Nicholas A. Hartell

Strong Activation of Parallel Fibers Produces Localized Calcium Transients and a Form of LTD That Spreads to Distant Synapses

The temporal and spatial changes in intracellular calcium levels during separate activation of parallel fiber (PF) and climbing fiber (CF) inputs to cerebellar Purkinje cells were studied. PF stimulation (1 Hz), at relatively high stimulus strengths, led to accumulations of calcium that were similar in peak levels to those following CF stimulation but that remained spatially localized. Such stimuli consistently induced a durable depression of PF synaptic transmission that partially occluded further depression by conventional conjunctive stimuli and that was independent of nitric oxide. This depression was accompanied by a reduction of synaptic efficacy in spatially isolated PF inputs to the same cell that was independent of postsynaptic calcium but that was mediated by nitric oxide. These data indicate that LTD comprises at least two separate processes and that parameters of PF stimulation that are capable of raising calcium levels in Purkinje cell dendrites are also able to induce long-term changes in synaptic efficacy.

Induction of cerebellar long-term depression requires activation of glutamate metabotropic receptors

In rat cerebellar slices, 500 microM (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) reversibly inhibited both dendritic and somatic increases in FLUO-3 fluorescence intensity induced by bath applications of 50-100 microM (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD). No effect of MCPG was observed on dendritically recorded excitatory postsynaptic potentials evoked by synaptic activation of either parallel or climbing fibres. Long-term depression of parallel fibre-Purkinje cell transmission, induced either by conjunctive activation of parallel and climbing fibres or by pairing parallel fibre stimulation with intradendritic injections of 8-BrcGMP, was not only prevented in the presence of MCPG but a robust long-term potentiation of responses consistently occurred. These data show that metabotropic glutamate receptor activation is necessary for the induction of LTD.

Nitric oxide is required for the induction and heterosynaptic spread of long-term potentiation in rat cerebellar slices

1 In the cerebellar cortex, brief, 8 Hz activation of parallel fibres (PFs) induces a cyclic adenosine 3′5’‐monophosphate (cAMP) and protein kinase A (PKA)‐dependent form of long‐term potentiation between PFs and Purkinje cells. 2 With 10 mm BAPTA in the recording pipette, potentiation evoked by raised frequency stimulation (RFS) to one of two, synaptically independent PF inputs to the same Purkinje cell did not remain input specific but consistently spread to synapses that did not receive RFS, up to the maximum distance tested of 168 μm. 3 LTP at activated and non‐activated sites was accompanied by a decrease in paired pulse facilitation (PPF). The PKA inhibitor H‐89 blocked both of these effects. Inhibition of nitric oxide synthase (NOS), either by 7‐nitro‐indazole (7‐NI) or NG‐nitro‐l‐arginine methyl ester (l‐NAME), completely prevented heterosynaptic potentiation and associated reduction in PPF. LTP at distant synapses was selectively prevented by the nitric oxide scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO). Inhibition of soluble guanylate cyclase or protein kinase G had no effect on either pathway. 4 Synaptic potentiation at PF‐PC synapses, induced by the adenylate cyclase activator forskolin, was also prevented by inhibition of NOS. Forskolin‐induced increases in mEPSC frequency were similarly prevented by NOS inhibition and mimicked by the NO donor spermine NONOate. 5 These results are consistent with the notion that heterosynaptic potentiation is of pre‐synaptic origin and dependent upon activation of cAMP/PKA and NO. Moreover, they suggest that cAMP/PKA activation stimulates NO production and this diffusible messenger facilitates pre‐synaptic transmitter release at synapses within a radius of upwards of 150 μm, through a mechanism that does not involve cGMP.

