Mitochondrial and intrinsic optical signals imaged during hypoxia and spreading depression in rat hippocampal slices

During hypoxia in the CA1 region of the rat hippocampus, spreading-depression-like depolarization (hypoxic spreading depression or HSD) is accompanied by both a negative shift of the extracellular DC potential (DeltaV(o)), and a sharp decrease in light transmittance (intrinsic optical signal or IOS). To investigate alterations in mitochondrial function during HSD and normoxic spreading depression (SD), we simultaneously imaged mitochondrial depolarization, using rhodamine-123 (R123) fluorescence, and IOS while monitoring extracellular voltage. Three major phases of the R123 signal were observed during hypoxia: a gradual, diffuse fluorescence increase, a sharp increase in fluorescence coincident with the HSD-related DeltaV(o), primarily in the CA1 region, and a plateau-like phase if reoxygenation is delayed after HSD onset, persisting until reoxygenation occurs. Two phases occurred following re-oxygenation: an abrupt and then slow decrease in fluorescence to near baseline and a slow secondary increase to slightly above baseline and a late recovery. Parallel phases of the IOS response during hypoxia were also observed though delayed compared with the R123 responses: an initial increase, a large decrease coincident with the HSD-related DeltaV(o), and a trough following HSD. After reoxygenation, there occurred a delayed increase in transmittance and then a slow decrease, returning to near baseline. When Ca(2+) was removed from the external medium, resulting in complete synaptic blockade, the mitochondrial response to hypoxia did not significantly differ from control (normal Ca(2+)) conditions. In slices maintained in low-chloride (2.4 mM) medium, a dramatic reversal in the direction of the IOS signal associated with HSD occurred, and the R123 signal during HSD was severely attenuated. Normoxic SD induced by micro-injection of KCl was also associated with a decrease in light transmittance and a sharp increase in R123 fluorescence but both responses were less pronounced than during HSD. Our results show two mitochondrial responses to hypoxia: an initial depolarization that appears to be caused by depressed electron transport due to lack of oxygen and a later, sudden, sharp depolarization linked to HSD. The depression of the second, sharp depolarization and the inversion of the IOS in low-chloride media suggest a role of Cl(-)-dependent mitochondrial swelling. Lack of effect of Ca(2+)-free medium on the R123 and IOS responses suggests that the protection against hypoxic damage by low Ca(2+) is not due to the prevention of mitochondrial depolarization.     

Intrinsic optical signals in rat hippocampal slices during hypoxia-induced spreading depression-like depolarization.

In interfaced rat hippocampal slices spreading depression (SD) and hypoxia-induced SD-like depolarization are associated with increased light reflectance and decreased light transmittance, indicating increased light scattering. By contrast, mild hypotonicity or electrical stimulation decrease light scattering, which is usually taken to be caused by cell swelling. This difference has been attributed to experimental conditions, but in our laboratory moderate osmotic challenge and SD produced opposite intrinsic optical signals (IOSs) in the same slice under identical conditions. To decide whether the SD-induced IOS is related to cell swelling, we investigated the effects of Cl(-) transport inhibitors and Cl(-) withdrawal on both light reflectance and transmittance, as well as on changes in interstitial volume and tissue electrical resistance. In normal [Cl(-)](o), early during hypoxia, there was a slight decrease in light reflectance paired with increase in transmittance. At the onset of hypoxic SD, coincident with the onset of cell swelling (restriction of TMA(+) space), the IOS signals suddenly inverted, indicating sharply increased scattering. The SD-related IOSs started in a single spot and spread out over the entire CA1 region without invading CA3. Application of 2 mM furosemide decreased IOS intensity. When [Cl(-)](o) was substituted by methylsulfate or gluconate, the SD-related reflectance increase and transmittance decrease were suppressed and replaced by opposite signals, indicating scattering decrease. Yet Cl(-) withdrawal did not prevent cell swelling measured as shrinkage of TMA(+) space. The SD-related increase of tissue electrical resistance was reduced when bath Cl(-) was replaced by methylsulfate and almost eliminated when replaced by gluconate. The TMA(+) signal is judged to be a more reliable indicator of interstitial space than tissue resistance. Neither application of cyclosporin A nor raising [Mg(2+)](o) depressed the SD-related reflectance increase, suggesting that Cl(-) flux through mitochondrial "megachannels" may not be a major factor in its generation. Fluoroacetate poisoning of glial cells (5 mM) accelerated SD onset and enhanced the SD-induced reflectance increase threefold. This suggests, first, that glial cells normally moderate the SD process and, second, that neurons are the predominant generators of the light-scattering increase. We conclude that light scattering by cerebral tissue can be changed by at least two different physical processes. Cell swelling decreases light scattering, whereas a second process increases scattering. During hypoxic SD the scattering increase masks the swelling-induced scattering decrease, but the latter is revealed when Cl(-) is removed. The scattering increase is Cl(-) dependent, nevertheless it is apparently not related to cell volume changes. Its underlying mechanism is as yet not clear; possible factors are discussed.     

Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation

Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation were recorded using darkfield infrared-videomicroscopy. Response amplitude was linearly related to stimulation intensity. The intensity of the optical signal reached its maximum 3 s after onset of stimulation and redecayed with a mean time constant of 23 ± 7.1 s. The optical signal had a columnar shape. The size of the column was independent from stimulation intensity with stimuli of medium amplitudes. The extent of the optical signal corresponded to the extent of the electrical activation. Changes in intrinsic optical properties may be a useful tool for the study of spread of excitation in neuronal tissue in vitro.     

alpha-Chloralose diminishes gamma oscillations in rat hippocampal slices

alpha-Chloralose is an anesthetic characterized by its ability to maintain animals in physiological conditions though immobilized and anesthetized. In addition, alpha-chloralose induces a loss of consciousness with little influence on either pain response or cardiovascular reflexes. The pharmacological mechanisms of alpha-chloralose's actions are poorly understood. In vitro experiments have demonstrated alpha-chloralose enhances GABA(A) receptor function, which may underlie its anesthetic effect. However, how alpha-chloralose affects hippocampal synaptic function and neuronal network synchronization is unknown. In the present study, we performed electrophysiological recordings to examine the effects of alpha-chloralose on synaptic transmission, tetanic stimulation-induced gamma oscillations (30-80 Hz) and neuronal receptor function in rat hippocampal slices and dissociated hippocampal CA1 pyramidal neurons. The results demonstrated that alpha-chloralose (30-100 microM) diminished tetanic stimulation-induced gamma oscillations without affecting single stimulation-induced field potential responses. In single, dissociated hippocampal CA1 pyramidal neurons, alpha-chloralose activated GABA(A) receptors at a high concentration while it potentiated GABA(A) receptor-mediated currents at low concentrations. However, alpha-chloralose did not affect glutamate-, glycine-, or ACh-induced currents. Slice-patch recordings revealed alpha-chloralose enhanced GABAergic leak current and prolonged the decay constant of spontaneous inhibitory postsynaptic currents (sIPSCs). It is concluded that alpha-chloralose suppresses hippocampal gamma oscillations without significantly affecting basic synaptic transmission or ionotropic glutamate, choline and glycine receptor function. Enhancement of GABAergic leak current and prolongation of GABAergic sIPSCs by alpha-chloralose likely underlie its disruption of neuronal network synchronization in the hippocampus.     

Pyroglutamyl-Asparagine amide normalizes long-term potentiation in rat hippocampal slices

Preapplication of peptide piracetam analogue pyroglutamyl-asparagine amide to rat hippocampal slices facilitates long-term potentiation of focal responses in the CA1 field after weak tetanization of the synaptic input (30 pulses, 100 Hz). This treatment normalized the development of long-term potentiation after standard tetanization (100 pulses, 100 Hz) impaired by ethanol. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 8, pp. 176–179, August, 2004 This work was supported by the Russian Foundation for Basic Research (grant No. 01-04-48304).     

Stereoselectivity of L-baclofen in hippocampal slices of the rat

Extra- and intracellular recording from hippocampal slices of the rat revealed the following effects when baclofen (BF) (0.1-10 microM) was added to the perfusion fluid: a block of synaptic potentials evoked by stimulation of stratum radiatum; a direct hyperpolarization and a conductance increase (for potassium ions) of CA1 pyramidal cells. All this activity was found in the L- none in the D-enantiomer. D-BF did not antagonize the action of L-BF.     

