Pyramidal Neurons In Hippocampal Slices Research Articles

BDNF Enhances Quantal Neurotransmitter Release and Increases the Number of Docked Vesicles at the Active Zones of Hippocampal Excitatory Synapses

Brain-derived neurotrophic factor (BDNF) is emerging as a key mediator of activity-dependent modifications of synaptic strength in the CNS. We investigated the hypothesis that BDNF enhances quantal neurotransmitter release by modulating the distribution of synaptic vesicles within presynaptic terminals using organotypic slice cultures of postnatal rat hippocampus. BDNF specifically increased the number of docked vesicles at the active zone of excitatory synapses on CA1 dendritic spines, with only a small increase in active zone size. In agreement with the hypothesis that an increased docked vesicle density enhances quantal neurotransmitter release, BDNF increased the frequency, but not the amplitude, of AMPA receptor-mediated miniature EPSCs (mEPSCs) recorded from CA1 pyramidal neurons in hippocampal slices. Synapse number, independently estimated from dendritic spine density and electron microscopy measurements, was also increased after BDNF treatment, indicating that the actions of BNDF on mEPSC frequency can be partially attributed to an increased synaptic density. Our results further suggest that all these actions were mediated via tyrosine kinase B (TrkB) receptor activation, established by inhibition of plasma membrane tyrosine kinases with K-252a. These results provide additional evidence of a fundamental role of the BDNF-TrkB signaling cascade in synaptic transmission, as well as in cellular models of hippocampus-dependent learning and memory.

PDZ Proteins Interacting with C-Terminal GluR2/3 Are Involved in a PKC-Dependent Regulation of AMPA Receptors at Hippocampal Synapses

We investigated the role of PDZ proteins (GRIP, ABP, and PICK1) interacting with the C-terminal GluR2 by infusing a ct-GluR2 peptide (“pep2-SVKI”) into CA1 pyramidal neurons in hippocampal slices using whole-cell recordings. Pep2-SVKI, but not a control or PICK1 selective peptide, caused AMPAR-mediated EPSC amplitude to increase in approximately one-third of control neurons and in most neurons following the prior induction of LTD. Pep2-SVKI also blocked LTD; however, this occurred in all neurons. A PKC inhibitor prevented these effects of pep2-SVKI on synaptic transmission and LTD. We propose a model in which the maintenance of LTD involves the binding of AMPARs to PDZ proteins to prevent their reinsertion. We also present evidence that PKC regulates AMPAR reinsertion during dedepression.

Potassium-induced enhancement of persistent inward current in hippocampal neurons in isolation and in tissue slices

Previous work suggested a role for the voltage-dependent persistent sodium current, I Na,P, in the generation of seizures and spreading depression (SD). Ordinarily, I Na,P is small in hippocampal neurons. We investigated the effect of raising external K + concentration, [K +] o, on whole-cell persistent inward current in freshly isolated hippocampal CA1 pyramidal neurons. I Na,P was identified by TTX-sensitivity and dependence on external Na + concentration. When none of the ion channels were blocked, I Na,P was not usually detectable, probably because competing K + current masked it, but after raising [K +] o I Na,P appeared, while K + currents diminished. With K + channels blocked, I Na,P could usually be evoked in control solution and raising [K +] o caused its reversible increase in most cells. The increase did not depend on external calcium [Ca 2+] o. In CA1 pyramidal neurons in hippocampal slices a TTX-sensitive persistent inward current was always recorded and when [K +] o was raised, it was reversibly enhanced. Strong depolarization evoked irregular current fluctuations, which were also augmented in high [K +] o. The findings support a role of potassium-mediated positive feedback in the generation of seizures and spreading depression.

Inositol 1,4,5-Trisphosphate (IP3)-Mediated Ca2+Release Evoked by Metabotropic Agonists and Backpropagating Action Potentials in Hippocampal CA1 Pyramidal Neurons

