Apelin-induced cardioprotection against ischaemia/reperfusion injury: roles of epidermal growth factor and Src.

AIM: Apelin, the ligand of the G-protein-coupled receptor (GPCR) APJ, exerts a post-conditioning-like protection against ischaemia/reperfusion injury through activation of PI3K-Akt-NO signalling. The pathway connecting APJ to PI3K is still unknown. As other GPCR ligands act through transactivation of epidermal growth factor receptor (EGFR) via a matrix metalloproteinase (MMP) or Src kinase, we investigated whether EGFR transactivation is involved in the following three features of apelin-induced cardioprotection: limitation of infarct size, suppression of contracture and improvement of post-ischaemic contractile recovery. METHOD: Isolated rat hearts underwent 30 min of global ischaemia and 2 h of reperfusion. Apelin (0.5 µm) was infused during the first 20 min of reperfusion. EGFR, MMP or Src was inhibited to study the pathway connecting APJ to PI3K. Key components of RISK pathway, namely PI3K, guanylyl cyclase or mitochondrial K+ -ATP channels, were also inhibited. Apelin-induced EGFR and phosphatase and tensing homolog (PTEN) phosphorylation were assessed. Left ventricular pressure and infarct size were measured. RESULTS: Apelin-induced reductions in infarct size and myocardial contracture were prevented by the inhibition of EGFR, Src, MMP or RISK pathway. The involvement of EGFR was confirmed by its phosphorylation. However, neither direct EGFR nor MMP inhibition affected apelin-induced improvement of early post-ischaemic contractile recovery, which was suppressed by Src and RISK inhibitors only. Apelin also increased PTEN phosphorylation, which was removed by Src inhibition. CONCLUSION: While EGFR and MMP limit infarct size and contracture, Src or RISK pathway inhibition suppresses the three features of cardioprotection. Src does not only transactivate EGFR, but also inhibits PTEN by phosphorylation thus playing a crucial role in apelin-induced cardioprotection.

Knock-down of synapsin alters cell excitability and action potential waveform by potentiating BK and voltage-gated Ca(2+) currents in Helix serotonergic neurons.

Synapsins (Syns) are an evolutionarily conserved family of presynaptic proteins crucial for the fine-tuning of synaptic function. A large amount of experimental evidences has shown that Syns are involved in the development of epileptic phenotypes and several mutations in Syn genes have been associated with epilepsy in humans and animal models. Syn mutations induce alterations in circuitry and neurotransmitter release, differentially affecting excitatory and inhibitory synapses, thus causing an excitation/inhibition imbalance in network excitability toward hyperexcitability that may be a determinant with regard to the development of epilepsy. Another approach to investigate epileptogenic mechanisms is to understand how silencing Syn affects the cellular behavior of single neurons and is associated with the hyperexcitable phenotypes observed in epilepsy. Here, we examined the functional effects of antisense-RNA inhibition of Syn expression on individually identified and isolated serotonergic cells of the Helix land snail. We found that Helix synapsin silencing increases cell excitability characterized by a slightly depolarized resting membrane potential, decreases the rheobase, reduces the threshold for action potential (AP) firing and increases the mean and instantaneous firing rates, with respect to control cells. The observed increase of Ca(2+) and BK currents in Syn-silenced cells seems to be related to changes in the shape of the AP waveform. These currents sustain the faster spiking in Syn-deficient cells by increasing the after hyperpolarization and limiting the Na(+) and Ca(2+) channel inactivation during repetitive firing. This in turn speeds up the depolarization phase by reaching the AP threshold faster. Our results provide evidence that Syn silencing increases intrinsic cell excitability associated with increased Ca(2+) and Ca(2+)-dependent BK currents in the absence of excitatory or inhibitory inputs.

Characterization and role of Helix contactin-related proteins in cultured Helix pomatia neurons.

