Participation of postsynaptic PKC in cerebellar long-term depression in culture.

Long-term depression (LTD) in the intact cerebellum is a decrease in the efficacy of the parallel fiber-Purkinje neuron synapse induced by coactivation of climbing fiber and parallel fiber inputs. In cultured Purkinje neurons, a similar depression can be induced by iontophoretic glutamate pulses and Purkinje neuron depolarization. This form of LTD is expressed as a depression of alpha-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA)-mediated current, and its induction is dependent on activation of metabotropic quisqualate receptors. The effect of inhibitors of protein kinase C (PKC) on LTD induction was studied. Inhibitors of PKC blocked LTD induction, while phorbol-12,13-diacetate (PDA), a PKC activator, mimicked LTD. These results suggest that PKC activation is necessary for the induction of cerebellar LTD.

Translocation of protein kinase C activity may mediate hippocampal long-term potentiation.

Protein kinase C activity in rat hippocampal membranes and cytosol was determined 1 minute and 1 hour after induction of the synaptic plasticity of long-term potentiation. At 1 hour after long-term potentiation, but not at 1 minute, protein kinase C activity was increased twofold in membranes and decreased proportionately in cytosol, suggesting translocation of the activity. This time-dependent redistribution of enzyme activity was directly related to the persistence of synaptic plasticity, suggesting a novel mechanism regulating the strength of synaptic transmission.

Long-term potentiation of glial synaptic currents in cerebellar culture.

Glial cells in the brain express neurotransmitter receptors and can respond appropriately to application of exogenous neurotransmitters such as glutamate. However, activation of receptors by endogenous, synaptically released transmitter has been difficult to demonstrate directly. Using cell-pair recording in cerebellar cultures from embryonic mouse, it is shown that activation of a cerebellar granule neuron can give rise to a rapid inward current in an adjacent glial cell. This current is mediated by activation of Ca2+-permeable AMPA/kainate receptors and is largely independent of glutamate reuptake or gap junctional coupling. Furthermore, prolonged stimulation of the granule neuron at 4 Hz can give rise to long-term potentiation (LTP) of the glial synaptic current that has similar properties to LTP of granule neuron-Purkinje neuron synaptic transmission--its induction is independent of postsynaptic depolarization, postsynaptic Ca2+ influx, or glutamate receptor activation but requires presynaptic Ca2+ influx. These findings suggest a model in which cerebellar LTP is both induced and expressed presynaptically and therefore may be detected by either neuronal or glial postsynaptic cells.

A protein synthesis-dependent late phase of cerebellar long-term depression.

Synthesis of new protein has been shown to be required for establishment of a late phase in hippocampal long-term potentiation. Whether a similar requirement is needed to produce the late phase of long-term depression (LTD) remains to be determined. Application of transcription inhibitors or of the translation inhibitor anisomycin, immediately, but not 30 min after glutamate/depolarization conjunction, attenuated a late phase of cerebellar LTD in culture. LTD, produced in a perforated outside-out macropatch of Purkinje neuron dendrite, which lacks nuclear material, returned to baseline values with a time course paralleling that observed with protein synthesis inhibitors in intact cultured Purkinje neurons. These findings suggest that there is a distinct late phase of cerebellar LTD that is dependent upon postsynaptic protein synthesis.

Phospholipase A2 controls the induction of short-term versus long-term depression in the cerebellar Purkinje neuron in culture.

Cerebellar long-term depression (LTD) may be reliably induced in the cultured Purkinje neuron when glutamate pulses and Purkinje neuron depolarization are applied together 6 times. When the number of these conjunctive stimuli was reduced to 2, a short-term depression (STD) lasting 20-40 min was induced in 4/12 cells. The enzyme phospholipase A2 cleaves membrane phospholipids causing liberation of free unsaturated fatty acids, which in turn synergistically activate protein kinase C when present with diacylglycerol and Ca. Application of free unsaturated fatty acids with 2 conjunctive stimuli resulted in an apparent conversion of STD cases to LTD. Application of phospholipase A2 inhibitors during 6 conjunctions converted LTD to STD. These findings suggest a model in which liberation of unsaturated fatty acids by phospholipase A2 contributes to a synergistic activation of protein kinase C, the full activation of which results in LTD induction, and the partial activation of which results in STD induction.

Long-term depression of the cerebellar climbing fiber--Purkinje neuron synapse.