We examined the properties of [Ca(2+)](i) changes that were evoked by backpropagating action potentials in pyramidal neurons in hippocampal slices from the rat. In the presence of the metabotropic glutamate receptor (mGluR) agonists t-ACPD, DHPG, or CHPG, spikes caused Ca(2+) waves that initiated in the proximal apical dendrites and spread over this region and in the soma. Consistent with previously described synaptic responses (Nakamura et al., 1999a), pharmacological experiments established that the waves were attributable to Ca(2+) release from internal stores mediated by the synergistic effect of receptor-mobilized inositol 1,4, 5-trisphosphate (IP(3)) and spike-evoked Ca(2+). The amplitude of the changes reached several micromoles per liter when detected with the low-affinity indicators fura-6F, fura-2-FF, or furaptra. Repetitive brief spike trains at 30-60 sec intervals generated increases of constant amplitude. However, trains at intervals of 10-20 sec evoked smaller increases, suggesting that the stores take 20-30 sec to refill. Release evoked by mGluR agonists was blocked by MCPG, AIDA, 4-CPG, MPEP, and LY367385, a profile consistent with the primacy of group I receptors. At threshold agonist concentrations the release was evoked only in the dendrites; threshold antagonist concentrations were effective only in the soma. Carbachol and 5-HT evoked release with the same spatial distribution as t-ACPD, suggesting that the distribution of neurotransmitter receptors was not responsible for the restricted range of regenerative release. Intracellular BAPTA and EGTA were approximately equally effective in blocking release. Extracellular Cd(2+) blocked release, but no single selective Ca(2+) channel blocker prevented release. These results suggest that IP(3) receptors are not associated closely with specific Ca(2+) channels and are not close to each other.

Microheterogeneity of calcium signalling in dendrites.

Transient changes in the intracellular concentration of free Ca2+ ([Ca2+]i) originating from voltage- or ligand-gated influx and by ligand- or Ca2+-gated release from intracellular stores, trigger or modulate many fundamental neuronal processes, including neurotransmitter release and synaptic plasticity. Of the intracellular compartments involved in Ca2+ clearance, the endoplasmic reticulum (ER) has received the most attention because it expresses Ca2+ pumps and Ca2+ channels, thus endowing it with the potential to act as both an intracellular calcium sink and store. We review here our ongoing work on the role of calcium sequestration into, and release from, ER cisterns and the role that this plays in the generation and termination of free [Ca2+]i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. These studies have been approached by combining parallel microfluorometric measurements of free cytosolic [Ca2+]i transients with energy-dispersive X-ray microanalytical measurements of total Ca content within specific dendritic compartments at the electron microscopy level. Our observations support the emerging realization that specific subsets of dendritic ER cisterns provide spatial and temporal microheterogeneity of Ca2+ signalling, acting not only as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca2+ influx, but also as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses.

Two-photon Na+ imaging in spines and fine dendrites of central neurons

Dendritic spines are assumed to be the smallest units of neuronal integration. Because of their miniature size, however, many of their functional properties are still unclear. New insights in spine physiology have been provided by two-photon laser-scanning microscopy which allows fluorescence imaging with high spatial resolution and minimal photodamage. For example, two-photon imaging has been employed successfully for the measurement of activity-induced calcium transients in individual spines. Here, we describe the first application of two-photon imaging to measure Na+ transients in spines and dendrites of CA1 pyramidal neurons in hippocampal slices. Whole-cell patch-clamped neurons were loaded with the Na(+)-indicator dye SBFI (sodium-binding benzofuran-isophthalate). In situ calibration of SBFI fluorescence with ionophores enabled the determination of the actual magnitude of the [Na+]i changes. We found that back-propagating action potentials (APs) evoked Na+ transients throughout the proximal part of the dendritic tree and adjacent spines. The action-potential-induced [Na+]i transients reached values of 4 mM for a train of 20 APs and monotonically decayed with a time constant of several seconds. These results represent the first demonstration of activity-induced Na+ accumulation in spines. Our results demonstrate that two-photon Na+ imaging represents a powerful tool for extending our knowledge on Na+ signaling in fine cellular subcompartments.

Correlated measurements of free and total intracellular calcium concentration in central nervous system neurons.