We report on the structural and functional properties of the Helix contactin-related proteins (HCRPs), a family of closely related glycoproteins previously identified in the nervous system of the land snail Helix pomatia through antibodies against the mouse F3/contactin glycoprotein. We focus on HCRP1 and HCRP2, soluble FNIII domains-containing proteins of 90 and 45 kD bearing consensus motifs for both N- and O-glycosylation. Using the anti-HCRPs serum, we find secreted HCRPs in Helix nervous tissue isotonic extracts and in culture medium conditioned by Helix ganglia. In addition, we demonstrate expression of HCRPs on neuronal soma and on neurite extensions. Functionally, in Helix neurons, the antisense HCRP2 mRNA counteracts neurite elongation, and the recombinant HCRP2 protein exerts a strong positive effect on neurite growth when used as substrate. These data point to HCRPs as novel neurite growth-promoting molecules expressed in invertebrate nervous tissue.

In vitro formation and activity-dependent plasticity of synapses between Helix neurons involved in the neural control of feeding and withdrawal behaviors.

Short-term activity-dependent synaptic plasticity has a fundamental role in short-term memory and information processing in the nervous system. Although the neuronal circuitry controlling different behaviors of land snails of the genus Helix has been characterized in some detail, little is known about the activity-dependent plasticity of synapses between identified neurons regulating specific behavioral acts. In order to study homosynaptic activity-dependent plasticity of behaviorally relevant Helix synapses independently of heterosynaptic influences, we sought to reconstruct them in cell culture. To this aim, we first investigated in culture the factors regulating synapse formation between Helix neurons, and then we studied the short-term plasticity of in vitro-reconstructed monosynaptic connections involved in the neural control of salivary secretion and whole-body withdrawal. We found that independently of extrinsic factors, cell-cell interactions are seemingly sufficient to trigger the formation of electrical and chemical synapses, although mostly inappropriate--in their type or association--with respect to the in vivo synaptic connectivity. The presence of ganglia-derived factors in the culture medium was required for the in vitro reestablishment of the appropriate in vivo-like connectivity, by reducing the occurrence of electrical connections and promoting the formation of chemical excitatory synapses, while apparently not influencing the formation of inhibitory connections. These heat-labile factors modulated electrical and chemical synaptogenesis through distinct protein tyrosine kinase signal transduction pathways. Taking advantage of in vitro-reconstructed synapses, we have found that feeding interneuron-efferent neuron synapses and mechanosensory neuron-withdrawal interneuron synapses display multiple forms of short-term enhancement-like facilitation, augmentation and posttetanic potentiation as well as homosynaptic depression. These forms of plasticity are thought to be relevant in the regulation of Helix feeding and withdrawal behaviors by inducing dramatic activity-dependent changes in the strength of input and output synapses of high-order interneurons with a crucial role in the control of Helix behavioral hierarchy.

Target-dependent modulation of neurotransmitter release in cultured Helix neurons involves adhesion molecules.

The secretory capabilities of the serotonergic neuron C1 of cerebral ganglion of Helix pomatia were markedly reduced when it was cultured in contact with the wrong target neuron, C3. When the neuron B2, one of its physiological targets, was micromanipulated within the network made of intermingled neurites originating from the axonal stumps of both C1 and C3 neurons, C1 increased the amount of the evoked transmitter release, which, after 30 min, reached the level observed when cocultured with the appropriate target. The removal of the appropriate target brought C1 back to the low release condition. By imaging C1 neurites with a fluorescent dye, morphological changes involving a local increase in the number of varicosities could be observed as early as 30 min after contact with the appropriate target. Monoclonal antibody 4E8 against apCAM, a family of Aplysia adhesion molecules, recognizes apCAM-like molecules of the Helix central nervous system on immunocytochemistry and Western blot analysis. The contact with the appropriate target previously incubated in a 4E8 solution, which did not interfere with its capacity to respond to serotonin, failed to increase the transmitter release of C1 cocultured in the presence of the wrong target, C3. These results suggest that the apCAM-like antigens bound to the target membrane participate in the molecular processes responsible for the assembly of the "release machinery" present in the functional presynaptic structure.

Intracellular injection of synapsin I induces neurotransmitter release in C1 neurons of Helix pomatia contacting a wrong target.

