Piergiorgio Strata

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. J. Comp. Neurol. 494:559–577, 2006.

Learning-related feedforward inhibitory connectivity growth required for memory precision

In the adult brain, new synapses are formed and pre-existing ones are lost, but the function of this structural plasticity has remained unclear. Learning of new skills is correlated with formation of new synapses. These may directly encode new memories, but they may also have more general roles in memory encoding and retrieval processes. Here we investigated how mossy fibre terminal complexes at the entry of hippocampal and cerebellar circuits rearrange upon learning in mice, and what is the functional role of the rearrangements. We show that one-trial and incremental learning lead to robust, circuit-specific, long-lasting and reversible increases in the numbers of filopodial synapses onto fast-spiking interneurons that trigger feedforward inhibition. The increase in feedforward inhibition connectivity involved a majority of the presynaptic terminals, restricted the numbers of c-Fos-expressing postsynaptic neurons at memory retrieval, and correlated temporally with the quality of the memory. We then show that for contextual fear conditioning and Morris water maze learning, increased feedforward inhibition connectivity by hippocampal mossy fibres has a critical role for the precision of the memory and the learned behaviour. In the absence of mossy fibre long-term potentiation in Rab3a−/− mice, c-Fos ensemble reorganization and feedforward inhibition growth were both absent in CA3 upon learning, and the memory was imprecise. By contrast, in the absence of adducin 2 (Add2; also known as β-adducin) c-Fos reorganization was normal, but feedforward inhibition growth was abolished. In parallel, c-Fos ensembles in CA3 were greatly enlarged, and the memory was imprecise. Feedforward inhibition growth and memory precision were both rescued by re-expression of Add2 specifically in hippocampal mossy fibres. These results establish a causal relationship between learning-related increases in the numbers of defined synapses and the precision of learning and memory in the adult. The results further relate plasticity and feedforward inhibition growth at hippocampal mossy fibres to the precision of hippocampus-dependent memories.

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.

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.

Synapse formation and clustering of neuroligin-2 in the absence of GABAA receptors

GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABAAα1 knockout (KO) mice express transiently the α3 subunit, leading to the assembly of functional GABAA receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the α3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABAA receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABAA receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.

Spontaneous Saccades and Gaze-Holding Ability in the Pigmented Rat. II. Effects of Localized Cerebellar Lesions

We have studied the effects of the ablation of the cerebellar vermal area corresponding to lobules VI – VIII and of the flocculus – paraflocculus of both sides on the spontaneous eye movements performed in the light and in the dark in head‐restrained pigmented rats. These effects have been compared with those already described for the inferior olive lesion. The cerebellar lesions were performed 1 week to 6 months in advance. Eye movements were recorded through a phase detection search coil apparatus. Following vermal topectomy, the main characteristics of the spontaneous saccades are unmodified. Following the ablation of the flocculus – paraflocculus there is no change in the saccadic main sequence. However, the spontaneous saccades in the dark present a postsaccadic drift made up of two components with different time courses, the first one being fast and the second one slow. The former is due in part to a mismatch between the phasic (the pulse) and the tonic (the step) components of the eye movements; the latter to the leakage of the neural integrator. In light only the first component is present and the eye maintains a steady position. The time constant of the neural integrator is considerably reduced to ∼600–900 ms from a value of ∼1600–4000 ms in the intact rats. The amplitude of the postsaccadic drift in the light depends on both the mismatch between the pulse and the step of innervation of the extraocular muscles and the increased leakiness of the neural integrator. The gain of the pulse to step transformation is reduced to ∼0.79 at all saccadic amplitudes and eccentricities and such a reduction is due to a decreased step amplitude, while the pulse amplitude remains unchanged. The contribution of the leakage of the neural integrator to the postsaccadic drift in the light is a function of the eccentricity with a slope of 0.23. The deficits described after flocculus–paraflocculus ablation are also very similar to those described following inferior olive lesion from a quantitative point of view. The possible mechanisms of the visually activated olivocerebellar system in the control of saccadic performance and in maintaining its calibration are discussed.

Role of glutamate δ-2 receptors in activity-dependent competition between heterologous afferent fibers

A principle that regulates detailed architecture in the brain is that active terminals have a competitive advantage over less active terminals in establishing synaptic connections. This principle is known to apply to fibers within a single neuronal population competing for a common target domain. Here we uncover an additional rule that applies when two neuronal populations compete for two contiguous territories. The cerebellar Purkinje cell dendrites have two different synaptic domains with spines innervated by two separate excitatory inputs, parallel fibers (PFs) and climbing fibers (CFs). Glutamate δ-2 receptors are normally present only on the PF spines where they are important for their innervation. After block of activity by tetrodotoxin, numerous new spines form in the CF domain and become innervated mainly by PFs; all spines, including those still innervated by the CFs, bear δ-2 receptors. Thus, in the absence of activity, PFs gain a competitive advantage over CFs. The entire dendritic arbor becomes a uniform territory with the molecular cues associated with the PFs. To access their proper territory and maintain synaptic contacts, CFs must be active and locally repress the cues of the competitor afferents.