Transient changes in the intracellular concentration of free calcium ([Ca2+])i) act as a trigger or modulator for a large number of important neuronal processes. Such transients can originate from voltage- or ligand-gated fluxes of Ca2+ into the cytoplasm from the extracellular space, or by ligand- or Ca2+(-)gated release from intracellular stores. Characterizing the sources and spatio-temporal patterns of [Ca2+]i transients is critical for understanding the role of different neuronal compartments in dendritic integration and synaptic plasticity. Optical imaging of fluorescent indicators sensitive to free Ca2+ is especially suited to studying such phenomena because this approach offers simultaneous monitoring of large regions of the dendritic tree in individual living central nervous system neurons. In contrast, energy-dispersive X-ray (EDX) microanalysis provides quantitative information on the amount and location of intracellular total, i.e., free plus bound, calcium (Ca) within specific subcellular dendritic compartments as a function of the activity state of the neuron. When optical measurements of [Ca2+]i transients and parallel EDX measurements of Ca content are used in tandem, and correlated simultaneously with electrophysiological measurements of neuronal activity, the combined information provides a relatively general picture of spatio-temporal neuronal total Ca fluctuations. To illustrate the kinds of information available with this approach, we review here results from our ongoing work aimed at evaluating the role of various Ca uptake, release, sequestration, and extrusion mechanisms in the generation and termination of [Ca2+]i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. Our observations support the long-standing speculation that the dendritic endoplasmic reticulum acts not only as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses, but also as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca2+ influx.

Long-term synaptic changes induced by intracellular tetanization of CA3 pyramidal neurons in hippocampal slices from juvenile rats

Minimal excitatory postsynaptic potentials were evoked in CA3 pyramidal neurons by activation of the mossy fibres in hippocampal slices from seven- to 16-day-old rats. Conditioning intracellular depolarizing pulses were delivered as 50- or 100-Hz bursts. A statistically significant depression and potentiation was induced in four and five of 13 cases, respectively. The initial state of the synapses influenced the effect: the amplitude changes correlated with the pretetanic paired-pulse facilitation ratio. Afferent (mossy fibre) tetanization produced a significant depression in four of six inputs, and no significant changes in two inputs. Quantal content decreased or increased following induction of the depression or potentiation, respectively, whereas no significant changes in quantal size were observed. Compatible with presynaptic maintenance mechanisms of both depression and potentiation, changes in the mean quantal content were associated with modifications in the paired-pulse facilitation ratios, coefficient of variation of response amplitudes and number of response failures. Cases were encountered when apparently “presynaptically silent” synapses were converted into functional synapses during potentiation or when effective synapses became “presynaptically silent” when depression was induced, suggesting respective changes in the probability of transmitter release. It is concluded that, in juvenile rats, it is possible to induce lasting potentiation at the mossy fibre–CA3 synapses by purely postsynaptic stimulation, while afferent tetanization is accompanied by long-lasting depression. The data support the existence not only of a presynaptically induced, but also a postsynaptically induced form of long-term potentiation in the mossy fibre–CA3 synapse. Despite a postsynaptic induction mechanism, maintenance of both potentiation and depression is likely to occur presynaptically.

Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated.

At excitatory synapses in the central nervous system, the number of glutamate molecules released from a vesicle is much larger than the number of postsynaptic receptors. But does release of a single vesicle normally saturate these receptors? Answering this question is critical to understanding how the amplitude and variability of synaptic transmission are set and regulated. Here we describe the use of two-photon microscopy to image transient increases in Ca2+ concentration mediated by NMDA (N-methyl-D-aspartate) receptors in single dendritic spines of CA1 pyramidal neurons in hippocampal slices. To test for NMDA-receptor saturation, we compared responses to stimulation with single and double pulses. We find that a single release event does not saturate spine NMDA receptors; a second release occurring 10 ms later produces approximately 80% more NMDA-receptor activation. The amplitude of spine NMDA-receptor-mediated [Ca2+] transients (and the synaptic plasticity which depends on this) may thus be sensitive to the number of quanta released by a burst of action potentials and to changes in the concentration profile of glutamate in the synaptic cleft.

Confocal laser scanning microscopy used to monitor intracellular Ca 2+ changes in hippocampal CA 1 neurons during energy deprivation

An increase in intracellular calcium during cerebral ischemia has been proposed as a common final pathway underlying the events leading to neuronal death. Intracellular calcium has been measured with ion selective electrodes during energy deprivation (ED) in hippocampal slices and with fluorescent techniques in neuronal cultures. In the present study, we describe a novel method to visualize and quantify changes in intracellular calcium in brain slices using Confocal Laser Scanning Microscopy (CLSM). CA 1 pyramidal neurons in hippocampal slices were filled by intracellular injection with a 1:2 mixture of the fluorescent dyes Fluo 3 and Fura Red. The neurons were then visualized using CLSM, and the ratio of the fluorescence from each probe used to quantify intracellular calcium concentrations before and during ED. The free intracellular calcium concentration was 60 nM prior to ED and increased to 24 μM during ED. These results demonstrates that CLSM and fluorescent probes can be used in functional neuronal networks in addition to cell cultures as previously described.

