Enhanced long-term potentiation during aging is masked by processes involving intracellular calcium stores

The contribution of Ca(2+) release from intracellular Ca(2+) stores (ICS) for regulation of synaptic plasticity thresholds during aging was investigated in hippocampal slices of old (22-24 mo) and young adult (5-8 mo) male Fischer 344 rats. Inhibition of Ca(2+)-induced Ca(2+) release by thapsigargin, cyclopiazonic acid (CPA), or ryanodine during pattern stimulation near the threshold for synaptic modification (5 Hz, 900 pulses) selectively induced long-term potentiation (LTP) to CA1 Schaffer collateral synapses of old rats. Increased synaptic strength was specific to test pathways and blocked by AP-5. Intracellular recordings demonstrated that ICS plays a role in the augmentation of the afterhyperpolarization (AHP) in old rats. The decrease in the AHP by ICS inhibition was reversed by the L-channel agonist, Bay K8644. Under conditions of ICS inhibition and a Bay K8644-mediated enhancement of the AHP, pattern stimulation failed to induce LTP, consistent with the idea that the AHP amplitude shapes the threshold for LTP induction. Finally, ICS inhibition was associated with an increase in the N-methyl-d-aspartate (NMDA) receptor component of synaptic transmission in old animals. This increase in the synaptic response was blocked by the calcineurin inhibitor FK506. The results reveal an age-related increase in susceptibility to LTP-induction that is normally inhibited by ICS and suggest that the age-related shift in Ca(2+) regulation and Ca(2+)-dependent synaptic plasticity is coupled to changes in cell excitability and NMDA receptor function through ICS.     

Functional properties of ryanodine receptors in hippocampal neurons change during early differentiation in culture

6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid ryanodine (BODIPY-ryanodine) binding and Ca(2+) imaging were used to study the properties of ryanodine receptors (RyRs) and cytoplasmic Ca(2+) (Ca) changes in neurons cultured from the embryonic rat hippocampus during the earliest stages of differentiation. Baseline Ca levels declined from 164 +/- 5 (SD) nM at early stages to 70 +/- 4 nM in differentiated neurons. Fluorescent BODIPY-ryanodine binding signals identified activated RyRs in somata, which were eliminated by removal of external Ca(2+) or by blockage of Ca(2+) entry through L-type but not N-type Ca(2+) channels. The GABA synthesis inhibitor 3-mercaptopropionic acid completely abolished ryanodine binding. Caffeine or K(+)-depolarization inhibited the activity of RyRs at very early stages of differentiation but had stimulatory effects at later stages after a network of processes had formed. BayK-8644 stimulated RyRs throughout all regions of all differentiating cells. The results suggest that in differentiating embryonic hippocampal neurons the activity of RyRs is maintained via Ca(2+) entering through L-type Ca(2+) channels. The mode of activation of L-type voltage-gated Ca(2+) channels with either membrane depolarization or specific pharmacological agents affects the coupled activity of RyRs differently as neurons differentiate processes and networks.     

Calcium-activated afterhyperpolarizations regulate synchronization and timing of epileptiform bursts in hippocampal CA3 pyramidal neurons

Calcium-activated potassium conductances regulate neuronal excitability, but their role in epileptogenesis remains elusive. We investigated in rat CA3 pyramidal neurons the contribution of the Ca(2+)-activated K(+)-mediated afterhyperpolarizations (AHPs) in the genesis and regulation of epileptiform activity induced in vitro by 4-aminopyridine (4-AP) in Mg(2+)-free Ringer. Recurring spike bursts terminated by prolonged AHPs were generated. Burst synchronization between CA3 pyramidal neurons in paired recordings typified this interictal-like activity. A downregulation of the medium afterhyperpolarization (mAHP) paralleled the emergence of the interictal-like activity. When the mAHP was reduced or enhanced by apamin and EBIO bursts induced by 4-AP were increased or blocked, respectively. Inhibition of the slow afterhyperpolarization (sAHP) with carbachol, t-ACPD, or isoproterenol increased bursting frequency and disrupted burst regularity and synchronization between pyramidal neuron pairs. In contrast, enhancing the sAHP by intracellular dialysis with KMeSO(4) reduced burst frequency. Block of GABA(A-B) inhibitions did not modify the abnormal activity. We describe novel cellular mechanisms where 1) the inhibition of the mAHP plays an essential role in the genesis and regulation of the bursting activity by reducing negative feedback, 2) the sAHP sets the interburst interval by decreasing excitability, and 3) bursting was synchronized by excitatory synaptic interactions that increased in advance and during bursts and decreased throughout the subsequent sAHP. These cellular mechanisms are active in the CA3 region, where epileptiform activity is initiated, and cooperatively regulate the timing of the synchronized rhythmic interictal-like network activity.     