In classic Marr-Albus-Ito models of cerebellar function, coactivation of the climbing fiber (CF) synapse, which provides massive, invariant excitation of Purkinje neurons (coding the unconditioned stimulus), together with a graded parallel fiber synaptic array (coding the conditioned stimulus) leads to long-term depression (LTD) of parallel fiber-Purkinje neuron synapses, underlying production of a conditioned response. Here, we show that the supposedly invariant CF synapse can also express LTD. Brief 5 Hz stimulation of the CF resulted in a sustained depression of CF EPSCs that did not spread to neighboring parallel fiber synapses. Like parallel fiber LTD, CF LTD required postsynaptic Ca2+ elevation, activation of group 1 mGluRs, and activation of PKC. CF LTD is potentially relevant for models of cerebellar motor control and learning and the developmental conversion from multiple to single CF innervation of Purkinje neurons.

Defining a minimal computational unit for cerebellar long-term depression.

In cerebellar long-term depression (LTD), conjunctive stimulation of parallel and climbing fiber inputs to a Purkinje neuron (PN) results in a selective depression of parallel fiber-PN synaptic strength. A similar phenomenon may be induced in the cultured PN when glutamate pulses and PN depolarization, which mimic the effects of parallel and climbing fibers, respectively, are coapplied. Here, we show that LTD can be induced in two very reduced preparations of the postsynaptic neuron; an acutely dissociated preparation and a perforated outside-out macropatch of dendritic membrane. LTD in these preparations retains properties of that seen in an intact cultured PN in that it is not induced by either glutamate pulses or depolarization alone and requires calcium influx, mGluR activation, and PKC activation for induction. As both of these preparations lack dendritic spine compartments, these findings suggest that the associative nature of LTD induction does not require this level of morphological organization.

Expression of cerebellar long-term depression requires postsynaptic clathrin-mediated endocytosis.

Cerebellar long-term depression (LTD) is a cellular model system of information storage that may underlie certain forms of motor learning. While cerebellar LTD is expressed as a selective modification of postsynaptic AMPA receptors, this might involve changes in receptor number/distribution, unitary conductance, kinetics, or glutamate affinity. The observation that GluR2-containing synaptic AMPA receptors could be internalized by regulated clathrin-mediated endocytosis suggested that this process could underlie LTD expression. To test this hypothesis, we postsynaptically applied dynamin and amphiphysin peptides that interfere with the clathrin endocytotic complex and found that they blocked LTD expression in cultured Purkinje neurons. In addition, induction of LTD and attenuation of AMPA responses by stimulation of clathrin-mediated endocytosis occluded each other. These findings suggest that the expression of cerebellar LTD requires clathrin-mediated internalization of postsynaptic AMPA receptors.

Induction of cerebellar long-term depression in culture requires postsynaptic action of sodium ions.

Cerebellar long-term depression (LTD) is a persistent attenuation of the parallel fiber-Purkinje neuron (PF-PN) synapse induced by conjunctive stimulation of PF and climbing fiber (CF) inputs. A similar phenomenon is seen in the voltage-clamped PN in tissue culture when iontophoretic quisqualate application and PN depolarization are substituted for PF and CF stimulation, respectively. In this model, LTD induction requires activation of both AMPA and metabotropic receptors, together with PN depolarization. We have sought to determine the role of the AMPA receptor in LTD induction. The AMPA receptor does not appear to exert its effect by directly gating Ca2+ influx. Replacement of external Na+ during quisqualate/depolarization conjunction with permeant ions caused a blockade of LTD induction, suggesting that Na+ influx through the AMPA-associated channel is necessary for this process. Similarly, pairing quisqualate pulses with depolarizing steps near ENa also failed to induce LTD. The present results indicate that postsynaptic Na+ influx is necessary for LTD induction. While a portion of the relevant Na+ influx is provided by voltage-gated channels, the AMPA-associated ion channel is most important in this regard.

Polarity of long-term synaptic gain change is related to postsynaptic spike firing at a cerebellar inhibitory synapse.

Long-term potentiation and depression (LTP and LTD) in excitatory synapses can coexist, the former being triggered by stimuli that produce strong postsynaptic excitation and the latter by stimuli that produce weaker postsynaptic excitation. It has not been determined whether these properties also apply to LTP and LTD in the inhibitory synapses between Purkinje neurons and the neurons of the deep cerebellar nuclei (DCN), a site that has been implicated in certain types of motor learning. DCN cells exhibit a prominent rebound depolarization (RD) and associated spike burst upon release from hyperpolarization. In these cells, LTP can be elicited by short, high-frequency trains of inhibitory postsynaptic potentials (IPSPs), which reliably evoke an RD. LTD is induced if the same protocol is applied with conditions where the amount of postsynaptic excitation is reduced. The polarity of the change in synaptic strength is correlated with the amount of RD-evoked spike firing during the induction protocol. Thus, some important computational principles that govern the induction of use-dependent change in excitatory synaptic efficacy also apply to inhibitory synapses.