The contact with the postsynaptic target induces structural and functional modifications in the serotonergic cell C1 of Helix pomatia. In previous studies we have found that the presence of a non-physiological target down-regulates the number of presynaptic varicosities formed by cultured C1 neurons and has a strong inhibitory effect on the action potential-evoked Ca(2+) influx and neurotransmitter release at C1 terminals. Since a large body of experimental evidence implicates the synapsins in the development and functional maturation of synaptic connections, we have investigated whether the injection of exogenous synapsin I into the presynaptic neuron C1 could affect the inhibitory effect of the wrong target on neurotransmitter release. C1 neurons were cultured with the wrong target neuron C3 for three to five days and then injected with either dephosphorylated or Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated Cy3-labeled synapsin I. The subcellular distribution of exogenous synapsin I, followed by fluorescence videomicroscopy, revealed that only synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II diffused in the cytoplasm and reached the terminal arborizations of the axon, while the dephosphorylated form did not diffuse beyond the cell body. Evoked neurotransmitter release was measured during C1 stimulation using a freshly dissociated neuron B2 (sniffer) micromanipulated in close contact with the terminals of C1. A three-fold increase in the amplitude of the sniffer depolarization with respect to the pre-injection amplitude (190+/-29% increase, n=10, P<0.006) was found 5 min after injection of Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated synapsin I that lasted for about 30 min. No significant change was observed after injection of buffer or dephosphorylated synapsin I. These data indicate that the presence of synapsin I induces a fast increase in neurotransmitter release that overcomes the inhibitory effect of the non-physiological target and suggest that the expression of synapsins may play a role in the modulation of synaptic strength and neural connectivity.

Influence of the target on distribution and functioning of the varicosities of Helix pomatia metacerebral cell C1 in dissociated cell culture.

The serotonergic metacerebral giant cell (C1) of Helix pomatia was isolated with its bifurcate axon and plated in culture under five conditions: (i) with no target; (ii) with the appropriate target B2 near the stump of the bigger branch (CBC); (iii) with B2 near the stump of the smaller branch (CC); (iv) with a wrong target (C3) near the stump of the CBC branch and (v) with B2 and C3 positioned near the CBC and CC stump, respectively. The counting of anti-serotonin antibody-labelled varicosities of the C1 neuron showed that the presence of the appropriate target in either axonal domain both down-regulated the number of varicosities of the contralateral neuritic field, and increased their average size, whereas the wrong target induced an overall reduction of the number of C1 neuron varicosities, and inhibited the evoked transmitter release. The action potential-evoked calcium concentration increase in the neuritic terminals of the C1 neuron cultured alone, or in presence of the appropriate target, reached a value significantly higher than that reached in presence of the wrong target. These results provide evidence that the postsynaptic neuron regulates both morphological and functional development of presynaptic terminals.

Synapsin-like molecules in Aplysia punctata and Helix pomatia: identification and distribution in the nervous system and during the formation of synaptic contacts in vitro.

The distribution and biochemical features of the synapsin-like peptides recognized in Aplysia and Helix by various antibodies directed against mammalian synapsins were studied. The peptides can be extracted at low pH and are digested by collagenase; further, they can be phosphorylated by both protein kinase A and Ca2+/calmodulin-dependent protein kinase II. In the ganglia of both snails, they are associated with the soma of most neurons and with the neuropil; punctate immunostaining is present along the neurites. Using cocultures of a Helix serotoninergic neuron and of its target cell, we analysed the redistribution of the synapsin-like peptides during the formation of active synaptic contacts. When the presynaptic neuron is plated in isolation, both synapsin and serotonin immunoreactivities are restricted to the distal axonal segments and to the growth cones; in the presence of the target, the formation of a chemical connection is accompanied by redistribution of the synapsin and serotonin immunoreactivities that concentrate in highly fluorescent round spots scattered along the newly grown neurites located close to the target cell. Almost every spot that is stained for serotonin is also positive for synapsin. In the presynaptic cell plated alone, the number of these varicosity-like structures is substantially stable throughout the whole period; by contrast, when the presynaptic cell synapses the target, their number increases progressively parallel to the increase in the mean amplitude of cumulative excitatory postsynaptic potentials recorded at the same times. The data indicate that mollusc synapsin-like peptides to some extent resemble their mammalian homologues, although they are not exclusively localized in nerve terminals and their expression strongly correlates with the formation of active synaptic contacts.

