Proteomic identification of a novel protein regulated in CA1 and CA3 hippocampal regions during intermittent hypoxia

The CA1 and CA3 regions of the hippocampus markedly differ in their susceptibility to hypoxia in general, and more particularly to the intermittent hypoxia (IH) that characterizes sleep apnea. We used proteomic analysis to build a database of proteins expressed in normoxic CA1 and CA3. The current hippocampus protein database identifies 106 proteins. A hypothetical protein with accession number AK006737 (gimid R:12839969) was strongly upregulated in the CA1, but not CA3 hippocampal region. Bioinformatic analysis revealed that the unknown protein contained a high stringency protein kinase e binding site. Domain analysis demonstrated the presence of a conserved sequence indicative of macrophage scavenger receptors. Using proteomic analysis we have previously demonstrated that acute (6 h) IH-mediated CA1 injury results from complex interactions between pathways involving increased metabolism, induction of stress-induced proteins and apoptosis, and ultimately disruption of structural proteins and cell integrity. The current findings identify a hypothetical protein that may play a key role in the response of CA1 to IH. These findings provide initial insights into mechanisms underlying differences in susceptibility to hypoxia in neural tissue and demonstrate how proteomic analysis can be used to generate new hypotheses, which define neuronal adaptation to IH.     

Different susceptibilities of long-term potentiations in CA3 and CA1 regions of guinea pig hippocampal slices to nootropic drugs

Effects of the nootropic drugs, piracetam and bifemelane, on long-term potentiation (LTP) of population spikes in the CA3 and CA1 regions of guinea pig hippocampal slices were investigated. Piracetam (10(-6) to 10(-4) M) and bifemelane (10(-8) to 10(-6) M) significantly augmented the LTP in the CA3 region. The effects of these drugs were inhibited by scopolamine (10(-6) M). However, the LTP in the CA1 region was not affected by piracetam and bifemelane even at highly effective concentrations (10(-5) and 10(-6) M, respectively). Thus, the LTP in the CA3 is more susceptible to nootropic drugs than in the CA1.     

Effects of desflurane and propofol on electrophysiological parameters during and recovery after hypoxia in rat hippocampal slice CA1 pyramidal cells

Cerebral ischemia is a major cause of death and disability and may be a complication of neurosurgery. Certain anesthetics may improve recovery after ischemia and hypoxia by altering electrophysiological changes during the insult. Intracellular recordings were made from CA1 pyramidal cells in hippocampal slices from adult rats. Desflurane or propofol was applied 10 min before and during 10 min of hypoxia (95% nitrogen, 5% carbon dioxide). None of the untreated CA1 pyramidal neurons, 46% of the 6% desflurane- and 38% of the 12% desflurane-treated neurons recovered their resting and action potentials 1 h after hypoxia (P<0.05). Desflurane (6% or 12%) enhanced the hypoxic hyperpolarization (4.9 or 4.7 vs. 2.6 mV), increased the time until the rapid depolarization (441 or 390 vs. 217 s) and reduced the level of depolarization at 10 min of hypoxia (−13.5 or −13.0 vs. −0.6 mV); these changes may be part of the mechanism of its protective effect. Either chelerythrine (5 μM), a protein kinase C inhibitor, or glybenclamide (5 μM), a K_ATP channel blocker, prevented the protective effect and the electrophysiological changes with 6% desflurane. Propofol (33 or 120 μM) did not improve recovery (0 or 0% vs. 0%) 1 h after 10 min of hypoxia; it did not significantly enhance the hypoxic hyperpolarization (3.6 or 3.1 vs. 2.6 mV) or increase the latency of the rapid depolarization (282 or 257 vs. 217 s). The average depolarization at 10 m of hypoxia with 33 μM propofol (−4.1 mV) was slightly but significantly different from that in untreated hypoxic tissue (−0.6 mV). Desflurane but not propofol improved recovery of the resting and action potentials in hippocampal slices after hypoxia, this improvement correlated with enhanced hyperpolarization and attenuated depolarization of the membrane potential during hypoxia. Our results demonstrate differential effects of anesthetics on electrophysiological changes during hypoxia.     