Slow afterhyperpolarization governs the development of NMDA receptor-dependent afterdepolarization in CA1 pyramidal neurons during synaptic stimulation

CA1 pyramidal neurons from animals that have acquired a hippocampus-dependent task show a reduced slow postburst afterhyperpolarization (sAHP). To understand the functional significance of this change, we examined and characterized the sAHP activated by different patterns of synaptic stimuli and its impact on postsynaptic signal integration. Whole cell current-clamp recordings were performed on rat CA1 pyramidal neurons, and trains of stratum radiatum stimuli varying in duration, frequency, and intensity were used to activate the AHP. At -68 mV, a short train of subthreshold stimuli (20-150 Hz) generated only the medium AHP. In contrast, just two suprathreshold stimuli >50 Hz triggered a prominent sAHP sensitive to bath-applications of isoproterenol, carbachol, or intracellularly applied BAPTA, suggesting that the underlying current is the Ca2+-activated K+ current, the sIAHP. The sAHP magnitude was positively related to stimulus train duration and frequency, consistent with its dependence on intracellular Ca2+ accumulation for activation. About 20% of neurons recorded did not have a sAHP. In response to high-frequency suprathreshold stimuli, these neurons developed a pronounced afterdepolarization (ADP) and multiple action potential firing. The ADP magnitude increased with successive stimuli and was positively related to stimulus intensity and frequency. It was sensitive to bath-applications of thapsigargin and nitrendipine, and abolished by d-AP5, indicating that it is supported by intracellular Ca2+ release, the L-type Ca2+ influx, and N-methyl-D-aspartate (NMDA) receptor-mediated influx. In the presence of D-AP5, we were unable to trigger an ADP with maximal stimulus intensity. Pharmacologically eliminating the sAHP allowed neurons to develop an ADP with the original stimulus train. We propose that the slow AHP acts to facilitate Mg2+ re-block of the activated NMDA receptors, thereby reducing temporal summation and preventing an NMDA receptor-dependent ADP during intense synaptic events. Neuromodulation of the sAHP may thus affect information throughput and regulate NMDA receptor-mediated plasticity.     

Relationships between intracellular calcium and afterhyperpolarizations in neocortical pyramidal neurons

We examined the effects of recent discharge activity on [Ca2+]i in neocortical pyramidal cells. Our data confirm and extend the observation that there is a linear relationship between plateau [Ca2+]i and firing frequency in soma and proximal apical dendrites. The rise in [Ca2+] activates K+ channels underlying the afterhyperpolarization (AHP), which consists of 2 Ca(2+)-dependent components: the medium AHP (mAHP) and the slow AHP (sAHP). The mAHP is blocked by apamin, indicating involvement of SK-type Ca(2+)-dependent K+ channels. The identity of the apamin-insensitive sAHP channel is unknown. We compared the sAHP and the mAHP with regard to: 1) number and frequency of spikes versus AHP amplitude; 2) number and frequency of spikes versus [Ca2+]i; 3) IAHP versus [Ca2+]i. Our data suggest that sAHP channels require an elevation of [Ca2+]i in the cytoplasm, rather than at the membrane, consistent with a role for a cytoplasmic intermediate between Ca2+ and the K+ channels. The mAHP channels appear to respond to a restricted Ca2+ domain.     

