branchlets, that are innervated by the parallel fibers. Although GluRdelta2 has no known channel function, it is presumed to be involved in the formation and stabilization of these synapses. After block of electrical activity by tetrodotoxin, GluRdelta2s appear in the postsynaptic densities of the proximal dendritic spines, which then lose their contact with climbing fibers and become ectopically innervated by parallel fibers. This phenomenon suggests that climbing fiber activity prevents GluRdelta2 targeting to proximal dendrites and that GluRdelta2s admitted to the postsynaptic density of the spine cause withdrawal of the silent climbing fiber. To test this hypothesis, we studied the distribution of GluRdelta2s in the rat cerebellum by immunoelectron microscopy during the recovery period that follows removal of the electrical block, and during the sprouting of climbing fibers that follows subtotal deletion of the parent inferior olivary neurons by administration of the drug 3-acetylpyridine. We found that after removal of the electrical block, the climbing fibers reinnervate proximal spines that bear GluRdelta2s and these subunits are successively repressed. Similarly, after subtotal lesion of the inferior olive, reinnervation of denervated Purkinje cells occurs on spines bearing GluRdelta2s. Thus, GluRdelta2s are not responsible for displacing silent climbing fibers. We propose instead that GluRdelta2s are associated with climbing fiber-to-Purkinje cell synapses, during development or at early stages of climbing fiber regeneration or sprouting, and are downregulated during the process of synapse maturation.

Animals↗

PubMed2003

Sodium imaging of climbing fiber innervation fields in developing mouse Purkinje cells.

Maturation of specific neuronal connections in the mature nervous system includes elimination of redundant synapses formed earlier during development. In the cerebellum of adult animals, each Purkinje cell (PC) is innervated by a single climbing fiber (CF). In early postnatal development each PC is innervated by multiple CFs and elimination of synapses formed by supernumerary CFs occurs until monoinnervation is established at around postnatal day 20 (P20) in mice. It is not clear whether multiple CFs, or only a single CF, translocate from the cell body of immature PCs to the developing dendrite and, in case several CFs translocate, whether they share or segregate their innervation fields. To localize CF innervation fields, we imaged changes in postsynaptic sodium concentration resulting from CF-mediated postsynaptic currents. We found that more than one CF translocates from an innervation field on the cell body of the PC to the developing dendrite and that these CFs share rather than segregate their innervation fields. We concluded that both the soma and the proximal dendrite of the PC are territories of competition for the developing CFs and that the overlapping of their termination fields may be the prerequisite for a local process of elimination of all but one CF, as previously demonstrated in the developing neuromuscular junction.

Aging↗

PubMed2003

Extrinsic regulation of injury/growth-related gene expression in the inferior olive of the adult rat.

Successful axon regeneration relies on the capability of the lesioned neurons to up-regulate a specific set of injury/growth-associated genes. In the adult central nervous system, the strength of the cell body response is generally related to the distance of the injury site from the perikaryon, being stronger for proximal lesions. Nevertheless, inferior olive (IO) cells react to injury and regenerate their axons even after distal transections. To investigate the mechanisms that regulate the IO growth properties, we examined the expression of injury/growth markers (nitric oxide synthase, growth-associated protein 43 and c-Jun) after target deletion or axotomy performed at different sites along the olivocerebellar pathway. Both axon injury and target loss disclose two subsets of IO neurons distributed within precise subnuclei: one subset up-regulates all markers in all conditions, whereas the other shows a mild c-Jun expression but remains unresponsive even after a very proximal axotomy. These observations indicate that distinct subpopulations of IO cells respond to different regulatory strategies. Unresponsive neurons appear insensitive to environmental positive or negative cues, suggesting that they are intrinsically unable to set up a cellular reaction to injury. In contrast, cell body changes in reactive neurons are elicited after the removal of retrogradely transported target-derived inhibitory signals. Target loss also induces degeneration of IO cells, whose survival remains partially dependent on Purkinje targets in adulthood. Thus, the intrinsic regenerative potential of a functionally homogeneous population is regulated by multiple mechanisms, specific for distinct neuronal subsets.

Animals↗

PubMed2002

Target-specific innervation of embryonic cerebellar transplants by regenerating olivocerebellar axons in the adult rat.

The reestablishment of topographically organized connections is a necessary prerequisite to obtain a full anatomical repair following brain injury. One system where such an issue can be addressed is the olivocerebellar system, where, normally, clusters of inferior olive neurons project to neurochemically heterogeneous Purkinje cell compartments defined by the expression of cell-specific markers, such as zebrin II. To assess whether adult injured olivocerebellar axons that regenerate into cerebellar transplants are able to establish target-specific innervation of grafted Purkinje cells, we made surgical transections in the white matter of adult rat cerebella and placed solid grafts from the embryonic cerebellar anlage into the lesion site. The transplanted tissue developed highly organized minicerebella, in which Purkinje cells were distributed into distinct clusters of zebrin II-immunopositive or -immunonegative neurons, mimicking the cortical compartments present in the normal adult cerebellum. Olivocerebellar axons, labeled by biotinylated dextran amine tracing, regenerated into the transplants where they formed discrete patches made of several terminal arbors impinging upon Purkinje cell dendrites. Among 401 such climbing fiber patches, 96% exclusively innervated Purkinje cells of either phenotype and stopped at the border of the zebrin II(+/-) Purkinje cell clusters, whereas only 4% were extended across this boundary and innervated both zebrin II-positive and -negative Purkinje cells. The results obtained support the view that the embryonic cerebellar tissue provides target-specific information that can be decoded by ingrowing adult olivocerebellar axons in order to establish appropriate innervation patterns with zebrin II-positive or -negative Purkinje cell compartments.

Animals↗

PubMed2002

Regenerative and survival capabilities of Purkinje cells overexpressing c-Jun.

Following axotomy, cerebellar Purkinje cells (PCs) do not elongate their axons, even in a favourable environment, and are resistant to death. They have no constitutive presence of common growth-associated proteins, such as GAP-43 and c-Jun. Previous experiments show that injured transgenic PCs overexpressing GAP-43 exhibit a profuse sprouting along the axon and at its severed end. Nevertheless, the lesioned axons are unable to regenerate either spontaneously or into growth-permissive environments. In addition, a considerable number of GAP-43 transgenic PCs degenerate after injury. c-Jun is an inducible transcription factor expressed in axotomized central neurons and regenerating peripheral neurons. It also contributes to programmed cell death during development. To test whether c-Jun could modify the response of PCs to axotomy or enhance the growth/death phenomena of GAP-43 Purkinje neurons, we generated transgenic mice overexpressing c-Jun in PCs. However, c-Jun upregulation did not affect the adult intact phenotype of these neurons and their regenerative and survival capabilities after axotomy. Also in the cross-bred GAP-43/c-Jun mice, c-Jun did not modify the response of GAP-43 PCs to axotomy. By contrast, in organotypic cultures of cerebellum taken from 9-day-old-pups, the survival capabilities of PCs overexpressing c-Jun decreased, in association with a consistent c-Jun phosphorylation. On the whole our data show that c-Jun alone is unable to trigger regenerative or degenerative phenomena in PCs and suggest that the cellular action of this early gene in developing and mature neurons strongly depends on interplaying intracellular signals.

Animals↗