Glycine potentiates NMDA responses in rat hippocampal CA1 neurons

When superfused onto rat hippocampal slices, glycine (0.1-0.5 mM) potentiated the depolarization induced by pressure application of NMDA in normal Krebs solution and the synaptic discharge evoked by stimulation of the Schaffer collateral-commissural inputs to the CA1 pyramidal neurons bathed in Mg2+-free media; the effects were not prevented by strychnine. In addition, glycine partially reversed the blocking effect of D-2-amino-5-phosphonovalerate (AP5) on N-methyl-D-aspartate (NMDA)-induced depolarization. These results show that glycine at relatively high concentrations potentiates the NMDA-mediated response in hippocampal slices.     

Properties of the visually driven neurons of cat's hippocampal regions CA 1 and CA 3

Neurons in areas CA 1 and 3 of cat's dorsal hippocampus were studied. Fifteen percent of the investigated cells were influenced by visual stimuli. Eighty five such neurons were investigated. The organization of their receptive fields was tested with stationary and moving visual stimuli. Twenty eight percent of neurons had small receptive fields (10-20 deg square). Forty one neurons responded to stationary flashing spots. They were ON-OFF, ON and OFF types with phasic (66%) and tonic (34%) characteristics. Seventy five responded to dark and bright stimuli moving across their receptive fields. Twenty five neurons were direction-sensitive and 21 responded better to the dark moving stimuli than to the bright ones. No significant differences in the response properties of neurons in the CA 1 and CA 3 fields were observed.     

The differential effects of volatile anesthetics on electrophysiological and biochemical changes during and recovery after hypoxia in rat hippocampal slice CA1 pyramidal cells

Two volatile agents, isoflurane and sevoflurane have similar anesthetic properties but different potencies; this allows the discrimination between anesthetic potency and other properties on the protective mechanisms of volatile anesthesia. Two times the minimal alveolar concentration of an anesthetic is approximately the maximally used clinical concentration of that agent; this concentration is 2% for isoflurane and 4% for sevoflurane. We measured the effects of isoflurane and sevoflurane on cornus ammonis 1 (CA1) pyramidal cells in rat hippocampal slices subjected to 10 min of hypoxia (95% nitrogen 5% carbon dioxide) and 60 min of recovery. Anesthetic was delivered to the gas phase using a calibrated vaporizer for each agent. At equipotent anesthetic concentrations, sevoflurane (4%) but not isoflurane (2%), enhanced the initial hyperpolarization (6.7 vs. 3.4 mV), delayed the hypoxic rapid depolarization (521 vs. 294 s) and reduced peak hypoxic cytosolic calcium concentration (203 vs. 278 nM). While both agents reduced the final membrane potential at 10 min of hypoxia compared with controls, 4% sevoflurane had a significantly greater effect than 2% isoflurane (-24.4 vs. -3.5 mV). The effect of these concentrations of isoflurane and sevoflurane was not different for sodium, potassium or ATP concentrations at 10 min of hypoxia, the only difference at 5 min of hypoxia was that ATP was better maintained with 4% sevoflurane (2.2 vs. 1.3 nmol/mg). If the same absolute concentration (4%) of isoflurane and sevoflurane is compared then the cellular changes during hypoxia are similar for both agents and they both improve recovery. We conclude that an anesthetic's absolute concentration and not its anesthetic potency correlates with improved recovery of CA1 pyramidal neurons. The mechanisms of sevoflurane-induced protection include delaying and attenuating the depolarization and the increase of cytosolic calcium and delaying the fall in ATP during hypoxia.     

