Sustained axon-glial signaling induces Schwann cell hyperproliferation, Remak bundle myelination, and tumorigenesis.

Type III neuregulins exposed on axon surfaces control myelination of the peripheral nervous system. It has been shown, for example, that threshold levels of type III beta1a neuregulin dictate not only the myelination fate of axons but also myelin thickness. Here we show that another neuregulin isoform, type III-beta3, plays a distinct role in myelination. Neuronal overexpression of this isoform in mice stimulates Schwann cell proliferation and dramatically enlarges peripheral nerves and ganglia-which come to resemble plexiform neurofibromas-but have no effect on myelin thickness. The nerves display other neurofibroma-like properties, such as abundant collagen fibrils and abundant dissociated Schwann cells that in some cases produce big tumors. Moreover, the organization of Remak bundles is dramatically altered; the small-caliber axons of each bundle are no longer segregated from one another within the cytoplasm of a nonmyelinating Schwann cell but instead are close packed and the whole bundle wrapped as a single unit, frequently by a compact myelin sheath. Because Schwann cell hyperproliferation and Remak bundle degeneration are early hallmarks of type I neurofibromatosis, we suggest that sustained activation of the neuregulin pathway in Remak bundles can contribute to neurofibroma development.

Inhibition of cAMP response element-binding protein reduces neuronal excitability and plasticity, and triggers neurodegeneration.

The cAMP-responsive element-binding protein (CREB) pathway has been involved in 2 major cascades of gene expression regulating neuronal function. The first one presents CREB as a critical component of the molecular switch that controls long-lasting forms of neuronal plasticity and learning. The second one relates CREB to neuronal survival and protection. To investigate the role of CREB-dependent gene expression in neuronal plasticity and survival in vivo, we generated bitransgenic mice expressing A-CREB, an artificial peptide with strong and broad inhibitory effect on the CREB family, in forebrain neurons in a regulatable manner. The expression of A-CREB in hippocampal neurons impaired L-LTP, reduced intrinsic excitability and the susceptibility to induced seizures, and altered both basal and activity-driven gene expression. In the long-term, the chronic inhibition of CREB function caused severe loss of neurons in the CA1 subfield as well as in other brain regions. Our experiments confirmed previous findings in CREB-deficient mutants and revealed new aspects of CREB-dependent gene expression in the hippocampus supporting a dual role for CREB-dependent gene expression regulating intrinsic and synaptic plasticity and promoting neuronal survival.

Enhanced CREB-dependent gene expression increases the excitability of neurons in the basal amygdala and primes the consolidation of contextual and cued fear memory.

Regulated expression of a constitutively active form of cAMP response element-binding protein (CREB), VP16-CREB, lowers the threshold for the late phase of long-term potentiation in the Schaffer collateral pathway in a de novo gene expression-independent manner, and increases the excitability and reduces afterhyperpolarization of neurons at the amygdala and the hippocampus. We explore the consequences of these changes on the consolidation of fear conditioning and find that the expression of VP16-CREB can bypass the requirement for de novo gene expression associated with long-term memory formation, suggesting that CREB-dependent gene expression is sufficient for fear memory consolidation.

Synapse-specific stabilization of plasticity processes: the synaptic tagging and capture hypothesis revisited 10 years later.

A decade ago, the synaptic tagging hypothesis was proposed to explain how newly synthesized plasticity products can be specifically targeted to active synapses. A growing number of studies have validated the seminal findings that gave rise to this model, as well as contributed to unveil and expand the range of mechanisms underlying late-associativity and neuronal computation. Here, we will review what it was learnt during this past decade regarding the cellular and molecular mechanisms underlying synaptic tagging and synaptic capture. The accumulated experimental evidence has widened the theoretical framework set by the synaptic tagging and capture (STC) model and introduced concepts that were originally considered part of alternative models for explaining synapse-specific long-term potentiation (LTP). As a result, we believe that the STC model, now improved and expanded with these new ideas and concepts, still represents the most compelling hypothesis to explain late-associativity in synapse-specific plasticity processes. We will also discuss the impact of this model in our view of the integrative capability of neurons and associative learning.

cAMP response element-binding protein-mediated gene expression increases the intrinsic excitability of CA1 pyramidal neurons.

To investigate the role of CREB-mediated gene expression on the excitability of CA1 pyramidal neurons, we obtained intracellular recordings from pyramidal neurons of transgenic mice expressing a constitutively active form of CREB, VP16-CREB, in a regulated and restricted manner. We found that transgene expression increased the neuronal excitability and inhibited the slow and medium afterhyperpolarization currents. These changes may contribute to the reduced threshold for LTP observed in these mice. When strong transgene expression was turned on for prolonged period of time, these mice also showed a significant loss of hippocampal neurons and sporadic epileptic seizures. These deleterious effects were dose dependent and could be halted, but not reversed by turning off transgene expression. Our experiments reveal a new role for hippocampal CREB-mediated gene expression, identify the slow afterhyperpolarization as a primary target of CREB action, provide a new mouse model to investigate temporal lobe epilepsy and associated neurodegeneration, and illustrate the risks of cell death associated to a sustained manipulation of this pathway. As a result, our study has important implications for both the understanding of the cellular bases of learning and memory and the consideration of therapies targeted to the CREB pathway.