17beta-estradiol benzoate decreases the AHP amplitude in CA1 pyramidal neurons

Disruption of Ca(2+) homeostasis is hypothesized to mediate several electrophysiological markers of brain aging. Recent evidence indicates that estradiol can rapidly alter Ca(2+)-dependent processes in neurons through nongenomic mechanisms. In the current study, electrophysiological effects of 17beta-estradiol benzoate (EB) on the Ca(2+)-activated afterhyperpolarization (AHP) were investigated using intracellular sharp electrode recording in hippocampal slices from ovariectomized Fischer 344 female rats. The AHP amplitude was enhanced in aged (22-24 mo) compared with young (5-8 mo) rats and direct application of EB (100 pM) reduced the AHP in aged rats. The age-related difference was due, in part, to the increased AHP amplitude of aged animals, since an EB-mediated decrease in the AHP could be observed in young rats when the extracellular Ca(2+) was elevated to increase the AHP amplitude. In aged rats, bath application of EB occluded the ability of the L-channel blocker, nifedipine (10 microM), to attenuate the AHP. The results support a role for EB in modifying hippocampal Ca(2+)-dependent processes in a manner diametrically opposite that observed during aging, possibly through L-channel inhibition.     

Effects of temperature on calcium transients and Ca2+-dependent afterhyperpolarizations in neocortical pyramidal neurons

In neocortical pyramidal neurons, the medium (mAHP) and slow AHP (sAHP) have different relationships with intracellular [Ca2+]. To further explore these differences, we varied bath temperature and compared passive and active membrane properties and Ca2+ transients in response to a single action potential (AP) or trains of APs. We tested whether Ca(2+)-dependent events are more temperature sensitive than voltage-dependent ones, the slow rise time of the sAHP is limited by diffusion, and temperature sensitivity differs between the mAHP and sAHP. The onset and decay kinetics of the sAHP were very temperature sensitive (more so than diffusion). We found that the decay time course of Ca2+ transients was also very temperature sensitive. In contrast, the mAHP (amplitude, time to peak, and exponential decay) and sAHP peak amplitude were moderately sensitive to temperature. The amplitudes of intracellular Ca2+ transients evoked either by a single spike or a train of spikes showed modest temperature sensitivities. Pyramidal neuron input resistance was increased by cooling. With the exception of threshold, which remained unchanged between 22 and 35 degrees C, action potential parameters (amplitude, half-width, maximum rates of rise and fall) were modestly affected by temperature. Collectively, these data suggest that temperature sensitivity was higher for the Ca(2+)-dependent sAHP than for voltage-dependent AP parameters or for the mAHP, diffusion of Ca2+ over distance cannot explain the slow rise of the sAHP in these cells, and the kinetics of the sAHP and mAHP are affected differently by temperature.     

Electrophysiological roles of L-type channels in different classes of guinea pig sympathetic neuron.

The electrophysiological consequences of blocking Ca(2+) entry through L-type Ca(2+) channels have been examined in phasic (Ph), tonic (T), and long-afterhyperpolarizing (LAH) neurons of intact guinea pig sympathetic ganglia isolated in vitro. Block of Ca(2+) entry with Co(2+) or Cd(2+) depolarized T and LAH neurons, reduced action potential (AP) amplitude in Ph and LAH neurons, and increased AP half-width in Ph neurons. The afterhyperpolarization (AHP) and underlying Ca(2+)-dependent K(+) conductances (gKCa1 and gKCa2) were reduced markedly in all classes. Addition of 10 microM nifedipine increased input resistance in LAH neurons, raised AP threshold in Ph and LAH neurons, and caused a small increase in AP half-width in Ph neurons. AHP amplitude and the amplitude and decay time constant of gKCa1 were reduced by nifedipine in all classes; the slower conductance, gKCa2, which underlies the prolonged AHP in LAH neurons, was reduced by 40%. Surprisingly, AHP half-width was lengthened by nifedipine in a proportion of neurons in all classes; despite this, neuron excitability was increased during a maintained depolarization. Nifedipine's effects on AHP half-width were not mimicked by 2 mM Cs(+) or 2 mM anthracene-9-carboxylic acid, a blocker of Cl(-) channels, and it did not modify transient outward currents of the A or D types. The effects of 100 microM Ni(2+) differed from those of nifedipine. Thus in Ph neurons, Ca(2+) entry through L-type channels during a single action potential contributes to activation of K(+) conductances involved in both the AP and AHP, whereas in T and LAH neurons, it acts only on gKCa1 and gKCa2. These results differ from the results in rat superior cervical ganglion neurons, in which L-type channels are selectively coupled to BK channels, and in hippocampal neurons, in which L-type channels are selectively coupled to SK channels. We conclude that the sources of Ca(2+) for activating the various Ca(2+)-activated K(+) conductances are distinct in different types of neuron.     