Eliprodil, a non-competitive, NR2B-selective NMDA antagonist, protects pyramidal neurons in hippocampal slices from hypoxic/ischemic damage

The N-methyl- d-aspartate (NMDA) subtype of glutamate receptor is one pathway through which excessive influx of calcium has been suggested to trigger ischemia-induced delayed neuronal death. NMDA receptors are heterooligomeric complexes comprised of both NR1 and NR2A-D subunits, in various combinations. NR2B-containing NMDA complexes exhibit larger, more prolonged conductances than those lacking this subunit. We tested the ability of the non-competitive, NR2B-selective NMDA antagonist eliprodil to (a) protect synaptic transmission in in vitro hippocampal slices from hypoxia, and (b) reduce ischemic delayed neuronal death in hippocampal organotypic slice cultures. Eliprodil markedly improved the recovery of Schaffer collateral-CA1 excitatory postsynaptic potentials following a 15 min hypoxic insult, with an EC50 of approximately 0.5 μM. In contrast to this functional protection, eliprodil did not reduce delayed death of CA1 pyramidal neurons in organotypic hippocampal slice cultures treated with severe hypoxia plus hypoglycemia, though it did potently protect CA3 pyramidal neurons in the same cultures. These data indicate that NMDA receptors containing NR2B subunits may play a role in long-term recovery of hippocampal synaptic function following ischemia/hypoxia. Furthermore, the selective protection of CA3, but not CA1, pyramidal neurons suggests that NR2B-containing NMDA receptors may preferentially contribute to an excitotoxic component of ischemia-induced delayed neuronal death.

P-1-139 - Preliminary study on controlled hypotension induced by nimodipine in cranio-cerebral surgery. A new trial

Young adult male albino rabbits were conditioned using a free field auditory conditioned stimulus (CS) and periorbital shock unconditioned stimulus (US) in a short delay eye blink paradigm. All rabbits received two80-trial training sessions. Intracellular recordings were made from hippocampal CA1 pyramidal neurons within brain slices prepared 24 h following the second training session. All 46 CA1 neurons included in the analysis had stable penetration, at least 70 mV impulse amplitudes and at least 40 MΩ input resistance. Recording and initial data analysis were done ‘blind’ regarding behavioral training performance of the rabbit from which the slices were prepared. The animals were separated into aHigh(86 ± 6%CRs, n = 12) andLow(12 ± 4%CRs, n = 10) Acquisiton group based on the number of blink CRs shown on the second training day(P < 0.001). CA1 pyramidal neurons from the High Acquisition group(n = 20) showed a significant reduction in the afterhypolarization (AHP) response following 4 impulses elicited by intracellular current injection as compared to neurons from the Low Acquisition group(n = 4). The mean maximal AHP amplitudes after 4 spikes were−2.9 ± 0.34mV and−0.4.0 ± 0.31mV, respectively, in the High and Low Acquisition groups(P < 0.01). The size of the AHP examined at 100 ms intervals during the first 1.7 s after the current pulse proved to be reduced in the High group both when evaluated for all points(F = 5.88,df= 1.44, P < 0.02) and for each of the individual time points (at leastP < 0.05). The mean AHP areas were1.6 ± 0.26 and2.6 ± 0.32mV·s, respectively(P < 0.02). These AHP differences were present in the absence of differences in mean resting potential(−69 ± 2.2mV and−70 ± 2.0mV), spike height(87 ± 1.9mV and87 ± 1.6mV), input assistance72 ± 4.8MΩ,and68 ± 3.5MΩ), and current required to elicit 4 action potentials(0.45 ± 0.05nA and0.43 ± 0.04nA), respectively, between the High and Low Acquisition groups. We have previously demonstrated conditioning-specific reductions in the AHP of CA1 pyramidal neurons in hippocampal slices taken from well-trained rabbits11. In the present experiments, all rabbits received the same number of CS-US pairings. But those which learned had CA1 pyramidal cells with significantly reduced AHPs. These data link the AHP reduction observed in vitro to conditioned response acquisition observed behaviorally. They also provide evidence that the AHP is not reduced merely as a non-specific consequences of training trial presentation. This ionic alteration, intrinsic to hippocampus for its expression, indeed intrinsic to the CA1 cells themselves, is stored in CA1 pyramidal cells after a symptotic learning occurs.

Ionic basis of the postsynaptic depolarizing GABA response in hippocampal pyramidal cells.