Bidirectional synaptic plasticity at nociceptive afferents in the rat central amygdala.

Glutamatergic inputs arising from the parabrachial nucleus to neurons in the lateral sector of the central amygdala were studied in vitro. Tetanic stimulation of these inputs led to LTP that did not require activation of NMDA receptors or a rise of postsynaptic calcium. LTP was accompanied by a reduction in the paired-pulse ratio, indicating that LTP results from an increase in transmitter release probability. Activation of adenylyl cyclase with forskolin potentiated these inputs with a similar reduction in paired-pulse facilitation and occluded LTP induction. LTP was inhibited by the protein kinase A blocker H89. Low-frequency stimulation led to LTD that required activation of postsynaptic NMDA receptors and a rise in postsynaptic calcium. There was no change in paired-pulse facilitation with LTD. LTD was blocked by protein phosphatase blockers calyculin and okadaic acid. We conclude that parabrachial inputs to the lateral sector of the central amygdala show presynaptic LTP that requires activation of a presynaptic protein kinase A via a calcium-dependent adenylyl cyclase while LTD at the same synapses is postsynaptic and requires a rise in postsynaptic calcium and activation of protein phosphatase.

Firing properties and connectivity of neurons in the rat lateral central nucleus of the amygdala.

Using whole cell recordings from acute slices of the rat amygdala, we have examined the physiological properties of and synaptic connectivity to neurons in the lateral sector of the central amygdala (CeA). Based on their response to depolarizing current injections, CeA neurons could be divided into three types. Adapting neurons fired action potentials at the start of the current injections at high frequency and then showed complete spike-frequency adaptation with only six to seven action potentials evoked with suprathreshold current injections. Late-firing neurons fired action potentials with a prolonged delay at threshold but then discharged continuously with larger current injections. Repetitive firers discharged at the start of the current injection at threshold and then discharged continuously with larger current injections. All three cells showed prolonged afterhyperpolarizations (AHPs) that followed trains of action potentials. The AHP was longer lasting with a larger slow component in adapting neurons. The AHP in all cell types contained a fast component that was inhibited by the SK channel blocker UCL1848. The slow component, not blocked by UCL1848, was blocked by isoprenaline and was significantly larger in adapting neurons. Blockade of SK channels increased the discharge frequency in late firers and regular-spiking neurons but had no effect on adapting neurons. Blockade of the slow AHP with isoprenaline had no effect on any cell type. All cells received a mixed glutamatergic and GABAergic input from a medial pathway. Electrical stimulation of the lateral (LA) and basolateral (BLA) nuclei evoked a large monosynaptic glutamatergic response followed by a disynaptic inhibitory postsynaptic potential. Activation of neurons in the LA and BLA by puffer application of glutamate evoked a small monosynaptic response in 13 of 55 CeA neurons. Local application of glutamate to the CeL evoked a GABAergic response in all cells. These results show that at least three types of neurons are present in the CeA that can be distinguished on their firing properties. The firing frequency of two of these cell types is determined by activation of SK channels. Cells receive a small input from the LA and BLA but may receive inputs that course through these nuclei en route to the CeA.

Development and subunit composition of synaptic NMDA receptors in the amygdala: NR2B synapses in the adult central amygdala.

NMDA receptors are well known to play an important role in synaptic development and plasticity. Functional NMDA receptors are heteromultimers thought to contain two NR1 subunits and two or three NR2 subunits. In central neurons, NMDA receptors at immature glutamatergic synapses contain NR2B subunits and are largely replaced by NR2A subunits with development. At mature synapses, NMDA receptors are thought to be multimers that contain either NR1/NR2A or NR1/NR2A/NR2B subunits, whereas receptors that contain only NR1/NR2B subunits are extrasynaptic. Here, we have studied the properties of NMDA receptors at glutamatergic synapses in the lateral and central amygdala. We find that NMDA receptor-mediated synaptic currents in the central amygdala in both immature and mature synapses have slow kinetics and are substantially blocked by the NR2B-selective antagonists (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propano and ifenprodil, indicating that there is no developmental change in subunit composition. In contrast, at synapses on pyramidal neurons in the lateral amygdala, whereas NMDA EPSCs at immature synapses are slow and blocked by NR2B-selective antagonists, at mature synapses their kinetics are faster and markedly less sensitive to NR2B-selective antagonists, consistent with a change from NR2B to NR2A subunits. Using real-time PCR and Western blotting, we show that in adults the ratio of levels of NR2B to NR2A subunits is greater in the central amygdala than in the lateral amygdala. These results show that the subunit composition synaptic NMDA receptors in the lateral and central amygdala undergo distinct developmental changes.