Muscarinic control of dendritic excitability and Ca(2+) signaling in CA1 pyramidal neurons in rat hippocampal slice.

The cholinergic system is critically involved in synaptic models of learning and memory by enhancing dendritic [Ca(2+)](i) signals. Diffuse cholinergic innervation suggests subcellular modulation of membrane currents and Ca(2+) signals. Here we use ion-selective microelectrodes to study spread of carbachol (CCh) after focal application into brain slice and subcellular muscarinic modulation of synaptic responses in CA1 pyramidal neurons. Proximal application of CCh rapidly blocked the somatic slow afterhyperpolarization (sAHP) following repetitive stimulation. In contrast, the time course of potentiation of the slow tetanic depolarization (STD) during synaptic input was slower and followed the time course of spread of CCh to the dendritic tree. With distal application, augmentation of the somatic STD and of dendritic Ca(2+) responses followed spread of CCh to the entire apical dendritic tree, whereas the sAHP was blocked only after spread of CCh to the proximal dendritic segment. In dendritic recordings, CCh blocked a small sAHP, augmented the STD, and rather reduced dendritic action potentials. Augmentation of dendritic Ca(2+) signals was highly correlated to augmentation of the STD. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV) blocked approximately 55% of the STD in control and during CCh application. In conclusion, muscarinic suppression of the proximal sAHP can augment firing and thereby Ca(2+) responses. Dendritic augmentation of the STD by blockade of the sAHP and direct enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated currents potentiates Ca(2+) signals even when firing is not affected due to suprathreshold input. In this way, subcellular muscarinic modulation may contribute to parallel information processing and storage by dendritic synapses of CA1 pyramidal neurons.     

Protein kinase A mediates the modulation of the slow Ca(2+)-dependent K(+) current, I(sAHP), by the neuropeptides CRF, VIP, and CGRP in hippocampal pyramidal neurons

We have studied modulation of the slow Ca(2+)-activated K(+) current (I(sAHP)) in CA1 hippocampal pyramidal neurons by three peptide transmitters: corticotropin releasing factor (CRF, also called corticotropin releasing hormone, CRH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). These peptides are known to be expressed in interneurons. Using whole cell voltage clamp in hippocampal slices from young rats, in the presence of tetrodotoxin (TTX, 0.5 microM) and tetraethylammonium (TEA, 5 mM), I(sAHP) was measured after a brief depolarizing voltage step eliciting inward Ca(2+) current. Each of the peptides CRF (100-250 nM), VIP (400 nM), and CGRP (1 microM) significantly reduced the amplitude of I(sAHP). Thus the I(sAHP) amplitude was reduced to 22% by 100 nM CRF, to 17% by 250 nM CRF, to 22% by 400 nM VIP, and to 40% by 1 microM CGRP. We found no consistent concomitant changes in the Ca(2+) current or in the time course of I(sAHP) for any of the three peptides, suggesting that the suppression of I(sAHP) was not secondary to a general suppression of Ca(2+) channel activity. Because each of these peptides is known to activate the cyclic AMP (cAMP) cascade in various cell types, and I(sAHP) is known to be suppressed by cAMP via the cAMP-dependent protein kinase (PKA), we tested whether the effects on I(sAHP) by CRF, VIP, and CGRP are mediated by PKA. Intracellular application of the PKA-inhibitor Rp-cAMPS significantly reduced the suppression of I(sAHP) by CRF, VIP, and CGRP. Thus with 1 mM Rp-cAMPS in the recording pipette, the average suppression of I(sAHP) was reduced from 78 to 26% for 100 nM CRF, from 83 to 32% for 250 nM CRF, from 78 to 30% for 400 nM VIP, and from 60 to 7% for 1 microM CGRP. We conclude that CRF, VIP, and CGRP suppress the slow Ca(2+)-activated K(+) current, I(sAHP), in CA1 hippocampal pyramidal neurons by activating the cAMP-dependent protein kinase, PKA. Together with the monoamine transmitters norepinephrine, serotonin, histamine, and dopamine, these peptide transmitters all converge on the cAMP cascade modulating I(sAHP).     

