Daniel Fulton

A single time-window for protein synthesis-dependent long-term memory formation after one-trial appetitive conditioning.

Protein synthesis is generally held to be essential for long‐term memory formation. Often two periods of sensitivity to blockade of protein synthesis have been described, one immediately after training and another several hours later. We wished to relate the timing of protein synthesis‐dependence of behavioural long‐term memory (LTM) formation to an electrophysiological correlate of the LTM memory trace. We used the snail Lymnaea because one‐trial appetitive conditioning of feeding using a chemical conditioned stimulus leads to a stable LTM trace that can be monitored behaviourally and then electrophysiologically in preparations made from the same animals. Anisomycin (an inhibitor of translation) injected 10 min after training blocked behavioural LTM formation. Actinomycin D (an inhibitor of transcription) was also effective at 10 min. When anisomycin, at doses shown to be effective in blocking central nervous system protein synthesis, was injected at 1, 2, 3, 4, 5 and 6 h after training there was no effect on recall. These results indicate that there is a single period of sensitivity to protein synthesis inhibition in Lymnaea lasting for between 10 min and 1 h after training with no evidence for a second window of sensitivity. An electrophysiological correlate of LTM was found to be sensitive to anisomycin injected 10 min after training. It is unusual to find only one period of protein synthesis‐dependence in detailed time‐course studies of LTM, and this suggests that the consolidation processes involving protein synthesis are relatively rapid in one‐trial appetitive conditioning and complete within 1 h of training.

The multiple roles of myelin protein genes during the development of the oligodendrocyte

It has become clear that the products of several of the earliest identified myelin protein genes perform functions that extend beyond the myelin sheath. Interestingly, these myelin proteins, which comprise proteolipid protein, 2′,3′-cyclic nucleotide 3′-phosphodiesterase and the classic and golli MBPs (myelin basic proteins), play important roles during different stages of oligodendroglial development. These non-myelin-related functions are varied and include roles in the regulation of process outgrowth, migration, RNA transport, oligodendrocyte survival and ion channel modulation. However, despite the wide variety of cellular functions performed by the different myelin genes, the route by which they achieve these many functions seems to converge upon a common mechanism involving Ca2+ regulation, cytoskeletal rearrangements and signal transduction. In the present review, the newly emerging functions of these myelin proteins will be described, and these will then be discussed in the context of their contribution to oligodendroglial development.

Golli Myelin Basic Proteins Regulate Oligodendroglial Progenitor Cell Migration through Voltage-Gated Ca2+ Influx

Migration of oligodendrocyte progenitor cells (OPCs) from proliferative zones to their final location in the brain is an essential step in nervous system development. Golli proteins, products of the myelin basic protein gene, can modulate voltage-gated Ca2+ uptake in OPCs during process extension and retraction. Given the importance of process extension/retraction on movement, the consequences of golli expression on OPC migration were examined in vivo and in vitro using time-lapse imaging of isolated OPCs and acute brain slice preparations from golli KO and golli J37 overexpressing mice (JOE). The results indicated that golli stimulated migration, and this enhanced motility was associated with increases in the activity of voltage operated Ca2+ channels (VOCCs). Activation of VOCCs by high K+ resulted in a significant increase in the migration speed of JOE OPCs versus control cells and golli-mediated modulation of OPC migration disappeared in the presence of VOCC antagonists. During migration, OPCs generated Ca2+ oscillations that were dependent on voltage-calcium influx and both the amplitude and frequency of these Ca2+ transients correlated positively with the rate of cell movement under a variety of pharmacological treatments. The Ca2+ transient amplitude and the rate of cell movement were significantly lower in KO cells and significantly higher in JOE cells suggesting that the presence of golli promotes OPC migration by increasing the size of voltage-mediated Ca2+ oscillations. These data define a new molecule that regulates Ca2+ homeostasis in OPCs, and are the first to demonstrate that voltage-gated Ca2+ channels can regulate an OPC function, such as migration.