In vivo single branch axotomy induces GAP-43–dependent sprouting and synaptic remodeling in cerebellar cortex

Plasticity in the central nervous system in response to injury is a complex process involving axonal remodeling regulated by specific molecular pathways. Here, we dissected the role of growth-associated protein 43 (GAP-43; also known as neuromodulin and B-50) in axonal structural plasticity by using, as a model, climbing fibers. Single axonal branches were dissected by laser axotomy, avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Despite the very small denervated area, the injured axons consistently reshape the connectivity with surrounding neurons. At the same time, adult climbing fibers react by sprouting new branches through the intact surroundings. Newly formed branches presented varicosities, suggesting that new axons were more than just exploratory sprouts. Correlative light and electron microscopy reveals that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites. By using an RNA interference approach, we found that downregulating GAP-43 causes a significant increase in the turnover of presynaptic boutons. In addition, silencing hampers the generation of reactive sprouts. Our findings show the requirement of GAP-43 in sustaining synaptic stability and promoting the initiation of axonal regrowth.

Interleukin-1β Alters Glutamate Transmission at Purkinje Cell Synapses in a Mouse Model of Multiple Sclerosis

Cerebellar deficit contributes significantly to disability in multiple sclerosis (MS). Several clinical and experimental studies have investigated the pathophysiology of cerebellar dysfunction in this neuroinflammatory disorder, but the cellular and molecular mechanisms are still unclear. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, proinflammatory cytokines, together with a degeneration of inhibitory neurons, contribute to impair GABAergic transmission at Purkinje cells (PCs). Here, we investigated glutamatergic transmission to gain insight into the pathophysiology of cerebellar dysfunction in EAE. Electrophysiological recordings from PCs showed increased duration of spontaneous excitatory postsynaptic currents (EPSCs) during the symptomatic phase of EAE, suggesting an alteration of glutamate uptake played by Bergmann glia. We indeed observed an impaired functioning of the glutamate-aspartate transporter/excitatory amino acid transporter 1 (GLAST/EAAT1) in EAE cerebellum caused by protein downregulation and in correlation with prominent astroglia activation. We have also demonstrated that the proinflammatory cytokine interleukin-1β (IL-1β), released by a subset of activated microglia/macrophages and infiltrating lymphocytes, was involved directly in such synaptic alteration. In fact, brief incubation of IL-1β in normal cerebellar slices replicated EAE modifications through a rapid GLAST/EAAT1 downregulation, whereas incubation of an IL-1 receptor antagonist (IL-1ra) in EAE slices reduced spontaneous EPSC alterations. Finally, EAE mice treated with intracerebroventricular IL-1ra showed normal glutamatergic and GABAergic transmissions, along with GLAST/EAAT1 normalization, milder inflammation, and reduced motor deficits. These results highlight the crucial role played by the proinflammatory IL-1β in triggering molecular and synaptic events involved in neurodegenerative processes that characterize neuroinflammatory diseases such as MS.

Inferior olive inactivation decreases the excitability of the intracerebellar and lateral vestibular nuclei in the rat.

In rats under sodium pentobarbitone anaesthesia the inferior olive region has been reversibly inactivated by applying a cooling probe to the ventral surface of the medulla. Unitary activity has been recorded from the fastigial, interpositus and Deiters nuclei. Identification of units was based on the presence of a dye spot, left by the recording micropipette. In the Deiters nucleus, an additional criterion of identification was the antidromic activation from spinal cord stimulation. Following cooling of the inferior olive of one side, we have observed suppression of the activity of all the fourteen Deiters neurones and of seventeen out of twenty neurones recorded from the intracerebellar nuclei. In two out of seven Deiters neurones tested the antidromic invasion elicited by spinal cord stimulation was suppressed. In rats, whose inferior olive was previously destroyed, cooling of the inferior olive region was not followed by the powerful depression of spike activity seen in the vestibular and cerebellar nuclei cells in the intact rats. These results indicate that the olivocerebellar system is very important in regulating the level of excitability of the subcerebellar structures and therefore in controlling both postural mechanisms and the processing of information relating to sensorimotor integration.