Redox modulation of synaptic responses and plasticity in rat CA1 hippocampal neurons

Effects of redox reagents on excitatory and inhibitory synaptic responses as well as on the bidirectional plasticity of α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) andN-methyl-d-aspartate (NMDA) receptor-mediated synaptic responses were studied in CA1 pyramidal neurons in rat hippocampal slices. The oxidizing agent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, 200 μM) did not affect AMPA, GABA_A or GABA_B receptor-mediated synaptic responses or the activation of presynaptic metabotropic receptors. However, DTNB irreversibly decreased (by approximately 50%) currents evoked by focal application of NMDA. DTNB also decreased the NMDA component of the EPSC. The reversal potential of NMDA currents and the Mg²⁺ block were not modified. In the presence of physiological concentrations of Mg²⁺ (1.3 mM), DTNB did not affect the NMDA receptor-dependent induction of long-term potentiation (LTP) or long-term depression (LTD) expressed by AMPA receptors. In contrast, DTNB fully prevented LTP and LTD induced and expressed by NMDA receptors. Plasticity of NMDA receptor-mediated synaptic responses could be reinstated by the reducing agenttris-(2-carboxyethyl) phosphine (TCEP, 200 μM). These results suggest that persistent, bidirectional changes in synaptic currents mediated by NMDA receptors cannot be evoked when these receptors are in an oxidized state, whereas NMDA-dependent LTP and LTD are still expressed by AMPA receptors. Our observations raise the possibility of developing therapeutic agents that would prevent persistent excitotoxic enhancement of NMDA receptor-mediated events without blocking long-term modifications of AMPA receptor-mediated synaptic responses, thought to underlie memory processes.     

Effects of a lidocaine derivative QX-572 on CA1 neuronal responses to electrical and chemical stimuli in a hippocampal slice

Activity of CA1 neurons was recorded in rat hippocampal slices with a lidocaine-derived QX-572-filled micropipette. The QX compound abolished Na action potentials as reported earlier. In addition it reduced markedly the burst slow afterhyperpolarization seen in these neurons in response to a depolarizing current pulse. It also modified responses to neurotransmitter substances associated with changes in K currents, acetylcholine and serotonin. QX-filled micropipettes can therefore provide some insight into mechanisms of action of certain neurotransmitters in the brain but they cannot be used as selective blockers of Na currents.     

Subcellular muscarinic enhancement of excitability and Ca2+-signals in CA1-dendrites in rat hippocampal slice

The cholinergic system is involved in Ca2+-dependent models of learning. To study subcellular modulation, we evoked 50-100 microm long dendritic Ca2+-responses by focal pressure application of glutamate. These Ca2+-responses were augmented by +70% by focally applied carbachol. This atropine-sensitive augmentation started within 1 s concurrent to an augmentation of the glutamate-evoked somatic depolarization and firing. Tetrodotoxin reduced the Ca2+-response to glutamate by 60-80% while, after having restored the Ca2+-signal by increasing the application of glutamate, its muscarinic augmentation was reduced from +73 to +30%. Lithium (2 mM, >2 h) slowed and reduced augmentation of Ca2+-signals and blocked augmentation of the glutamate-evoked depolarization and firing, but not suppression of the slow after-hyperpolarization following repetitive discharge. Thus, several mechanisms contribute to muscarinic augmentation of Ca2+-signals.     

Rapid and slow swelling during hypoxia in the CA1 region of rat hippocampal slices.

