Long-Term Synaptic Changes Induced in the Cerebellar Cortex by Fear Conditioning

AbstractTo 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

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

Impaired Sprouting and Axonal Atrophy in Cerebellar Climbing Fibres following In Vivo Silencing of the Growth-Associated Protein GAP-43

The adult mammalian central nervous system has a limited ability to establish new connections and to recover from traumatic or degenerative events. The olivo-cerebellar network represents an excellent model to investigate neuroprotection and repair in the brain during adulthood, due to its high plasticity and ordered synaptic organization. To shed light on the molecular mechanisms involved in these events, we focused on the growth-associated protein GAP-43 (also known as B-50 or neuromodulin). During development, this protein plays a crucial role in growth and in branch formation of neurites, while in the adult it is only expressed in a few brain regions, including the inferior olive (IO) where climbing fibres (CFs) originate. Following axotomy GAP-43 is usually up-regulated in association with regeneration. Here we describe an in vivo lentiviral-mediated gene silencing approach, used for the first time in the olivo-cerebellar system, to efficiently and specifically downregulate GAP-43 in rodents CFs. We show that lack of GAP-43 causes an atrophy of the CF in non-traumatic conditions, consisting in a decrease of its length, branching and number of synaptic boutons. We also investigated CF regenerative ability by inducing a subtotal lesion of the IO. Noteworthy, surviving CFs lacking GAP-43 were largely unable to sprout on surrounding Purkinje cells. Collectively, our results demonstrate that GAP-43 is essential both to maintain CFs structure in non-traumatic condition and to promote sprouting after partial lesion of the IO

GluRδ2 Expression in the Mature Cerebellum of Hotfoot Mice Promotes Parallel Fiber Synaptogenesis and Axonal Competition

Glutamate receptor delta 2 (GluRdelta2) is selectively expressed in the cerebellum, exclusively in the spines of the Purkinje cells (PCs) that are in contact with parallel fibers (PFs). Although its structure is similar to ionotropic glutamate receptors, it has no channel function and its ligand is unknown. The GluRdelta2-null mice, such as knockout and hotfoot have profoundly altered cerebellar circuitry, which causes ataxia and impaired motor learning. Notably, GluRdelta2 in PC-PF synapses regulates their maturation and strengthening and induces long term depression (LTD). In addition, GluRdelta2 participates in the highly territorial competition between the two excitatory inputs to the PC; the climbing fiber (CF), which innervates the proximal dendritic compartment, and the PF, which is connected to spiny distal branchlets. Recently, studies have suggested that GluRdelta2 acts as an adhesion molecule in PF synaptogenesis. Here, we provide in vivo and in vitro evidence that supports this hypothesis. Through lentiviral rescue in hotfoot mice, we noted a recovery of PC-PF contacts in the distal dendritic domain. In the proximal domain, we observed the formation of new spines that were innervated by PFs and a reduction in contact with the CF; ie, the pattern of innervation in the PC shifted to favor the PF input. Moreover, ectopic expression of GluRdelta2 in HEK293 cells that were cocultured with granule cells or in cerebellar Golgi cells in the mature brain induced the formation of new PF contacts. Collectively, our observations show that GluRdelta2 is an adhesion molecule that induces the formation of PF contacts independently of its cellular localization and promotes heterosynaptic competition in the PC proximal dendritic domain

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

Intensive remodeling of Purkinje cell spines after climbing fibers deafferentation does not involve MAPK and Akt activation

Subtotal lesion of the inferior olive (IO) achieved by treating experimental animals with 3-acetylpyridine (3AP) induces partial Purkinje cells (PCs) deafferentation that leads to PC hyperactivity and new spine formation. Coincidentally, the olivary terminals belonging to the few survived olivary neurons undergo an extensive collateral sprouting resulting in reinnervation of the neighboring denervated PCs. We obtained chemical deafferentation of PCs in adult rats (body weight, 120-170 g; age, 35-40 days) by a single intraperitoneal injection of 3AP (65 mg/kg body weight), and as early as 3 days after 3AP treatment, important morphological changes could be observed on PCs. Mitogen-activated protein kinase (MAPK) cascades and more specifically extracellular signal-regulated kinases 1/2 (ERK1/2) play a critical role in the signaling events underlying synaptic plasticity. For instance, long-term depression (LTD) in the adult hippocampus and long-term potentiation (LTP) in cerebellum both involve ERK activation. Since PCs deprived of their climbing fibers (CFs) afferents initiate an intensive remodeling of the spines and rapid recall of the remaining CFs, it prompted us to see whether the observed phenomena correlated with MAPK and Akt activation. Immunohistochemistry and Western blotting were done at various time points after 3AP application (from 24 h to 6 days), as the exact dynamics of CF loss is not precisely known. As judged by Western blotting, there was no increase of activated ERK in the cerebellum. However, immunohistochemistry revealed increased ERK phosphorylation in the "pinceaux" of basket cells in 3AP animals. Similarly, stress-activated protein kinase(SAPK)/c-Jun N-terminal kinase (JNK), p38 MAPK, and Akt activation were also studied by means of Western blotting and immunohistochemistry. Upon 3AP treatment no changes in phosphorylation status could be seen in the different kinases subjected to analysis. Our results suggest that activation of MAPK and Akt cascades is not essential in this model of neuronal plasticity

Basolateral Amygdala Inactivation Impairs Learning- Induced Long-Term Potentiation in the Cerebellar Cortex

Learning to fear dangerous situations requires the participation of basolateral amygdala (BLA). In the present study, we provide evidence that BLA is necessary for the synaptic strengthening occurring during memory formation in the cerebellum in rats. In the cerebellar vermis the parallel fibers (PF) to Purkinje cell (PC) synapse is potentiated one day following fear learning. Pretraining BLA inactivation impaired such a learning-induced long-term potentiation (LTP). Similarly, cerebellar LTP is affected when BLA is blocked shortly, but not 6 h, after training. The latter result shows that the effects of BLA inactivation on cerebellar plasticity, when present, are specifically related to memory processes and not due to an interference with sensory or motor functions. These data indicate that fear memory induces cerebellar LTP provided that a heterosynaptic input coming from BLA sets the proper local conditions. Therefore, in the cerebellum, learninginduced plasticity is a heterosynaptic phenomenon that requires inputs from other regions. Studies employing the electrically-induced LTP in order to clarify the cellular mechanisms of memory should therefore take into account the inputs arriving from other brain sites, considering them as integrative units. Based on previous and the present findings, we proposed that BLA enables learning-related plasticity to be formed in the cerebellum in order to respond appropriately to new stimuli or situations