Neuropeptide localization in varicosities of Aplysia sensory neurons is regulated by target and neuromodulators evoking long-term synaptic plasticity.

The synapses between the sensory neuron (SN) and motor neuron of Aplysia undergo long-term functional and structural modulation with appropriate behavioral training or with applications of specific neuromodulators. Expression of molecules within the presynaptic terminals may be regulated in parallel with the changes evoked by the neuromodulators. We examined with immunocytochemical methods whether the level of sensorin, the SN-specific neuropeptide, is modulated in SN varicosities by the location of interaction with the target motor cell L7 and by applications of either 5-HT that evoke long-term facilitation or FMRFamide that evoke long-term depression of Aplysia sensorimotor connections in vitro. A significantly higher proportion of SN varicosities are sensorin positive when they are in contact with the proximal axons of L7 compared to varicosities of the same SNs in contact with distal L7 neurites. Both 5-HT and FMRFamide evoked changes in the efficacy and structure of sensorimotor connections that are accompanied by changes in the frequency of sensorin-positive varicosities contacting the axons of L7. More preexisting SN varicosities are stained after 5-HT, and fewer preexisting SN varicosities are stained after FMRFamide. These results suggest that the postsynaptic target and the neuromodulators not only regulate overall structure but also regulate the level of SN neuropeptide at synaptic sites.

Aplysia hemolymph promotes neurite outgrowth and synaptogenesis of identified Helix neurons in cell culture.

Hemolymph of adult Aplysia californica significantly affects neurite outgrowth of identified neurons of the land snail Helix pomatia. The metacerebral giant cell (MGC) and the motoneuron C3 from the cerebral ganglion and the neuron B2 from the buccal ganglion of H. pomatia were isolated by enzymatic and mechanical dissociation and plated onto poly-L-lysine-coated dishes either containing culture medium conditioned by Helix ganglia, or pre-treated with Aplysia hemolymph. To determine the extent of neuronal growth we measured the neurite elongation and the neuritic field of cultured neurons at different time points. Aplysia hemolymph enhances the extent and rate of linear outgrowth and the branching domain of Helix neurons. With the hemolymph treatment the MGC neuron more consistently forms specific chemical synapses with its follower cell B2, and these connections are more effective than those established in the presence of the conditioned medium.

A novel intermediate stage in the transition between short- and long-term facilitation in the sensory to motor neuron synapse of aplysia.

A major difference between short- and long-term memory is that long-term memory is dependent on new protein synthesis. Long-term memory can be further subdivided into a transient, initial phase that is readily susceptible to disruption and a later, more stable and persistent stage. To analyze this transition on the cellular level, we have examined the steps whereby short-term facilitation is converted to a long-term form in the sensorimotor connection of the Aplysia gill-withdrawal reflex. We found that stable long-term facilitation (at 24 hr) requires a higher concentration (100 nM) of serotonin (5-HT) than does short-term facilitation (10 nM). By using low concentrations of 5-HT, which do not produce long-term facilitation, we now have been able to explore the intermediate phases between the short- and long-term processes. By this means we have uncovered a new transient phase that involves three mechanistically different mechanisms--covalent modification, translation, and transcription--each of which can be recruited as a function of the concentration of 5-HT.

cAMP and arachidonic acid simulate long-term structural and functional changes produced by neurotransmitters in Aplysia sensory neurons.

The efficacy of the synapses between the sensory and motor cells of Aplysia, as well as the number of presynaptic sensory cell varicosities in vitro, can undergo long-term increases and decreases, respectively, following application of the facilitatory modulator serotonin or the inhibitory modulator FMRFamide. We here report that cAMP and arachidonic acid, two second messenger systems mediating some of the short-term actions of serotonin and FMRFamide on sensory cells, reproduce some of the long-term changes in the structure of the sensory cells, and these structural changes in turn parallel the long-term changes in the functional effectiveness of the synapses. cAMP enhances the strength of the connections between the sensory and motor cells and increases the number of sensory varicosities. Conversely, arachidonic acid decreases the strength of the connections and decreases the number of sensory varicosities. Thus, each of the modulatory neurotransmitters may activate the same intracellular second messenger system to establish both short and long lasting functional changes in synaptic efficacy, as well as to produce enduring structural changes in neuron connectivity.