Multiple Kinase Pathways Regulate Voltage-Dependent Ca2+ Influx and Migration in Oligodendrocyte Precursor Cells

It is becoming increasingly clear that voltage-operated Ca2+ channels (VOCCs) play a fundamental role in the development of oligodendrocyte progenitor cells (OPCs). Because direct phosphorylation by different kinases is one of the most important mechanisms involved in VOCC modulation, the aim of this study was to evaluate the participation of serine–threonine kinases and tyrosine kinases (TKs) on Ca2+ influx mediated by VOCCs in OPCs. Calcium imaging revealed that OPCs exhibited Ca2+ influx after plasma membrane depolarization via L-type VOCCs. Furthermore, VOCC-mediated Ca2+ influx declined with OPC differentiation, indicating that VOCCs are developmentally regulated in OPCs. PKC activation significantly increased VOCC activity in OPCs, whereas PKA activation produced the opposite effect. The results also indicated that OPC morphological changes induced by PKC activation were partially mediated by VOCCs. Our data clearly suggest that TKs exert an activating influence on VOCC function in OPCs. Furthermore, using the PDGF response as a model to probe the role of TK receptors (TKr) on OPC Ca2+ uptake, we found that TKr activation potentiated Ca2+ influx after membrane depolarization. Interestingly, this TKr modulation of VOCCs appeared to be essential for the PDGF enhancement of OPC migration rate, because cell motility was completely blocked by TKr antagonists, as well as VOCC inhibitors, in migration assays. The present study strongly demonstrates that PKC and TKrs enhance Ca2+ influx induced by depolarization in OPCs, whereas PKA has an inhibitory effect. These kinases modulate voltage-operated Ca2+ uptake in OPCs and participate in the modulation of process extension and migration.

Regulation of Store-Operated and Voltage-Operated Ca2+ Channels in the Proliferation and Death of Oligodendrocyte Precursor Cells by Golli Proteins

OPCs (oligodendrocyte precursor cells) express golli proteins which, through regulation of Ca2+ influx, appear to be important in OPC process extension/retraction and migration. The aim of the present study was to examine further the role of golli in regulating OPC development. The effects of golli ablation and overexpression were examined in primary cultures of OPCs prepared from golli-KO (knockout) and JOE (golli J37-overexpressing) mice. In OPCs lacking golli, or overexpressing golli, differentiation induced by growth factor withdrawal was impaired. Proliferation analysis in the presence of PDGF (platelet-derived growth factor), revealed that golli enhanced the mitogen-stimulated proliferation of OPCs through activation of SOCCs (store-operated Ca2+ channels). PDGF treatment induced a biphasic increase in OPC intracellular Ca2+, and golli specifically increased Ca2+ influx during the second SOCC-dependent phase that followed the initial release of Ca2+ from intracellular stores. This store-operated Ca2+ uptake appeared to be essential for cell division, since specific SOCC antagonists completely blocked the effects of PDGF and golli on OPC proliferation. Additionally, in OPCs overexpressing golli, increased cell death was observed after mitogen withdrawal. This phenomenon could be prevented by exposure to VOCC (voltage-operated Ca2+ channel) blockers, indicating that the effect of golli on cell death involved increased Ca2+ influx through VOCCs. The results showed a clear effect of golli on OPC development and support a role for golli in modulating multiple Ca2+-regulatory events through VOCCs and SOCCs. Our results also suggest that PDGF engagement of its receptor resulting in OPC proliferation proceeds through activation of SOCCs.

Neuronal activity and AMPA-type glutamate receptor activation regulates the morphological development of oligodendrocyte precursor cells.