Differences in Transmission Properties and Susceptibility to Long-Term Depression Reveal Functional Specialization of Ascending Axon and Parallel Fiber Synapses to Purkinje Cells

An understanding of the patterns of mossy fiber transmission to Purkinje cells, via granule cell axons, is fundamental to models of cerebellar cortical signaling and processing. Early theories assumed that mossy fiber input is widely disseminated across the cerebellar cortex along beams of parallel fibers, which spread for several millimeters across the cerebellar cortex. Direct evidence for this has, however, proved controversial, leading to the development of an alternative hypothesis that mossy fiber inputs to the cerebral cortex are in fact vertically organized such that the ascending segment of the granule axon carries a greater synaptic weight than the parallel fiber segment. Here, we report that ascending axon synapses are selectively resistant to cerebellar long-term depression and that they release transmitter with higher mean release probabilities and mean quantal amplitudes than parallel fiber synapses. This novel specialization of synapses formed by different segments of the same axon not only explains the reported patterns of granule cell→ Purkinje cell transmission across the cerebellar cortex but also reveals an additional level of functionality and complexity of cerebellar processing. Consequently, ascending axon synapses represent a new element of cortical signal processing that should be distinguished from parallel fiber synapses in future experimental and theoretical studies of cerebellar function.

Parallel fiber plasticity

Cerebellar long-term depression (LTD) is classically observed when climbing fibers, originating from the inferior olive, and parallel fibers, axons of granule cells, are activated repetitively and synchronously. On the basis that the climbing fiber signals errors in motor performance, LTD provides a mechanism of learning whereby inappropriate motor signals, relayed to the cerebellar cortex by parallel fibers, are selectively weakened through their repeated, close temporal association with climbing fiber activity. LTD therefore provides a cellular substrate for error-driven motor learning in the cerebellar cortex. In recent years, it has become apparent that depression at this synapse can also occur without the need for concurrent climbing fiber activation provided the parallel fibers are activated in such a way as to mobilize calcium within the Purkinje cell. A form of long-term potentiation (LTP) has also been uncovered at this synapse, which similarly relies only upon parallel fiber activation. In brain slice preparations and contrary to expectation, each of these forms of parallel fiber induced plasticity, as well as classical LTD, does not remain confined to activated parallel fibers as previously thought, but both depression and potentiation have the capacity to spread to neighboring parallel fiber synapses several tens of microns away from the activated fibers. Here, the cellular mechanisms responsible for the induction and heterosynaptic spread of parallel fiber LTP and LTD are compared to those involved in classical LTD and the physiological implications that the heterosynaptic spread of plasticity may have on cerebellar signal processing are discussed.

An evaluation of the synapse specificity of long-term depression induced in rat cerebellar slices

1 Whole‐cell excitatory postsynaptic currents (EPSCs) were recorded from single Purkinje cells (PCs) in rat cerebellar slices in response to alternate activation of two separate sets of parallel fibres (PF1 and PF2). Pairing the stimulation of one input (PF1) with PC depolarisation at 1 Hz for 5 min produced varied effects, including a long‐term depression (LTD) of subsequent responses, a medium‐term potentiation, or no change relative to baseline levels (n= 14). In all but two cases PF2 responses mirrored those in PF1, in both direction and magnitude even though this second pathway was not specifically activated during pairing. 2 Increasing the stimulus strength to evoke larger amplitude EPSCs (> 1000 pA) dramatically increased the proportion of cells that underwent LTD in both PF1 and PF2. LTD in both pathways was postsynaptic calcium dependent. PC depolarisation alone (n= 7) or PF1 stimulation paired with PC hyperpolarisation (n= 6) failed to induce LTD at either site. 3 Pairing PF1 stimulation with climbing fibre (CF) activation at 1 Hz for 5 min produced LTD in the majority of cells regardless of the strength of PF stimulation. LTD under these conditions was not, however, input specific, even at the lowest stimulus strengths. 4 With EPSCs greater than 1000 pA in amplitude, depression was apparent in both pathways even when the duration of PF1 pairing with depolarisation was limited to 1 min. Full expression of LTD in PF2 required stimulation of this pathway to be resumed within a distinct temporal window of conjunctive pairing with PF1. Introducing a delay of 20 min before resumption of PF2 activation preserved the input specificity of synaptic depression. 5 We conclude that pairing either PC depolarisation or CF activation with stimulation of a discrete set of PFs produces LTD that spreads to adjacent synapses on the same PC.