Effects of inferior olive inactivation and lesion on the activity of medial vestibular neurons in the rat.

In anaesthetized rats, the unitary activity from the medial vestibular nucleus had been recorded during horizontal sinusoidal rotation in the absence of visual stimulation. In the first series of experiments, the inferior olivary nuclei were selectively destroyed by means of 3-acetylpyridine. Unitary activity was recorded three to five days or one month after the lesion. A few days after the lesion, the average spontaneous activity, as well as the peak-to-peak amplitude of the modulation of the medial vestibular neurons during sinusoidal rotation, were significantly lower compared to those recorded in intact rats, and to those recorded one month after the lesion. In the second series of experiments, during reversible cooling of the inferior olive region of one side, in the contralateral medial vestibular nuclei 57% of units underwent a clear decrease in firing rate accompanied by a decrease in the amplitude of modulation. In rats whose inferior olivary nuclei had been destroyed by means of 3-acetylpyridine one month before, or whose cerebellum had been removed, there were few units that showed a decrease of the firing rate and modulation amplitude on cooling the same olivary region. Our experiments show that silencing the activity of the inferior olive causes a decrease both in the spontaneous firing rate and in the amplitude of the response of the vestibular neurons to natural labyrinthine stimulation. These results support the hypothesis that the inferior olive, by changing its firing rate, may regulate on-line the gain of reflexes which are under cerebellar control.

Inhibitors of protein and RNA synthesis block structural changes that accompany long-term heterosynaptic plasticity in Aplysia.

Synaptic connections between the sensory and motor neurons of Aplysia in culture undergo long-term facilitation in response to serotonin (5-HT) and long-term depression in response to FMRFamide. These long-term functional changes are dependent on the synthesis of macromolecules during the period in which the transmitter is applied and are accompanied by structural changes. There is an increase and a decrease, respectively, in the number of sensory neuron varicosities in response to 5-HT and FMRFamide. To determine whether macromolecular synthesis is also required for the structural changes, we examined in parallel the effects of inhibitors of protein (anisomycin) or RNA (actinomycin D) synthesis on the structural and functional changes. We have found that anisomycin and actinomycin D block both the enduring alterations in varicosity number and the long-lasting changes in synaptic potential. These results indicate that macromolecular synthesis is required for expression of the long-lasting structural changes in the sensory cells and that this synthesis is correlated with the long-term functional modulation of sensorimotor synapses.

Roles of PKA and PKC in facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses in Aplysia sensory neurons.

Two second messenger pathways, one that uses the cAMP-dependent protein kinase A (PKA), the other that uses protein kinase C (PKC), have been found to contribute to the short-term presynaptic facilitation of the connections between the sensory neurons in Aplysia and their target cells, the interneurons and motor neurons of the gill-withdrawal reflex. To study their relative contributions as a function of the previous history of the neuron's activity, we have examined the effects of inhibiting PKA (using Rp-cAMPS) and PKC (using H7) on the short-term facilitation of spontaneous release as well as of the evoked release induced by serotonin at nondepressed, partially depressed, and highly depressed synapses. Our results suggest that whereas activation of PKA is sufficient to trigger the facilitation of nondepressed synapses, activation of both PKA and PKC is required to facilitate depressed synapses, with the contribution of PKC becoming progressively more important as synaptic transmission becomes more depressed.

Target-dependent structural changes in sensory neurons of Aplysia accompany long-term heterosynaptic inhibition.

FMRFamide evokes both short-term and long-term inhibition of synapses between mechanosensory and motor neurons in Aplysia. We report here, using dissociated cell culture and low-light epifluorescence video microscopy, that depression lasting 24 hr of sensorimotor synapses evoked by four brief applications of FMRFamide is accompanied by a significant loss of sensory cell varicosities and neurites. These structural changes in the sensory cells require the presence of the target motor cell L7. Because the loss of structures known to contain transmitter release sites correlates significantly with the changes in the amplitude of the excitatory postsynaptic potential in L7, our results suggest that the structural changes evoked by FMRFamide reflect a loss of synaptic contacts. Thus, long-term depression parallels long-term facilitation of the sensorimotor synapse produced by serotonin in that both forms of heterosynaptic plasticity involve target-dependent modulation of the number of presynaptic varicosities.