Neuronal endoplasmic reticulum acts as a single functional Ca2+ store shared by ryanodine and inositol-1,4,5-trisphosphate receptors as revealed by intra-ER [Ca2+] recordings in single rat sensory neurones

We addressed the fundamentally important question of functional continuity of endoplasmic reticulum (ER) Ca(2+) store in nerve cells. In cultured rat dorsal root ganglion neurones we measured dynamic changes in free Ca(2+) concentration within the ER lumen ([Ca(2+)](L)) in response to activation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs). We found that both receptors co-exist in these neurones and their activation results in Ca(2+) release from the ER as judged by a decrease in [Ca(2+)](L). Depletion of Ca(2+) stores following an inhibition of sarco(endoplasmic)reticulum Ca(2+)-ATPase by thapsigargin or cyclopiazonic acid completely eliminated Ca(2+) release via both InsP(3)Rs and RyRs. Similarly, when the store was depleted by continuous activation of InsP(3)Rs, activation of RyRs (by caffeine or 0.5 microM ryanodine) failed to produce Ca(2+) release, and vice versa, when the stores were depleted by activators of RyRs, the InsP(3)-induced Ca(2+) release disappeared. We conclude that in mammalian neurones InsP(3)Rs and RyRs share the common continuous Ca(2+) pool associated with ER.     

Distribution of inositol-1,4,5-trisphosphate receptor isotypes and ryanodine receptor isotypes during maturation of the rat hippocampus

Activation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) can lead to the release of Ca(2+) from intracellular stores and propagating Ca(2+) waves. Previous studies of these proteins in neurons have focused on their distribution in adult tissue, whereas, recent functional studies have examined neural tissue extracted from prenatal and young postnatal animals. In this study we examined the distribution of InsP(3)R isotypes 1-3 and RyR isotypes 1-3 in rat hippocampus during postnatal maturation, with a focus on InsP(3)R1 because it is most prominent in the hippocampus. InsP(3)R1 was observed in pyramidal cells and granule cells, InsP(3)R2 immunoreactivity was observed in perivascular astrocytes and endothelial cells, and InsP(3)R3 immunoreactivity was detected in axon terminals located in stratum pyramidale of CA1 and microvessels in stratum radiatum. RyR1 immunolabeling was enriched in CA1, RyR2 was most intense in CA3 and the dentate gyrus, and RyR3 immunolabeling was detected in all subfields of the hippocampus, but was most intense in stratum lacunosum-moleculare. During maturation from 2 to 10 weeks of age there was a shift in InsP(3)R1 immunoreactivity from a high density in the proximal apical dendrites to a uniform distribution along the dendrites. Independent of age, InsP(3)R1 immunoreactivity was observed to form clusters within the primary apical dendrite and at dendritic bifurcations of pyramidal neurons. As CA1 pyramidal neurons matured, InsP(3)R1 was often co-localized with the Ca(2+) binding protein calbindin D-28k. In contrast, InsP(3)R1 immunolabel was never co-localized with calbindin D-28k immunopositive interneurons located outside of stratum pyramidale or with parvalbumin, typically found in hippocampal basket cells, suggesting that InsP(3)R1s do not play a role in internal Ca(2+) release in these interneurons. These findings should help to interpret past functional studies and inform future studies examining the characteristics and consequences of InsP(3)R-mediated internal Ca(2+) release and Ca(2+) waves in hippocampal neurons.     