A late phase of cerebellar long-term depression requires activation of CaMKIV and CREB.

Recently, it has been shown that cerebellar LTD has a late phase that may be blocked by protein synthesis inhibitors. To understand the mechanisms underlying the late phase, we interfered with the activation of transcription factors that might couple synaptic activation to protein synthesis. Particle-mediated transfection of cultured Purkinje neurons with an expression vector encoding a dominant inhibitory form of CREB resulted in a nearly complete blockade of the late phase. Kinases that activate CREB were inhibited, and LTD was assessed. Inhibition of PKA or the MAPK/RSK cascades were without effect on the late phase, while constructs designed to interfere with CaMKIV function attenuated the late phase. These results indicate that the activation of CaMKIV and CREB are necessary to establish a late phase of cerebellar LTD.

Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins.

Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. Recently, it was shown that AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ domain-containing proteins GRIP/ABP and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuated LTD induction by glutamate/depolarization pairing, as did antibodies directed against the PDZ domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ domain-containing proteins.

Impaired cerebellar long-term potentiation in type I adenylyl cyclase mutant mice.

Activation of adenylyl cyclase and the consequent production of cAMP is a process that has been shown to be central to invertebrate model systems of information storage. In the vertebrate brain, it has been suggested that a presynaptic cascade involving Ca influx, cAMP production, and subsequent activation of cAMP-dependent protein kinase is necessary for induction of long-term potentiation (LTP) at the cerebellar parallel fiber-Purkinje cell synapse. We have used mutant mice in which the major Ca-sensitive adenylyl cyclase isoform of cerebellar cortex (type I) is deleted to show that this results in an approximately 65% reduction in cerebellar Ca-sensitive cyclase activity and a nearly complete blockade of cerebellar LTP assessed using granule cell-Purkinje cell pairs in culture. This blockade is not accompanied by alterations in a number of basal electrophysiological parameters and may be bypassed by application of an exogenous cAMP analog, suggesting that it results specifically from deletion of the type I adenylyl cyclase.

A long-term depression of AMPA currents in cultured cerebellar Purkinje neurons.

Cerebellar long-term depression (LTD) is a model of synaptic plasticity in which conjunctive stimulation of parallel fiber and climbing fiber inputs to a Purkinje neuron induces a persistent depression of the parallel fiber-Purkinje neuron synapse. We report that an analogous phenomenon may be elicited in the cultured mouse Purkinje neuron when iontophoretic glutamate application and depolarization of the Purkinje neurons are substituted for parallel fiber and climbing fiber stimulation, respectively. The induction of LTD in these cerebellar cultures requires activation of both ionotropic (AMPA) and metabotropic quisqualate receptors, together with depolarization in the presence of external Ca2+. This postsynaptic alteration is manifest as a depression of glutamate or AMPA currents, but not aspartate or NMDA currents. These results strengthen the contention that the expression of cerebellar LTD is at least in part postsynaptic and provide evidence that activation of both ionotropic and metabotropic quisqualate receptors are necessary for LTD induction.

Expression of a protein kinase C inhibitor in Purkinje cells blocks cerebellar LTD and adaptation of the vestibulo-ocular reflex.

Cerebellar long-term depression (LTD) is a model system for neuronal information storage that has an absolute requirement for activation of protein kinase C (PKC). It has been claimed to underlie several forms of cerebellar motor learning. Previous studies using various knockout mice (mGluR1, GluRdelta2, glial fibrillary acidic protein) have supported this claim; however, this work has suffered from the limitations that the knockout technique lacks anatomical specificity and that functional compensation can occur via similar gene family members. To overcome these limitations, a transgenic mouse (called L7-PKCI) has been produced in which the pseudosubstrate PKC inhibitor, PKC[19-31], was selectively expressed in Purkinje cells under the control of the pcp-2(L7) gene promoter. Cultured Purkinje cells prepared from heterozygous or homozygous L7-PKCI embryos showed a complete blockade of LTD induction. In addition, the compensatory eye movements of L7-PKCI mice were recorded during vestibular and visual stimulation. Whereas the absolute gain, phase, and latency values of the vestibulo-ocular reflex and optokinetic reflex of the L7-PKCI mice were normal, their ability to adapt their vestibulo-ocular reflex gain during visuo-vestibular training was absent. These data strongly support the hypothesis that activation of PKC in the Purkinje cell is necessary for cerebellar LTD induction, and that cerebellar LTD is required for a particular form of motor learning, adaptation of the vestibulo-ocular reflex.

Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors.