1. Whole cell voltage-clamp recording with recording pipette solutions of differing ionic composition was used to determine the ionic basis of the depolarizing gamma-aminobutyric acid (GABA) response. In the presence of 4-aminopyridine and excitatory amino acid receptor blockers, giant GABA-mediated postsynaptic currents (GPSCs) were recorded from CA3 pyramidal neurons in hippocampal slices from adult guinea pigs. With the GABAB component blocked, the GPSC was composed of an initial outward current (GABAA component) that peaked at 115 ms followed by a late inward current (GABAD component) that peaked at 400-600 ms. 2. Reduction of the intracellular concentration of potassium ([K+]i)resulted in no significant change in the reversal potential of the GABAD component of the GPSC, indicating that it is not a nonspecific cation current. 3. The HCO3- permeability of the channel mediating the GABAD response was assessed by using recording pipette solutions containing three different concentrations of bicarbonate ([HCO3-], 19, 49, and 102 mM). The reversal potential of the GABAD response shifted in the depolarizing direction as the HCO3- equilibrium potential was shifted in the depolarizing direction, indicating that the channel mediating the GABAD response is permeable to HCO3-. The reversal potential of the GABAD response was more sensitive to changes in recording pipette [HCO3-] than the reversal potential of the GABAA response, indicating that the GABAD response is carried by HCO3- to a greater extent than the GABAA response. 4. The outward current-inward current sequence of the biphasic GPSC was reversed to an inward current-outward current sequence by using a high [Cl-]/low [HCO3-] recording pipette solution (40 mM Cl-/6 mM HCO3-), indicating that the GABAA component is more sensitive to changes in [Cl-]i, and the GABAD component is more sensitive to changes in [HCO3-]i. 5. These data indicate that the GABAD component of the GPSC is predominantly carried by HCO3-. While this result supports the recently propsed chloride accumulation model, the model in its present form cannot explain the inward current-outward current polarity sequence of the GPSC recorded with the high [Cl-]/low [HCO3-] intracellular solution. The data obtained using that solution reveal the need for a more expansive chloride accumulation/ depletion model or for a model utilizing two distinct ionotropic GABA channels with different anion permeability ratios to account for the biphasic nature of the GPSC.

Acute and chronic actions of ethanol on CA1 hippocampal responses to serotonin

The effects of acute or chronic ethanol on serotonin (5-HT)-induced membrane hyperpolarization and inhibition of the slow Ca2+-dependent after hyperpolarization (sAHP) were recorded in rat CA1 pyramidal neurons in hippocampal slices using sharp intracellular electrodes. 5-HT (1–100 μM) caused concentration-dependent hyperpolarization of the membrane that was not altered by simultaneous 30 mM ethanol treatment, but blunted by 10 μM buspirone, a weak 5-HT1A agonist. 5-HT (1–30 μM) also partially inhibited (≈ 40%) the sAHP following a burst of five or more action potentials. Initially ethanol (30 mM) alone did not alter the sAHP, but over a period of 38 min, a slow increase in amplitude (≈ 40%) was observed. 5-HT-mediated inhibition of the sAHP was significantly greater with ethanol present, regardless of the length of exposure. Pyramidal neurons in hippocampal slices prepared from ethanol-dependent animals showed no obvious signs of withdrawal related hyperexcitability and neither concentration-dependent membrane hyperpolarization nor sAHP inhibition caused by 5-HT were significantly changed from responses in controls. These results suggest that hyperpolarizing responses to 5-HT in hippocampal CA1 pyramidal neurons are functionally resistant to acute or chronic ethanol treatment. 5-HT-mediated inhibition of the sAHP is enhanced by ethanol acutely, but does not show an adaptive change as a result of ethanol dependence.