Ca(2+) entry through L-type Ca(2+) channels helps terminate epileptiform activity by activation of a Ca(2+) dependent afterhyperpolarisation in hippocampal CA3

In CA3 neurons of disinhibited hippocampal slice cultures the slow afterhyperpolarisation, following spontaneous epileptiform burst events, was confirmed to be Ca(2+) dependent and mediated by K(+) ions. Apamin, a selective blocker of the SK channels responsible for part of the slow afterhyperpolarisation reduced, but did not abolish, the amplitude of the post-burst afterhyperpolarisation. The result was an increased excitability of individual CA3 cells and the whole CA3 network, as measured by burst duration and burst frequency. Increases in excitability could also be achieved by strongly buffering intracellular Ca(2+) or by minimising Ca(2+) influx into the cell, specifically through L-type (but not N-type) voltage operated Ca(2+) channels. Notably the L-type Ca(2+) channel antagonist, nifedipine, was more effective than apamin at reducing the post-burst afterhyperpolarisation. Nifedipine also caused a greater increase in network excitability as determined from measurements of burst duration and frequency from whole cell and extracellular recordings. N-methyl D-aspartate receptor activation contributed to the depolarisations associated with the epileptiform activity but Ca(2+) entry via this route did not contribute to the activation of the post-burst afterhyperpolarisation.We suggest that Ca(2+) entry through L-type channels during an epileptiform event is selectively coupled to both apamin-sensitive and -insensitive Ca(2+) activated K(+) channels. Our findings have implications for how the route of Ca(2+) entry and subsequent Ca(2+) dynamics can influence network excitability during epileptiform discharges.     

Group I mGluRs increase excitability of hippocampal CA1 pyramidal neurons by a PLC-independent mechanism

Previous studies have implicated phospholipase C (PLC)-linked Group I metabotropic glutamate receptors (mGluRs) in regulating the excitability of hippocampal CA1 pyramidal neurons. We used intracellular recordings from rat hippocampal slices and specific antagonists to examine in more detail the mGluR receptor subtypes and signal transduction mechanisms underlying this effect. Application of the Group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) suppressed slow- and medium-duration afterhyperpolarizations (s- and mAHP) and caused a consequent increase in cell excitability as well as a depolarization of the membrane and an increase in input resistance. Interestingly, with the exception of the suppression of the mAHP, these effects were persistent, and in the case of the sAHP lasting for more than 1 h of drug washout. Preincubation with the specific mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), reduced but did not completely prevent the effects of DHPG. However, preincubation with both MPEP and the mGluR1 antagonist LY367385 completely prevented the DHPG-induced changes. These results demonstrate that the DHPG-induced changes are mediated partly by mGluR5 and partly by mGluR1. Because Group I mGluRs are linked to PLC via G-protein activation, we also investigated pathways downstream of PLC activation, using chelerythrine and cyclopiazonic acid to block protein kinase C (PKC) and inositol 1,4,5-trisphosphate-(IP(3))-activated Ca(2+) stores, respectively. Neither inhibitor affected the DHPG-induced suppression of the sAHP or the increase in excitability nor did an inhibitor of PLC itself, U-73122. Taken together, these results argue that in CA1 pyramidal cells in the adult rat, DHPG activates mGluRs of both the mGluR5 and mGluR1 subtypes, causing a long-lasting suppression of the sAHP and a consequent persistent increase in excitability via a PLC-, PKC-, and IP(3)-independent transduction pathway.     

Coexistence of functional IP(3) and ryanodine receptors in vagal sensory neurons and their activation by ATP