Sleep deprivation impairs long-term potentiation in rat hippocampal slices

To determine if 12-h sleep deprivation disrupts neural plasticity, we compared long-term potentiation (LTP) in five sleep-deprived and five control rats. Thirty minutes after tetanus population spike amplitude increased 101 +/- 15% in 16 slices from sleep deprived rats and 139 +/- 14% in 14 slices from control rats. This significant (P < 0.05) reduction of LTP, the first demonstration that the sleep deprivation protocol impairs plasticity in adult rats, may be due to several factors. Reduced LTP may indicate that sleep provides a period of recuperation for cellular processes underlying neural plasticity. Alternatively, the stress of sleep deprivation, as indicated by elevated blood corticosterone levels, or other non-sleep-specific factors of deprivation may contribute to the LTP reduction.     

Phenylethylidenehydrazine, a novel GABA-transaminase inhibitor, reduces epileptiform activity in rat hippocampal slices

Phenylethylidenehydrazine (PEH), an analog of the monoamine oxidase inhibitor, beta-phenylethylhydrazine (phenelzine), inhibits the gamma-aminobutyric acid (GABA) catabolic enzyme GABA-transaminase and increases brain levels of GABA. GABA is the predominant fast inhibitory transmitter counteracting glutamatergic excitation, and increased neural GABA could influence a wide range of synaptic and circuit properties under both physiologic and pathophysiologic conditions. To examine the scope of these effects, we applied PEH (or vehicle) to rat hippocampal slices and measured basal glutamatergic transmission, synaptic plasticity, and epileptiform activity using extracellular field and whole cell patch clamp recordings. In vitro pre-treatment with PEH (100 microM) increased the GABA content of hippocampal slices by approximately 60% over vehicle-treated controls, but it had no effect on basal field excitatory postsynaptic potentials, tonic GABA currents, paired-pulse facilitation, or long-term potentiation. In contrast, pre-incubation with PEH caused a dose- and time-dependent reduction in epileptiform burst frequency induced by superfusion with Mg2+-free or high-K+ artificial cerebrospinal fluid. Thus, the inhibitory effects of PEH are state-dependent: hyper-excitation during epileptiform bursting was reduced, whereas synaptic transmission and plasticity were unaffected.     

Size does matter: generation of intrinsic network rhythms in thick mouse hippocampal slices

Rodent hippocampal slices of < or = 0.5 mm thickness have been widely used as a convenient in vitro model since the 1970s. However, spontaneous population rhythmic activities do not consistently occur in this preparation due to limited network connectivity. To overcome this limitation, we develop a novel slice preparation of 1 mm thickness from adult mouse hippocampus by separating dentate gyrus from CA3/CA1 areas but preserving dentate-CA3-CA1 connectivity. While superfused in vitro at 32 or 37 degrees C, the thick slice exhibits robust spontaneous network rhythms of 1-4 Hz that originate from the CA3 area. Via assessing tissue O2, K+, pH, synaptic, and single-cell activities of superfused thick slices, we verify that these spontaneous rhythms are not a consequence of hypoxia and nonspecific experimental artifacts. We suggest that the thick slice contains a unitary circuitry sufficient to generate intrinsic hippocampal network rhythms and this preparation is suitable for exploring the fundamental properties and plasticity of a functionally defined hippocampal "lamella" in vitro.     

Real-time multisite observation of glutamate release in rat hippocampal slices

A multichannel glutamate sensor was fabricated that consists of enzyme modified electrodes and has a high sensitivity and selectivity to glutamate. We placed a rat hippocampal slice on the sensor and monitored the current at four electrodes resulting from the stimulation with muscimol, a gamma-aminobutyric acid(A) (GABA(A)) receptor agonist. We obtained different glutamate concentration increases at the different positions, suppressed by bicuculline, a GABA(A) receptor antagonist. This demonstrated that the sensor can monitor the glutamate released via GABA(A) receptors pathways, and the difference in the concentrations may indicate differences in the distribution of GABA(A) receptor as well as diverse receptor functions. This multichannel sensor may be useful for non-invasive, real-time monitoring of glutamate distribution, which would make it a valuable tool for pharmacological analysis.     