Acute and chronic actions of ethanol on CA1 hippocampal responses to serotonin

The effects of acute or chronic ethanol on serotonin (5-HT)-induced membrane hyperpolarization and inhibition of the slow Ca 2+-dependent after hyperpolarization (sAHP) were recorded in rat CA1 pyramidal neurons in hippocampal slices using sharp intracellular electrodes. 5-HT (1–100 μM) caused concentration-dependent hyperpolarization of the membrane that was not altered by simultaneous 30 mM ethanol treatment, but blunted by 10 μM buspirone, a weak 5-HT 1A agonist. 5-HT (1–30 μM) also partially inhibited (≈ 40%) the sAHP following a burst of five or more action potentials. Initially ethanol (30 mM) alone did not alter the sAHP, but over a period of 38 min, a slow increase in amplitude (≈ 40%) was observed. 5-HT-mediated inhibition of the sAHP was significantly greater with ethanol present, regardless of the length of exposure. Pyramidal neurons in hippocampal slices prepared from ethanol-dependent animals showed no obvious signs of withdrawal related hyperexcitability and neither concentration-dependent membrane hyperpolarization nor sAHP inhibition caused by 5-HT were significantly changed from responses in controls. These results suggest that hyperpolarizing responses to 5-HT in hippocampal CA1 pyramidal neurons are functionally resistant to acute or chronic ethanol treatment. 5-HT-mediated inhibition of the sAHP is enhanced by ethanol acutely, but does not show an adaptive change as a result of ethanol dependence.

Ca2+ release from intracellular stores induced by afferent stimulation of CA3 pyramidal neurons in hippocampal slices.

1. Ca2+ imaging and simultaneous intracellular recording were performed on CA3 pyramidal neurons in hippocampal slice cultures and standard acute slices. Both fura-2 and a dextran conjugate of fura-2 (MW = 10,000) were used in the Ca2+ measurements to control for compartmentalization artifacts. Experiments were performed under conditions giving minimal ligand- and voltagegated Ca2+ influx, with the use of competitive and noncompetitive antagonists of ionotropic glutamate receptors and steady-state depolarization, respectively. 2. Tetanic stimulation of stratum lucidum evoked dendritic Ca2+ transients with rapid onset that were blocked by the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (2-5 microM), but not by the competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-50 microM). Zn(2+)-containing mossy fiber terminals (assessed by Timm's staining) and postsynaptic structures (thorny excrescences) are preserved in s. lucidum of hippocampal slice cultures. 3. A Ca2+ store loading protocol, consisting of brief repolarizations followed by steady depolarization, primed most of the neurons so that a subsequent tetanus gave a Ca2+ increase in the presence of MK-801 that was reported by both fura-2 and the dextran conjugate. The onset of the Ca2+ increase was significantly delayed (by 2-3 s) with respect to the MK-801-sensitive increase, and often had a different spatial pattern within the neuron. Response characteristics were similar in slice cultures and acute slices. 4. The delayed Ca2+ increase showed a steep rundown with subsequent stimuli, but was restored by further priming by the Ca2+ store loading paradigm. Postsynaptic currents evoked by the tetani under these conditions were not correlated with the magnitude of the delayed Ca2+ transients. 5. Delayed Ca2+ increases were observed in 44% of the neurons dialyzed with normal intracellular solution at room temperature. The success rate of observing delayed Ca2+ transients was increased to 86% in neurons maintained at 30 degrees C, and dialyzed with an inhibitor of the inositol-triphosphate-3-kinase. 6. The delayed Ca2+ transients could not be initiated after inhibition of endosomal Ca(2+)-ATPase-mediated uptake by thapsigargin. 7. Both fura-2 and the dextran conjugate reported increases in resting Ca2+ levels after the loading protocols, that were absent after priming in thapsigargin, and decreases in resting Ca2+ levels after successive tetani in MK-801, suggesting that the Ca2+ changes were largely cytosolic. 8. The present results support the hypothesis that these synaptically mediated, delayed Ca2+ transients represent release from intracellular Ca2+ stores that can be loaded and depleted repeatedly, and are evoked by presynaptic release of endogenous neurotransmitter.

Age-dependent reduction of hippocampal LTP in mice lacking N-methyl- d-aspartate receptor ϵ1 subunit

The effects of targeted disruption of the N-methyl- d-aspartate (NMDA) receptor ε1 subunit gene were studied during the postnatal development of ϵ1-disrupted mutant mice. Using the mice at the ages of 2–3, 5–6 and 9–10 weeks, we examined NMDA receptor channel-mediated synaptic currents and long-term potentiation (LTP) in CAI pyramidal neurons of hippocampal slices. NMDA receptor channel currents, expressed as the ratios to non-NMDA receptor channel currents, decreased with the age in both wild-type and mutant mice, but the values in the mutant mice were approximately half of those of the wild-type mice at all ages examined. The LTP in the mutant mice was also reduced, but in contrast to the NMDA receptor channel currents, the extent of the reduction in the LTP was age-dependent. The reduction was marginal at the age of 2–3 weeks, and became progressively prominent to adulthood, with the potentiation being 26 % of that of the wild-type mice at 9–10 weeks.