Intracellular photorelease of caged D-myo-inositol 1,4,5-trisphosphate (IP(3)), caffeine application, and immunofluorescence confocal microscopy were used to determine that D-myo-inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) coexist in rabbit vagal sensory nodose ganglion neurons (NGNs). ATP, an extracellular physiological signaling molecule, consistently evoked robust transient increases in cytosolic free Ca(2+) concentration (Ca(2+) transients). ATP applied in Ca(2+)-free physiological saline elicited Ca(2+) transients that averaged approximately 70% of the amplitude of transients evoked in the presence of extracellular Ca(2+). The component of the ATP-evoked Ca(2+) transient that was independent of extracellular Ca(2+) corresponds to Ca(2+) release from intracellular stores. This release component was sensitive to the pharmacological antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), U73122, neomycin, and heparin (13.5-15 kD), indicating that P2 purinoreceptors (P2Y) and the IP(3) signaling pathway are required for ATP-evoked Ca(2+) release. Additionally, a portion of ATP-evoked Ca(2+) release was inhibited by ryanodine, a selective blocker of RyRs. The ryanodine-insensitive component (approximately 70%) of ATP-evoked Ca(2+) release corresponds to IP(3)-induced Ca(2+) release via IP(3)Rs, while the ryanodine-sensitive component (approximately 30%) corresponds to consequent Ca(2+)-induced Ca(2+) release (CICR) via RyRs. These results indicate that functional IP(3)Rs and RyRs coexist in nodose neurons and that both IP(3)-induced Ca(2+) release and CICR can be activated by ATP.     

Ryanodine-sensitive stores regulate the excitability of AH neurons in the myenteric plexus of guinea-pig ileum

Myenteric afterhyperpolarizing (AH) neurons are primary afferent neurons within the gastrointestinal tract. Stimulation of the intestinal mucosa evokes action potentials (AP) that are followed by a slow afterhyperpolarization (AHP(slow)) in the soma. The role of intracellular Ca(2+) ([Ca(2+)](i)) and ryanodine-sensitive Ca(2+) stores in modulating the electrical activity of myenteric AH neurons was investigated by recording membrane potential and bis-fura-2 fluorescence from 34 AH neurons. Mean resting [Ca(2+)](i) was approximately 200 nM. Depolarizing current pulses that elicited APs evoked AHP(slow) and an increase in [Ca(2+)](i), with similar time courses. The amplitudes and durations of AHP(slow) and the Ca(2+) transient were proportional to the number of evoked APs, with each AP increasing [Ca(2+)](i) by approximately 50 nM. Ryanodine (10 microM) significantly reduced both the amplitude and duration (by 60%) of the evoked Ca(2+) transient and AHP(slow) over the range of APs tested (1-15). Calcium-induced calcium release (CICR) was graded and proportional to the number of APs, with each AP triggering a rise in [Ca(2+)](i) of approximately 30 nM Ca(2+) via CICR. This indicates that CICR amplifies Ca(2+) influx. Similar changes in [Ca(2+)](i) and AHP(slow) were evoked by two APs in control and six APs in ryanodine. Thus, the magnitude of the change in bulk [Ca(2+)](i) and not the source of the Ca(2+) is the determinant of the magnitude of AHP(slow). Furthermore, lowering of free [Ca(2+)](i), either by reducing extracellular Ca(2+) or injecting high concentrations of Ca(2+) buffer, induced depolarization, increased excitability, and abolition of AHP(slow). In addition, activation of synaptic input to AH neurons elicited a slow excitatory postsynaptic potential (sEPSP) that was completely blocked in ryanodine. These results demonstrate the importance of [Ca(2+)](i) and CICR in sensory processing in AH neurons. Activity-dependent CICR may be a mechanism to grade the output of AH neurons according to the intensity of sensory input.     

Roles of ryanodine and inositol triphosphate receptors in regulation of plateau potentials in turtle spinal motoneurons

Generation of plateau potentials in spinal motoneurons depends on activation of voltage sensitive L-type Ca(2+) channels. These channels are facilitated by metabotropic receptors known to promote release of Ca(2+) from intracellular stores. The aim of this study is to determine if Ca(2+)-release receptors in the endoplasmic reticulum (ER) that are sensitive to ryanodine (RyRs) and to inositol triphosphate receptors (IP(3)Rs) contribute to the generation of plateau potentials. The effects of antagonists to RyRs, IP(3)Rs and phospholipase C (PLC) were tested on discharge patterns associated with plateau potentials in motoneurons in slices from the spinal cord of the turtle. Plateau-related discharge patterns, un-facilitated or facilitated by agonists for group I glutamate metabotropic receptors, muscarine-sensitive cholinergic receptors or L-type Ca(2+) channels were inhibited by blockade of RyRs. In contrast, antagonists of IP(3)Rs or PLC preferentially inhibited plateau-related discharge patterns when facilitated by activation of metabotropic receptors but in only half of the cells when promoted in the absence of metabotropic facilitators. Our findings show that RyRs and IP(3)Rs regulate the generation of plateau potentials in motoneurons and suggest that RyRs may be directly involved with activation of the plateau potential.     