Group I metabotropic glutamate receptors (mGluRs) activate PI turnover and thereby trigger intracellular calcium release. Previously, we demonstrated that mGluRs form natural complexes with members of a family of Homer-related synaptic proteins. Here, we present evidence that Homer proteins form a physical tether linking mGluRs with the inositol trisphosphate receptors (IP3R). A novel proline-rich "Homer ligand" (PPXXFr) is identified in group 1 mGluRs and IP3R, and these receptors coimmunoprecipitate as a complex with Homer from brain. Expression of the IEG form of Homer, which lacks the ability to cross-link, modulates mGluR-induced intracellular calcium release. These studies identify a novel mechanism in calcium signaling and provide evidence that an IEG, whose expression is driven by synaptic activity, can directly modify a specific synaptic function.

Beyond parallel fiber LTD: the diversity of synaptic and non-synaptic plasticity in the cerebellum.

In recent years, it has become clear that motor learning, as revealed by associative eyelid conditioning and adaptation of the vestibulo-ocular reflex, contributes to the well-established cerebellar functions of sensorimotor integration and control. Long-term depression of the parallel fiber-Purkinje cell synapse (which is often called 'cerebellar LTD') is a cellular phenomenon that has been suggested to underlie these forms of learning. However, it is clear that parallel fiber LTD, by itself, cannot account for all the properties of cerebellar motor learning. Here we review recent electrophysiological experiments that have described a rich variety of use-dependent plasticity in cerebellum, including long-term potentiation (LTP) and LTD of excitatory and inhibitory synapses, and persistent modulation of intrinsic neuronal excitability. Finally, using associative eyelid conditioning as an example, we propose some ideas about how these cellular phenomena might function and interact to endow the cerebellar circuit with particular computational and mnemonic properties.

Cellular mechanisms of long-term depression in the cerebellum.

Cerebellar long-term depression is a persistent, input-specific attenuation of the parallel fiber-Purkinje neuron synapse induced by co-activation of parallel fibers and climbing fibers. This phenomenon endows the Purkinje neuron with a powerful associative computational ability. Recent investigations have provided strong evidence that two mechanisms, Ca2+ influx via voltage-gated channels, and stimulation of protein kinase C via metabotropic receptor activation, are required for induction of long-term depression. In addition, two other mechanisms, Na+ influx via AMPA receptors, and stimulation of a nitric oxide/cGMP cascade may also be involved in this process.

Selective decline in protein F1 phosphorylation in hippocampus of senescent rats.

Certain forms of neuronal plasticity have been found to be expressed through alterations in brain protein phosphorylation, and its regulation by protein kinase activity. Of interest in this regard is the possibility that the decline in neuronal plasticity and cognitive function that occurs in advanced age may result in part from altered phosphorylation of specific proteins. As a first attempt to identify age-related changes in phosphoproteins, we assayed in vitro phosphorylation of proteins in hippocampus, cerebellum, entorhinal cortex, and frontal cortex from Fischer-344 rats of 5 months, 11 months, and 25 months of age. Compared to the middle-aged animals, the aged rats showed a selective 46% decline in phosphorylation of the 47 kDa protein (F1) in hippocampus, with no change in the phosphorylation of other proteins measured in this structure. Aged animals also showed decreased phosphorylation relative to young animals. No age-related change was observed in any protein band for the other brain areas examined. Since protein F1 is phosphorylated by protein kinase C (PKC), the cytosolic and membrane distribution of this enzyme was compared across age groups. The activity of PKC in hippocampus did not change across age. The explanation of this age-related decline in protein F1 phosphorylation is likely to be a decline in the substrate protein itself. The results are discussed in terms of protein F1's possible role in age-related decline of hippocampal synaptic plasticity.

A newly discovered protein kinase C activator (oleic acid) enhances long-term potentiation in the intact hippocampus.

Protein kinase C can be activated by oleate, an unsaturated fatty acid. Since protein kinase C is activated by long-term potentiation, we wished to determine whether iontophoretic ejection of oleate into the intact hippocampal dentate gyrus of urethane-anesthetized rats would cause an enhancement of the response potentiated by high frequency stimulation of the perforant path. Oleate ejection did significantly enhance the persistence of the potentiated response. Moreover, a growth of the response beyond the initial potentiation was seen. Arachidonate, which stimulates protein kinase C to a lesser degree, had a significant preservation effect, but no effect on growth of the response. After vehicle and elaidate (trans-stereoisomer of oleate) ejections, the potentiated response decayed to baseline values. In addition, the persistence of the potentiated response observed two hours after its induction was positively correlated with the ability of an unsaturated fatty acid to activate protein kinase C in vitro. The present results support the proposal that protein kinase C activation enhances synaptic strength. It is suggested that one mechanism for this activation may be PLA2-mediated release of oleate.