G protein-coupled receptors mediate a fast excitatory postsynaptic current in CA3 pyramidal neurons in hippocampal slices

Synaptic activation in the presence of competitive (D,L-APV,CNQX) and noncompetitive (MK-801,GYKI-52466) ionotropic glutamate receptor antagonists induced fast (10-90% rise time of 15-30 msec) postsynaptic responses in CA3 pyramidal neurons from acute and cultured hippocampal slices. Postsynaptic currents were studied extensively in slice cultures, and displayed a linear current-voltage relationship, with a reversal potential between 0 mV and +10 mV, suggesting the activation of a nonselective cationic conductance. Inhibition of the GTPase cycle by intracellular perfusion with the nonhydrolyzable analog of GDP, GDP beta S, blocked the fast postsynaptic responses evoked in ionotropic antagonists, as well as baclofen-mediated outward K+ currents, known to be mediated by G protein-coupled GABAB receptors. Intracellular perfusion with GDP beta S did not affect the AMPA/kainate component of the synaptic currents. Irreversible activation of G proteins by intracellular perfusion with the nonhydrolyzable analog of GTP, GMP-PNP, occluded the baclofen responses, and evoked an inward current, consistent with the synaptically mediated conductance. Incubation of the slice cultures in pertussis toxin for 72 hr blocked baclofen-induced outward K+ currents, while the fast postsynaptic currents remained. The metabotropic glutamate receptor (mGluR) agonists 1S,3R-ACPD and 1S,3S-ACPD induced an inward current in the presence of the ionotropic antagonists, and occluded the fast EPSCs. The fast EPSCs were partially blocked by the mGluR antagonists L-AP3 and (+)MCPG, but there was differential antagonists sensitivity in two pathways stimulated (CA3 stratum radiatum vs CA3 stratum oriens). These data suggest that fast postsynaptic responses evoked in the presence of ionotropic glutamate receptor antagonists are mediated by G protein-coupled mGluRs linked to nonselective cationic channels.

Kinetics and Mg2+ block of N-methyl-D-aspartate receptor channels during postnatal development of hippocampal CA3 pyramidal neurons.

Voltage-dependent magnesium block of N-methyl-D-aspartate-activated channels, and the N-methyl-D-aspartate component of excitatory synaptic currents were studied in CA3 pyramidal neurons of hippocampal slices from immature (postnatal day 3-8) and adult (postnatal day 25-60) rats. In all neurons studied the kinetics of single-channel openings in cell-attached and inside-out configurations was strongly modulated by extracellular Mg2+, in a voltage-dependent manner. No age-dependent difference in the Mg2+ sensitivity of N-methyl-D-aspartate channels was observed. At physiological concentrations of external Mg2+ (1.3 mM), N-methyl-D-aspartate components of excitatory synaptic currents measured from immature and adult rats displayed a similar voltage-dependence. In immature neurons (postnatal day 3-6), the N-methyl-D-aspartate component of excitatory postsynaptic currents decay time-course was a single-exponential with an average time-constant of about 300 ms. In neurons from older animals the decay was described by a double-exponential function with both a fast component (tau f, 54-130 ms) and a slow component (tau s, 275-400 ms). With age, the contribution of the fast component increased and the decay time-course of the N-methyl-D-aspartate component of excitatory postsynaptic currents accelerated. It is concluded that the Mg2+ block of N-methyl-D-aspartate channels in CA3 pyramidal neurons does not change with development, but the kinetic properties of N-methyl-D-aspartate receptor channels are developmentally regulated.

Synaptic activation of voltage-gated channels in the dendrites of hippocampal pyramidal neurons.

Activation of dendritic voltage-gated ion channels by local synaptic input was tested by simultaneous dendrite-attached patch-clamp recordings and whole-cell somatic voltage recordings made from CA1 pyramidal neurons in hippocampal slices. Schaffer collateral stimulation elicited subthreshold excitatory postsynaptic potentials (EPSPs) that opened voltage-gated sodium and calcium channels in the apical dendrites. The EPSP-activated sodium channels opened near the peak of the EPSP, whereas low voltage-activated calcium channels opened near the EPSP peak and during the decay phase. Dendritic high voltage-activated channels required somatic action potential generation or suprathreshold synaptic trains for activation. Dendritic voltage-gated channels are, therefore, likely to participate in dendritic integration of synaptic events.