Differential Susceptibility to Synaptic Plasticity Reveals a Functional Specialization of Ascending Axon and Parallel Fiber Synapses to Cerebellar Purkinje Cells

Granule cell axons, via their parallel fibers, form synapses with Purkinje cells across large areas of the cerebellar cortex. Evidence for uniform transmission along parallel fibers to Purkinje cells is controversial, however, leading to speculation that the ascending axonal segment plays a dominant role in cerebellar processing. We have compared the relative susceptibilities of ascending axon and parallel fiber synaptic inputs to several forms of synaptic plasticity. We demonstrate that ascending axon synapses have a limited capability to undergo forms of long-term depression and potentiation compared with parallel fiber synapses. These results demonstrate that these two segments of the same axon play fundamentally different roles in cerebellar signaling, and, as such, the synapses formed between granule cells and Purkinje cells should not be treated as a homogenous population.

Intercellular action of nitric oxide increases cGMP in cerebellar Purkinje cells

cGMP is thought to play a role in cerebellar signalling yet its production within Purkinje cells has never been detected. In the present study, the hydrolysis of a fluorescent substrate analogue, 2′-O-anthranyloyl cyclic GMP, by type 5 phosphodiesterase was monitored within Purkinje cells in slices and in culture. Nitric oxide, either endogenously released from adjacent neurons or pharmacologically applied, accelerated the rate of hydrolysis in a manner that was dependent on soluble guanylyl cyclase, demonstrating that nitric oxide triggers cyclic GMP production in Purkinje cells, which in turn activates type 5 phosphodiesterase. We conclude that NO acts as an intercellular messenger in the cerebellar cortex and that parallel fibre terminals are a probable source of nitric oxide.

An intrinsically fluorescent dendrimer as a nanoprobe of cell transport

Dendrimers, spherical or quasi-spherical synthetic polymers in the nano-size range, have found useful applications as prospective carriers in drug and gene delivery. The investigation of dendrimer uptake by cells has been previously achieved by the incorporation of a fluorescent dye to the dendrimer either by chemical conjugation or by physical interaction. Here we describe the synthesis of two intrinsically fluorescent lysine based cationic dendrimers which lack a fluorophore, but which has sufficient fluorescence intensity to be detected at low concentrations. The nomenclature used to describe our compounds results in, for example the 6th generation dendrimer being notated as Gly–Lys63(NH2)64; Gly denotes that the compound has a glycine in the core coupled to 63 lysine branching units (Lys63) and that the surface has 64 free amino groups (NH2)64. The use of these dendrimers in probing transport avoids the need for fluorescent tagging with its attendant problems. The uptake of Gly–Lys63(NH2)64 into Caco-2 cells was followed using confocal microscopy. Being cationic, it first adsorbs to the cell surface, enters the cytoplasm and reaches the nucleus within 35–45 min. Estimates of the diffusion coefficient of the dendrimer within the cell cytoplasm leads to a value of 6.27 ( ± 0.49) × 10− 11 cm2s− 1, which is up to 1000 times lower than the diffusion coefficient of the dendrimer in water. Intrinsically fluorescent dendrimers of different size and charge are useful probes of transport in cells.

Receptors, second messengers and protein kinases required for heterosynaptic cerebellar long-term depression

Raising the frequency and intensity of stimulation to one of two sets of parallel fibre synaptic inputs to cerebellar Purkinje cells results in a localised calcium influx and a long-term depression (LTD) of parallel fibre-Purkinje cell responses. Although the calcium influx remains spatially constrained, depression spreads heterosynaptically to distant sites. Inhibition of the synthetic enzyme for cGMP, guanylate cyclase, did not significantly affect the overall level of calcium-dependent synaptic depression observed at the site of raised stimulation (test site), but it entirely prevented synaptic depression at the distant (control) site. Inhibition of protein kinase G produced identical results. In contrast, protein kinase A inhibition had no effect. Selective inhibition of either metabotropic glutamate receptors (mGluRs), protein kinase C (PKC) or tyrosine protein kinase (PTK) blocked depression at both sites equally effectively. These data reveal that two, inter-dependent cellular pathways capable of inducing cerebellar LTD exist. The levels of PF stimulation required to induce heterosynaptic depression were similar to those used routinely in more widely accepted models of LTD. The data predict that cerebellar long-term depression will not be input specific at the single cell level under those conditions of PF-activation that give rise to NO/cGMP production.