Selective short- and long-term effects of serotonin, small cardioactive peptide, and tetanic stimulation on sensorimotor synapses of Aplysia in culture.

Synapses between the sensory and motor cells of Aplysia can be enhanced by heterosynaptic or homosynaptic stimulation. We have used the isolated sensorimotor synapse of Aplysia in cell culture to explore short- and long-term heterosynaptic facilitation produced by 2 facilitatory transmitters and compared these to homosynaptic facilitation produced by posttetanic potentiation. We found that brief application of 5-HT or small cardioactive peptide (SCP) evokes comparable short-lasting enhancement of nondepressed sensorimotor synapses. The effect evoked by SCP diverges from that of 5-HT when the sensorimotor synapse is first depressed by low-frequency homosynaptic stimulation. Whereas 5-HT facilitates sensorimotor synapses whether or not they are depressed, SCP has little or no effect on synapses that have been depressed by more than 75%. The 2 transmitters also differ in producing long-term facilitation. Whereas repeated applications of 5-HT evoke long-term facilitation of the synapses, SCP applications do not. To determine whether these failures to facilitate could be overcome by increasing levels of cAMP, we applied SCP in the presence of phosphodiesterase inhibitors, which resulted in SCP evoking both short- and long-term changes comparable to that of 5-HT. Homosynaptic facilitation by post-tetanic potentiation differed from heterosynaptic facilitation in that tetanic stimulation failed to evoke long-lasting changes in the synapse. These results support recent findings that 5-HT is a critical neuromodulator in behavioral sensitization and dishabituation and suggest that critical levels of cAMP may be required for long- and short-term facilitation of depressed synapses.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term habituation of the acoustic startle response: role of the cerebellar vermis.

The cerebellar vermis has been recognized as a key region of a circuit essential for long-term habituation of the acoustic startle response in rats. The removal of this neuronal structure before training prevents the build-up of this long-term behavioral change. Our data show that, when the same lesion is performed after training for long-term habituation, the learned behavior is not affected. These results indicate that the cerebellar vermis is essential for the acquisition, but not for the retention of long-term habituation of the startle response.

Lesions of the inferior olive do not affect long- or short-term habituation of the acoustic startle response in rats.

Short- and long-term habituation of the acoustic startle response were assessed in a group of inferior olive-lesioned rats. Neither short- and long-term habituation, nor the performance of the reflex, were affected by the lesion. Since the cerebellar vermis is essential for long-term habituation of this reflex, we suggest that climbing fibres are not involved in this form of learning, which would therefore be mediated by the other cerebellar input, presumably the mossy fibres.

Long-term heterosynaptic inhibition in Aplysia.

Synaptic transmission between mechanosensory and motor neurons of the gill withdrawal reflex in Aplysia can undergo both short-term and long-term modulation. One form of short-term synaptic depression lasting minutes can be evoked by the peptide Phe-Met-Arg-Phe-amide (FMRFamide), and is mediated by the lipoxygenase pathway of arachidonic acid. We report here using cell culture, that the same monosynaptic sensory-to-motor component of the gill withdrawal reflex can also undergo long-term synaptic depression lasting 24 h after five applications of FMRFamide over a 2-h period. The long-term depression evoked by FMRFamide is transmitter-specific. Dopamine or low-frequency stimulation of sensory neurons, which also produce short-lasting synaptic depression in vivo, failed to evoke a long-term change. As is the case for long-term presynaptic facilitation of this connection with serotonin, the long-term depression, but not the short-term, can be blocked when applications of FMRFamide are given in the presence of anisomycin, a reversible inhibitor of protein synthesis. Thus, heterosynaptic depression parallels heterosynaptic facilitation in having a long-term as well as a short-term form, and in both cases the long-term modulation requires the synthesis of gene products not essential for the short-term changes.