The role of swelling in hypoxic/ischemic neuronal injury is incompletely understood. We investigated the extent and time course of cell swelling during hypoxia, and recovery of cell volume during reoxygenation, in the CA1 region of rat hippocampal slices in vitro. Cell swelling was measured optically and compared with simultaneous measurements of the extracellular DC potential, extracellular [K+], and synaptic transmission in the presence and absence of hypoxic depolarization. Hypoxia-induced swelling consisted of rapid and/or slow components. Rapid swelling was observed frequently and always occurred simultaneously with hypoxic depolarization. Additionally, rapid swelling was followed by a prolonged phase of swelling that was approximately 15 times slower. Less frequently, slow swelling occurred independently, without either hypoxic depolarization or a preceding rapid swelling. For slices initially swelling rapidly, recovery of both cell volume and the slope of field excitatory postsynaptic potentials were best correlated with the duration of hypoxia (r = 0.77 and 0.87, respectively). This was also the case for slices initially swelling slowly (r = 0.70 and 0.58, respectively). In contrast, the degree of recovery of cell volume was the same at 30 or 60 min of reoxygenation, indicating that prolonging the duration of reoxygenation within these limits was ineffective in improving recovery. Spectral measurements indicated that the hypoxia-induced changes in light transmittance were related to changes in cell volume and not changes in the oxidation state of mitochondrial cytochromes. The persistent impairment of synaptic transmission in slices swelling slowly (i.e., without hypoxic depolarization) indicates that swelling may play a role in this injury and that hypoxic depolarization is not required. Additionally, the correlation between the degree of recovery of cell volume and the degree of recovery of synaptic transmission during reoxygenation supports a role for swelling in hypoxic neuronal injury.     

Somatostatin acts in CA1 and CA3 to reduce hippocampal epileptiform activity

Although the peptide somatostatin (SST) has been speculated to function in temporal lobe epilepsy, its exact role is unclear, as in vivo studies have suggested both pro- and anticonvulsant properties. We have shown previously that SST has multiple inhibitory cellular actions in the CA1 region of the hippocampus, suggesting that in this region SST should have antiepileptic actions. To directly assess the effect of SST on epileptiform activity, we studied two in vitro models of epilepsy in the rat hippocampal slice preparation using extracellular and intracellular recording techniques. In one, GABA-mediated neurotransmission was inhibited by superfusion of the GABAA receptor antagonist bicuculline. In the second, we superfused Mg2+-free artificial cerebrospinal fluid to remove the Mg2+ block of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We show here that SST markedly reduces the intensity of evoked epileptiform afterdischarges and the frequency of spontaneous bursts in both CA1 and CA3. SST appears to act additively in the two regions to suppress the transmission of epileptiform events through the hippocampus. We further examined SST's actions in CA3 and found that SST dramatically reduced the frequency of paroxysmal depolarizing shifts (PDSs) recorded intracellularly in current clamp, as well as increasing the threshold for evoking "giant" excitatory postsynaptic currents (EPSCs), large polysynaptically mediated EPSCs that are the voltage-clamp correlate of PDSs. We also examined the actions of SST on pharmacologically isolated EPSCs generated at both mossy fiber (MF) and associational/commissural (A/C) synapses. SST appears to act specifically to reduce recurrent excitation between CA3 neurons because it depresses A/C- but not MF-evoked EPSCs. SST also increased paired-pulse facilitation of A/C EPSCs, suggesting a presynaptic site of action. Reciprocal activation of CA3 neurons through A/C fibers is critical for generation of epileptiform activity in hippocampus. Thus SST reduces feedforward excitation in rat hippocampus, acting to "brake" hyperexcitation. This is a function unique from that described for other hippocampal neuropeptides, which affect more standard neurotransmission. Our results suggest that SST receptors could be a unique, selective clinical target for treatment of limbic seizures.     

Cumulative effects of glutamate microstimulation on Ca(2+) responses of CA1 hippocampal pyramidal neurons in slice