Myelination is initiated when oligodendrocyte precursor cells (OPC) contact target axons. Neuronal activity promotes myelination through actions that may involve OPC AMPA and NMDA glutamate receptors (AMPAR, NMDAR). Therefore, activity and AMPAR/NMDAR activation are predicted to promote the morphological development of OPC. AMPAR can regulate OPC development, but this analysis was not performed in situ and the role of action potentials was not examined. Hence, the influence of activity and AMPAR on OPC morphology and development remain untested in the CNS where axon‐glial interactions are preserved. Data on NMDAR are mixed with conflicting results from in vitro and in vivo work. To gain a fuller understanding of activity‐dependent OPC development in situ, we explored the role of AMPAR and NMDAR in cerebellar slice cultures that permit the study of endogenous OPC development and myelination. The structure of individual OPC was resolved from cells labeled with membrane targeted GFP. Morphological data were then validated against assays of OPC development. Blocking either activity or AMPAR impaired the morphological development of OPC and promoted proliferation and differentiation. Increasing the pool of oligodendrocytes by blocking activity or AMPAR failed to promote myelination. Instead both myelination and the expression of myelin basic protein were reduced by these treatments suggesting that full differentiation to a myelinating phenotype did not occur. Blocking NMDAR left OPC proliferation, differentiation and morphology unchanged. These data indicate an important role for AMPAR but not NMDAR in mediating the activity‐dependent signals that regulate OPC morphology, development and myelination. GLIA 2015;63:1021–1035

Modulation of Canonical Transient Receptor Potential Channel 1 in the Proliferation of Oligodendrocyte Precursor Cells by the Golli Products of the Myelin Basic Protein Gene

Golli proteins, products of the myelin basic protein gene, function as a new type of modulator of intracellular Ca2+ levels in oligodendrocyte progenitor cells (OPCs). Because of this, they affect a number of Ca2+-dependent functions, such as OPC migration and process extension. To examine further the Ca2+ channels regulated by golli, we studied the store-operated Ca2+ channels (SOCCs) in OPCs and acute brain slice preparations from golli knock-out and golli-overexpressing mice. Our results showed that pharmacologically induced Ca2+ release from intracellular stores evoked a significant extracellular Ca2+ entry after store depletion in OPCs. They also indicated that, under these pharmacological conditions, golli promoted activation of Ca2+ influx by SOCCs in cultured OPCs as well as in tissue slices. The canonical transient receptor potential family of Ca2+ channels (TRPCs) has been postulated to be SOCC subunits in oligodendrocytes. Using a small interfering RNA knockdown approach, we provided direct evidence that TRPC1 is involved in store-operated Ca2+ influx in OPCs and that it is modulated by golli. Furthermore, our data indicated that golli is probably associated with TRPC1 at OPC processes. Additionally, we found that TRPC1 expression is essential for the effects of golli on OPC proliferation. In summary, our data indicate a key role for golli proteins in the regulation of TRPC-mediated Ca2+ influx, a finding that has profound consequences for the regulation of multiple biological processes in OPCs. More important, we have shown that extracellular Ca2+ uptake through TRPC1 is an essential component in the mechanism of OPC proliferation.

Voltage‐operated Ca2+ and Na+ channels in the oligodendrocyte lineage

It is becoming increasingly clear that expression of Ca2+ and Na+ channels in the OL lineage is highly regulated and may be functionally related to different stages of development and myelination. Characterization of the mechanisms of voltage‐dependent Ca2+ and Na+ entry are important because changes in intracellular Ca2+ and Na+ are central to practically all cellular activities. In nonexcitable cells, voltage‐dependent Ca2+ influx plays a key role in several important processes, including proliferation, apoptosis, and cell migration. It has been demonstrated that Ca2+ signaling is essential in the development and functioning of OLs. For example, Ca2+ uptake is required for the initiation of myelination, and perturbation of Ca2+ homeostasis, e.g., overwhelming influxes of Ca2+, leads to demyelination. Although OL progenitor cell Na+ channels are present at a much lower density, their physiological properties appear to be indistinguishable from those recorded in neurons. Interestingly, recent data indicate that, as with neurons, some white matter OPCs possess the ability to generate Na+‐dependent action potentials. This Mini‐Review focuses on the mechanisms of Ca2+ and Na+ signaling in cells within the OL lineage mediated by voltage‐operated ion channels, with a particular focus on the relevance of these voltage‐dependent currents to oligodendroglial development, myelination, and demyelination. Overall, it is clear that cells in the OL lineage exhibit remarkable plasticity with regard to the expression of voltage‐gated Ca2+ and Na+ channels and that perturbation of Ca2+ and Na+ homeostasis likely plays an important role in the pathogenesis underlying demyelinating diseases.