Distribution of slow AHP channels on hippocampal CA1 pyramidal neurons

This work was designed to localize the Ca(2+)-activated K(+) channels underlying the slow afterhyperpolarization (sAHP) in hippocampal CA1 pyramidal cells. Cell-attached patches on the proximal 100 microm of the apical dendrite contained K(+) channels, but not sAHP channels, activated by backpropagating action potentials. Amputation of the apical dendrite approximately 30 microm from the soma, while simultaneously recording the sAHP whole cell current at the soma, depressed the sAHP amplitude by only approximately 30% compared with control. Somatic cell-attached and nucleated patches did not contain sAHP current. Amputation of the axon >/=20 microm from the soma had little effect on the amplitude of the sAHP recorded in cortical pyramidal cells. By this process of elimination, it is suggested that sAHP channels may be concentrated in the basal dendrites of CA1 pyramids.     

Distinct intracellular calcium profiles following influx through N- versus L-type calcium channels: role of Ca2+-induced Ca2+ release

Selective activation of neuronal functions by Ca(2+) is determined by the kinetic profile of the intracellular calcium ([Ca(2+)](i)) signal in addition to its amplitude. Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca(2+) current and the resulting rise and decay of [Ca(2+)](i) in differentiated pheochromocytoma (PC12) cells. We show that equal amounts of Ca(2+) entering through N-type and L-type voltage-gated Ca(2+) channels result in significantly different [Ca(2+)](i) temporal profiles. When the contribution of N-type channels was reduced by omega-conotoxin MVIIA treatment, a faster [Ca(2+)](i) decay was observed. Conversely, when the contribution of L-type channels was reduced by nifedipine treatment, [Ca(2+)](i) decay was slower. Potentiating L-type current with BayK8644, or inactivating N-type channels by shifting the holding potential to -40 mV, both resulted in a more rapid decay of [Ca(2+)](i). Channel-specific differences in [Ca(2+)](i) decay rates were abolished by depleting intracellular Ca(2+) stores with thapsigargin or by blocking ryanodine receptors with ryanodine, suggesting the involvement of Ca(2+)-induced Ca(2+) release (CICR). Further support for involvement of CICR is provided by the demonstration that caffeine slowed [Ca(2+)](i) decay while ryanodine at high concentrations increased the rate of [Ca(2+)](i) decay. We conclude that Ca(2+) entering through N-type channels is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR provides a mechanism whereby the kinetics of intracellular Ca(2+) leaves a fingerprint of the route of entry, potentially encoding the selective activation of a subset of Ca(2+)-sensitive processes within the neuron.     

Caloric restriction prevents aging-associated changes in spike-mediated Ca2+ accumulation and the slow afterhyperpolarization in hippocampal CA1 pyramidal neurons

In hippocampal pyramidal neurons from aged animals voltage-gated Ca2+ entry and the slow, post-burst afterhyperpolarization are enhanced. As a result, there is a decrease in neuronal excitability and, in turn, an alteration in synaptic plasticity. Restricting the caloric intake of a rodent is a well-known paradigm for increasing lifespan and ameliorating a number of neurodegenerative features of aging, including deficits in synaptic plasticity and cognition. Here we show in rat CA1 pyramidal neurons from aged animals (18-20 months old) that a restricted diet prevents the enhancement of dendritic spike-mediated Ca2+ accumulation. In contrast, no significant changes in the rates of Ca2+ recovery were observed suggesting that Ca2+ clearance mechanisms are not affected by aging or caloric restriction. Lastly, we found that caloric restriction also prevented the aging-associated increase in the slow, post-burst afterhyperpolarization. Our results suggest that caloric restriction-sensitive changes in Ca2+ accumulation and membrane excitability may in part account for the protective effects of dietary restriction on synaptic plasticity and learning deficits in aged animals.