Glutamate stimulation of hippocampal CA1 neurons in slice was delivered via iontophoresis from a microelectrode. Five pulses (approximately 5 muA, 10 s duration, repeated at 1 min intervals) were applied with the electrode tip positioned in the stratum radiatum near the dendrites of a neuron filled with the Ca(2+) indicator fura-2. A single stimulus set produced Ca(2+) elevations that ranged from several hundred nM to several microM and that, in all but a few neurons, recovered within 1 min of stimulus termination. Subsequent identical stimulation produced Ca(2+) elevations that outlasted the local glutamate elevations by several minutes as judged by response recoveries in neighboring cells or in other parts of the same neuron. These long responses ultimately recovered but persisted for up to 10 min and were most prominent in the mid and distal dendrites. Recovery was not observed for responses that spread to the soma. The elevated Ca(2+) levels were accompanied by membrane depolarization but did not appear to depend on the depolarization. High-resolution images demonstrated responsive areas that involved only a few mu(m) of dendrite. Our results confirm the previous general findings from isolated and cell culture neurons that glutamate stimulation, if carried beyond a certain range, results in long-lasting Ca(2+) elevation. The response characterized here in mature in situ neurons was significantly different in terms of time course and reversibility. We suggest that the extended Ca(2+) elevations might serve not only as a trigger for delayed neuron death but, where more spatially restricted, as a signal for local remodeling in dendrites.     

Hyperpolarizing responses to application of glutamate in hippocampal CA1 pyramidal neurons

Micropressure injection of glutamate onto the apical dendrites of hippocampal CA1 pyramidal cells usually produces a fast rising, brief depolarization. However, hyperpolarizing responses with longer durations (300-500 ms) can be produced over a range of drug electrode locations. These hyperpolarizations can be reversed with intracellular injection of hyperpolarizing current. Localized application of glutamate in the stratum radiatum produces a depolarizing response in intracellularly recorded CA1 interneurons. Previous studies have shown that the dendrites of GABA-ergic basket cell interneurons extend into the stratum radiatum and are involved in mediating feedforward inhibition of pyramidal neurons. The glutamate-induced hyperpolarizations observed in pyramidal neurons are probably due to direct excitation of dendrites of interneurons, which in turn produce a synaptic inhibition in pyramidal cells.     

Inactivation of a slow Ca2+ current in CA1 neurones of the adult rat hippocampal slice

CA1 neurones of the adult rat hippocampal slice preparation were voltage clamped at or near -40 mV membrane potential using a single electrode clamp method. Depolarizing voltage commands from a holding potential of -40 mV elicited voltage-dependent inward Ca2+ currents comprising a fast and a slow component. The latter one was investigated for its susceptibility to inactivation, which was maximally expressed at around 0 mV membrane potential. When extracellular Ca2+ was replaced by Ba2+, inward currents became much larger and were followed by long tail currents. Similar data were observed in neurones injected with the Ca2+ chelator BAPTA. It is suggested that inactivation of the slow Ca2+ current depends at least partly on the levels of intracellular free Ca2+ in hippocampal neurones.     

Behavior-dependent paired-pulse responses in the hippocampal CA1 region

Summary Paired-pulses of 20–100 ms interpulse interval (IPI) were delivered to the Schaffer collaterals/commissural fibers in order to excite the apical dendrites of the hippocampal CA1 region in freely behaving rats. Significant differences were observed for the paired-pulse responses during different behavioral states. The responses recorded during awake immobility (IMM), and slow-wave sleep (SWS) were similar, but as a group, were different from those during walking (WLK) and rapid-eye-movement sleep (REM). During WLK and REM, the population spike evoked by the second pulse (P2) at IPI of 30 and 50 ms, was greatly facilitated as compared to the population spike evoked by the first pulse (P1), i.e. P2 > P1. During IMM and SWS and using moderate stimulus intensities, P2 was generally smaller than P1 (paired-pulse suppression) at IPI of 30 and 50 ms. The P2/P1 relation with behavior was not caused by the slight variations of P1 with behavior. In addition, paired-pulse facilitation of the population excitatory postsynaptic potentials (EPSP) was relatively small and not significantly dependent on behavior. Behavioral dependence of the paired-pulse responses was not generally found for IPI of 20 or 100 ms. It is concluded that paired-pulse facilitation at 30–50 ms IPI can best be explained by EPSP facilitation combined with a behaviorally dependent disinhibition.