Glycolysis prevents anoxia-induced synaptic transmission damage in rat hippocampal slices

Prolonged anoxia can cause permanent damage to synaptic transmission in the mammalian CNS. We tested the hypothesis that lack of glucose is the major cause of irreversible anoxic transmission damage, and that anoxic synaptic transmission damage could be prevented by glycolysis in rat hippocampal slices. The evoked population spike (PS) was extracellularly recorded in the CA1 pyramidal cell layer after stimulation of the Schaffer collaterals. When the slice was superfused with artificial cerebrospinal fluid (ACSF) containing 4 mM glucose, following 10 min anoxia, the evoked PS did not recover at all after 60 min reoxygenation. When superfusion ACSF contained 10 mM glucose with or without 0.5 mM alpha-cyano-4-hydroxycinnate (4-CIN), after 60 min reoxygenation the evoked PS completely recovered following 10 min anoxia. When superfusion ACSF contained 20 mM glucose with or without 1 mM sodium cyanide (NaCN), after 60 min reoxygenation the evoked PS completely recovered even following 120 min anoxia. In contrast, when superfusion ACSF contained 4 mM glucose, following 10 min 1 mM NaCN chemical anoxia alone, without anoxic anoxia, the evoked PS displayed no recovery after 60 min reoxygenation. Moreover, when 16 mM mannitol and 16 sodium L-lactate were added into 4 mM glucose ACSF, following 10 min anoxia the evoked PS failed to recover at all after 60 min reoxygenation. The results indicate that elevated glucose concentration powerfully protected the synaptic transmission against anoxic damage, and the powerful protection is due to anaerobic metabolism of glucose and not a result of the higher osmolality in higher glucose ACSF. We conclude that lack of glucose is the major cause of anoxia-induced synaptic transmission damage, and that if sufficient glucose is supplied, glycolysis could prevent this damage in vitro.     

The properties of carbachol-induced beta oscillation in rat hippocampal slices

The rhythmical and pharmacological properties of carbachol-induced beta oscillation were studied using rat hippocampal slices. With the application of 30 microM carbachol, beta-range oscillations with frequencies of 13-20 Hz were recorded from the CA3 region. An AMPA receptor antagonist, CNQX, diminished the oscillations. An NMDA receptor antagonist, APV, significantly suppressed the pre-established beta oscillations. The pre-application of APV blocked the start of the carbachol-induced beta oscillations. When bicuculline (BIC), a GABAA receptor antagonist, was applied to the pre-established beta oscillations, the frequency decreased to the theta-range. When 5 microM BIC was applied with 30 microM carbachol, the beta oscillations did not start; instead, theta-like activities were induced. It has been reported that carbachol in hippocampal slices can induce theta-like activities, which are not modulated by BIC, while BIC's facilitating the start of the activities. The results of the present study suggest that the GABAA receptor-mediated inhibitory transmission modulates the beta oscillation and that the transmission is needed for the start process of the oscillations. Therefore, the start and generation mechanisms of carbachol-induced beta oscillation will be different from those of carbachol-induced theta-like activities.     

Simultaneous multi-site recordings of neural activity with an inline multi-electrode array and optical measurement in rat hippocampal slices

Understanding neuronal network function requires multi-site recording. An optical imaging method using voltage-sensitive dyes (VSD) is one such suitable method. It is also important to complement this technique with methods that measure other physiological parameters of neuronal network activity. We aimed to develop a multi-channel electrode method that would easily permit the simultaneous optical imaging of neural activity in vitro. There are several multi-electrode systems; some of these are commercially available, but they are not easy to use and are also expensive. We developed a novel, less expensive, stainless steel inline electrode array that utilizes the standard etching method of circuit board manufacturing. This method, along with widely available computer drawing software, allowed us to produce electrode arrays with uniform quality in electrical characteristics and shapes. The electrode array was designed so that it could easily substitute for single electrode systems without modifying the conventional set-up used for electrophysiological measurements and optical recordings. Simultaneous measurement of evoked neural activities in area CA1 of the rat hippocampal slice with our optical imaging method using the VSD Di-4-ANEPPS is demonstrated in this paper. The electrode array was also used as a stimulating electrode, and the evoked response was measured by the optical recording method. The results demonstrate the advantages of simultaneous recordings obtained by these complementary multi-site recording methods in vitro.     