Regulation of L-type Ca++ currents and process morphology in white matter oligodendrocyte precursor cells by golli-myelin proteins

The golli myelin basic proteins are expressed in oligodendroglial precursor cells (OPCs) where they play a role in regulating Ca2+ homeostasis. During depolarization, they influence process outgrowth and migration through their action on voltage‐operated Ca2+ channels (VOCCs). To identify ion channels that are modulated by golli, we examined the electrophysiological properties of VOCCs in OPCs in the white matter of golli knock‐out and control mice. OPCs exhibited two distinct Ca2+ channels, which were distinguished by their voltage dependence and pharmacological profiles and which exhibited many of the hallmarks of LVA/T‐type and HVA/L‐type Ca2+ channels. The density of high‐voltage‐activated (HVA) currents was reduced in OPCs recorded in golli‐KO tissue, while low‐voltage‐activated (LVA) currents remained unaltered in these cells. These data indicate that golli exerts an exclusive influence on L‐type Ca2+ channels in OPCs. Oligodendrocytes (OLs) also displayed LVA and HVA currents, although the density of these currents was much reduced at this developmental stage. These currents were not altered in golli‐KO OLs showing the influence of golli on L‐type Ca2+ channels is restricted to a specific time‐window during the course of oligodendroglial development. The actions of golli on OPC L‐type Ca2+ channels were accompanied by changes in process morphology, including a reduction in process complexity and the appearance of enlarged varicosities that decorated these cellular processes. These data on L‐type Ca2+ channels and process development provide in situ evidence for the influence of golli on VOCCs, and offer an explanation for the hypomyelination observed in the brains of golli‐KO mice.

Time-window for sensitivity to cooling distinguishes the effects of hypothermia and protein synthesis inhibition on the consolidation of long-term memory.

The effects of hypothermia on memory formation have been examined extensively, and while it is clear that post-training cooling interferes with the process of consolidation, the nature of the temperature sensitive processes disrupted in this way remain poorly defined. Post-training manipulations that disrupt consolidation tend to be effective during specific time-windows of sensitivity, the timing and duration of which are directly related to the mechanism through which the treatment induces amnesia. As such, different treatments that target the same basic processes should be associated with similar time-windows of sensitivity. Using this rationale we have investigated the possibility that cooling induced blockade of long-term memory (LTM) stems from the disruption of protein synthesis. By varying the timing of post-training hypothermia we have determined the critical period during which cooling disrupts the consolidation of appetitive long-term memory in the pond snail Lymnaea. Post-training hypothermia was found to disrupt LTM only when applied immediately after conditioning, while delaying the treatment by 10 min left the 24 h memory trace intact. This brief (<10 min) window of sensitivity differs from the time-window we have previously described for the protein synthesis inhibitor anisomycin, which was effective during at least the first 30 min after conditioning [Fulton, D., Kemenes, I., Andrew, R. J., & Benjamin, P. R. (2005). A single time-window for protein synthesis-dependent long-term memory formation after one-trial appetitive conditioning. European Journal of Neuroscience, 21, 1347-1358]. We conclude that hypothermia and protein synthesis inhibition exhibit distinct time-windows of effectiveness in Lymnaea, a fact that is inconsistent with the hypothesis that cooling induced amnesia occurs through the direct disruption of macromolecular synthesis.