Excitatory synaptic transmission in the lateral and central amygdala.

The amygdala plays a major role in the acquisition and expression of fear conditioning. NMDA receptor-dependent synaptic plasticity within the basolateral amygdala has been proposed to underlie the acquisition and possible storage of fear memories. Here the properties of fast glutamatergic transmission in the lateral and central nuclei of the amygdala are presented. In the lateral amygdala, two types of neurons, interneurons and projection neurons, could be distinguished by their different firing properties. Glutamatergic inputs to interneurons activated AMPA receptors with inwardly rectifying current-voltage relations (I-Vs), whereas inputs to projection neurons activated receptors that had linear I-Vs, indicating that receptors on interneurons lack GluR2 subunits. Inputs to projection neurons formed dual component synapses with both AMPA and NMDA components, whereas at inputs to interneurons, the contribution of NMDA receptors was very small. Neurons in the central amygdala received dual component glutamatergic inputs that activated AMPA receptors with linear I-Vs. NMDA receptor-mediated EPSCs had slow decay time constants in the central nucleus. Application of NR2B selective blockers ifenprodil or CP-101,606 blocked NMDA EPSCs by 70% in the central nucleus, but only by 30% in the lateral nucleus. These data show that the distribution of glutamatergic receptors on amygdalar neurons is not uniform. In the lateral amygdala, interneurons and pyramidal neurons express AMPA receptors with different subunit compositions. Synapses in the central nucleus activate NMDA receptors that contain NR1 and NR2B subunits, whereas synapses in the lateral nucleus contain receptors with both NR2A and NR2B subunits.

Responses to sympathomimetics in rat sensory neurones after nerve transection.

Noradrenaline activation of sensory somata that project in damaged peripheral nerves has been postulated to underlie sympathetically-mediated pain. Intracellular recordings from some neurones with myelinated axons in acutely isolated rat dorsal root ganglia showed small prolonged depolarizations to brief applications of 0.1-5 mM noradrenaline whether or not the spinal nerve had been transected. Similar responses were evoked to noradrenaline when phentolamine was present, and also to 1-5 mM catechol, but not 1 mM clonidine, implying the responses were not adrenoceptor-mediated. In extracellular recordings from similar preparations after sciatic transection, many spontaneously active myelinated dorsal root axons were excited by noradrenaline and other sympathomimetics. Silent axons in injured or control ganglia did not respond. Thus, non-specific depolarizations may activate neurones that are hyperexcitable after a lesion but activation of neuronal alpha-adrenoceptors by sympathetically-released noradrenaline seems unlikely.

Postnatal changes in membrane properties of mice trigeminal ganglion neurons.

Intracellular recordings from neurons in the mouse trigeminal ganglion (TG) in vitro were used to characterize changes in membrane properties that take place from early postnatal stages (P0-P7) to adulthood (>P21). All neonatal TG neurons had uniformly slow conduction velocities, whereas adult neurons could be separated according to their conduction velocity into Adelta and C neurons. Based on the presence or absence of a marked inflection or hump in the repolarization phase of the action potential (AP), neonatal neurons were divided into S- (slow) and F-type (fast) neurons. Their passive and subthreshold properties (resting membrane potential, input resistance, membrane capacitance, and inward rectification) were nearly identical, but they showed marked differences in AP amplitude, AP overshoot, AP duration, rate of AP depolarization, rate of AP repolarization, and afterhyperpolarization (AHP) duration. Adult TG neurons also segregated into S- and F-type groups. Differences in their mean AP amplitude, AP overshoot, AP duration, rate of AP depolarization, rate of AP repolarization, and AHP duration were also prominent. In addition, axons of 90% of F-type neurons and 60% of S-type neurons became faster conducting in their central and peripheral branch, suggestive of axonal myelination. The proportion of S- and F-type neurons did not vary during postnatal development, suggesting that these phenotypes were established early in development. Membrane properties of both types of TG neurons evolved differently during postnatal development. The nature of many of these changes was linked to the process of myelination. Thus myelination was accompanied by a decrease in AP duration, input resistance (R(in)), and increase in membrane capacitance (C). These properties remained constant in unmyelinated neurons (both F- and S-type). In adult TG, all F-type neurons with inward rectification were also fast-conducting Adelta, suggesting that those F-type neurons showing inward rectification at birth will evolve to F-type Adelta neurons with age. The percentage of F-type neurons showing inward rectification also increased with age. Both F- and S-type neurons displayed changes in the sensitivity of the AP to reductions in extracellular Ca(2+) or substitution with Co(2+) during the process of maturation.