Evoked potential changes in rat hippocampal slices under helium pressure

Summary High pressures of helium affect the physiology of the central nervous system in animals and humans. We examined these effects in rat hippocampal slices. The in vitro preparation displayed a reversible reduction in postsynaptic and antidromic field potentials of CA1 pyramidal cells, but no significant change in the amplitude of the afferent volley. Although the subliminal synaptic response of CA1 neurons was depressed, the ability of these cells to produce population spikes was enhanced. These changes resembled those previously found in vivo in the rat hippocampus. The present results support the hypothesis of a helium pressure-induced depolarization of hippocampal neurons. Other possible mechanisms are discussed.     

High-frequency magnetic stimulation induces long-term potentiation in rat hippocampal slices

Recent reports indicate that the exposure of brain tissues to transcranial magnetic stimulation induces persistent changes in neuronal activity and influences hippocampal synaptic plasticity. However, the modulation of synaptic efficiency by magnetic stimulation in vitro is still unclear. In the present study, we investigated whether high-frequency magnetic stimulation (HFMS) can induce long-term potentiation (LTP) in rat hippocampal slices in vitro. During baseline recording and after HFMS, field excitatory postsynaptic potentials (fEPSPs) were recorded within the CA1 stratum radiatum in response to electrical stimulation of the Schaffer collateral inputs. For LTP induction, HFMS was delivered through a circular coil positioned closely above the slices using two different paradigms (A: 10 trains of 20 pulses at 100 Hz with 1 s intervals, 5 repetitions with 10 s intervals; B: 3 trains of 100 pulses at 100 Hz with 20 s intervals). The intensity of the magnetic stimulus was adjusted to 60–75 A/μs. After application of HFMS, electrically evoked CA1 fEPSPs were enhanced showing significant levels of LTP by both paradigms (A: 142 ± 9% of baseline, n = 6; B: 129 ± 7%, n = 8). Furthermore, HFMS-induced LTP induced by paradigm A was prevented by the presence of the selective N-methyl-d-aspartate receptor (NMDAR) blocker D-AP5 (50 μM) in the bath solution (95 ± 6% of the baseline, n = 6; p < 0.01 compared to control condition without D-AP5). Further, the lack of changes in paired-pulse ratio and the afferent fiber volleys exclude presynaptic involvement in HFMS-induced LTP. In summary, we have demonstrated that HFMS can induce NMDAR-dependent LTP in the CA1 region in vitro.     

Heterogeneous spatial patterns of long-term potentiation in rat hippocampal slices

Although LTP (long-term potentiation) of synaptic transmission has received much attention as a model for learning and memory, its function within a neural circuit context remains poorly understood. To monitor LTP over an extensive circuit, we imaged responses in hippocampal slices using a voltage-sensitive dye. Following theta-burst stimulation, evoked optical signals showed an increase that lasted 40 min or more. Weak stimuli only potentiated the local area around the stimulating electrode, but stronger stimuli induced LTP over a wide area with a complex and non-uniform spatial pattern. The expression of LTP showed distinct peaks and valleys that depended on which axons were activated. Interestingly, the spatial distribution of LTP bore no relation to the spatial distribution of single-shock responses, but closely resembled the distribution of postsynaptic spikes evoked by theta bursts. Thus, postsynaptic spikes during induction constitute a critical determinant for the expression of LTP in intact circuits.     

Kainic acid inhibits cholecystokinin release from rat hippocampal slices

Antagonistic interactions between cholecystokinin (CCK) and nanomolar concentrations of kainic acid (KA) have been reported in area CA3 of the rat hippocampal slice. This study tested the possibility that kainic acid inhibits the release of CCK. Elevated K⁺ was found to release CCK from hippocampal slices in a Ca²⁺-dependent manner. KA, at concentrations as low as 100 nM, inhibited this release by about one-third. Because CCK appears to exert a net inhibitory effect on the firing of CA3 pyramidal cells, the epileptogenic action of KA may be explained, in part, by the depression of CCK release.