An electrophysiological analysis of oxygen and pressure on synaptic transmission

The effect of oxygen at high pressure (OHP), helium at 150 PSIG and 100% oxygen at ambient pressure on excitatory synaptic transmission was studied using the lobster walking leg neuromuscular preparation. Both 100% oxygen at 150 PSIG (7135 mm Hg oxygen) and helium at 150 PSIG (7000 mm Hg helium plus 135 mm Hg oxygen) produced a significant decrease in the amplitude of the junction potential (Vejp). The decrease in Vejp induced by OHP, however, was greater than with pressure alone. OHP also produced a significant decrease in short term facilitation. Exposure to 100% oxygen at ambient pressure produced a transient increase in Vejp and a large increase in frequency of miniature junction potentials. In each case the change in Vejp was due to changes in presynaptic release of transmitter since quantal content per fiber (M') was shown to decrease for OHP and helium at 150 PSIG and to transiently rise with 100% oxygen at ambient pressure. In addition, the response to exogenously applied glutamate (the putative neurotransmitter) was not affected by OHP, 150 PSIG helium or 100% oxygen at ambient pressure. This further indicates a presynaptic site of action.     

Repeated exposure to hyperbaric oxygen sensitizes rats to oxygen-induced seizures

Repeated exposure to increased partial pressure of oxygen (Po₂) is the standard of care for several medical conditions. The side-effects of repeated exposure to hyperbaric oxygen (HBO), however, are not well defined. Previous studies have demonstrated that acute exposure of rats to HBO causes hypothermia that precedes convulsions. In the present studies, rats that were repeatedly exposed to 100% oxygen at 4 atmospheres absolute (ATA) pressure developed convulsions earlier than naive controls. There was also a trend toward less hypothermia in the rats repeatedly exposed to oxygen. The purpose of this study was to test the hypothesis that repeated exposure to HBO increases sensitivity to convulsions induced by HBO and to determine if the time to onset of convulsions is affected by the hypothermia caused by exposure to HBO. Rats were repeatedly exposed to 2 ATA oxygen for a total of 10 days. After 72 h, these rats were challenged by exposure to 100% oxygen at 4 ATA pressure. Rats repeatedly exposed to HBO had convulsions significantly earlier than the naive controls (84±8min compared to 147±11min), and they developed significantly less hypothermia. Control studies suggested that the decrease in the degree of hypothermia was caused by both repeated exposure to oxygen and adaptation to the mild restraint used during oxygen re-exposures. Adaptation to restraint eliminated the hypothermia induced by oxygen but did not change the time to onset of convulsions. Increased sensitivity to convulsions was present after five exposures to 2 ATA oxygen and persisted for 10 days after the last 2 ATA oxygen re-exposure. Kindling is an animal model of epilepsy which is caused by repeated exposure to subthreshold doses of convulsant stimuli. This repeated dosing causes an increase in the sensitivity of the animal to the convulsant stimuli that can persist for weeks. The increased sensitivity of rats to convulsions during subsequent HBO challenge that is induced by repeated exposures to subconvulsant ‘doses’ of HBO (2 ATA) is similar to kindling. At present the mechanism that causes either kindling or the increased sensitivity to seizures induced by HBO are unknown, and therefore may not be the same.     

Modulation of GABAergic transmission by endogenous glutamate in the rat supraoptic nucleus

The presence of group III metabotropic glutamate receptors on GABAergic terminals in the supraoptic nucleus suggests that the level of glutamate in the extracellular space may regulate synaptic strength at inhibitory synapses. To test this hypothesis we examined the consequences of increasing ambient glutamate on GABA-mediated synaptic activity in supraoptic neurons. The concentration of the excitatory amino acid in the extracellular space was increased pharmacologically by blocking glutamate transporters. Inhibition of the astrocyte-specific GLT-1 glutamate transporter led to a reversible decrease in evoked inhibitory postsynaptic current amplitude. This modulation had a presynaptic origin as revealed by analysis of paired-pulse ratio and miniature inhibitory currents. Furthermore, blocking group III metabotropic glutamate receptors with the specific antagonist MAP4 prevented the depression of GABAergic transmission induced by glutamate transporter blockade. Thus, presynaptic metabotropic glutamate receptors located on inhibitory terminals in the supraoptic nucleus appear to sense changes in ambient glutamate and modify GABA release accordingly. However, it seems that such changes need to reach a certain magnitude because the discrete deficit in glutamate clearance which occurs in the supraoptic nucleus of lactating rats is not sufficient to modulate GABA-mediated transmission. These results suggest that ambient glutamate contributes to the modulation of synaptic efficacy not only at glutamatergic synapses but also at inhibitory GABAergic synapses.     

Hyperbaric oxygen and cerebral physiology

Hyperbaric oxygen (HBO) therapy is defined by the Undersea and Hyperbaric Medical Society (UHMS) as a treatment in which a patient intermittingly breathes 100% oxygen under a pressure that is greater than the pressure at sea level [a pressure greater than 1 atmosphere absolute (ATA)]. HBO has been shown to be a potent means to increase the oxygen content of blood and has been advocated for the treatment of various ailments, including air embolism, carbon monoxide poisoning, wound healing and ischemic stroke. However, definitive established mechanisms of action are still lacking. This has led to uncertainty among clinicians, who have understandingly become hesitant in regard to using HBO therapy, even in situations where it could prove beneficial. Therefore, this review will summarize the literature regarding the effects of HBO on brain oxygenation, cerebral blood flow and intracranial pressure in both the healthy and injured brains, as well as discuss how changes in these three factors can impart protection.     

Ethanol alters synaptic activity in cultured spinal cord neurons

The acute effects of ethyl alcohol on mammalian central neurons were investigated using electrophysiological techniques and an in vitro model system, cultured fetal mouse spinal cord neurons. Intracellular recordings were made from the cultured neurons to evaluate the effect of alcohol (10-100 mM) on membrane potential, membrane permeability, amplitude of the action potential, sensitivity of the neurons to putative neurotransmitters and the process of synaptic transmission. Alcohol was applied by superfusion; putative amino acid neurotransmitters were applied by micropressure ejection. The most dramatic effect of alcohol on the spinal cord neurons was a reduction in the spontaneous activity (excitatory and inhibitory synaptic potentials and action potentials) and the glutamate evoked synaptic activity. Alcohol doses as low as 20-30 mM, concentrations which reflect blood levels during intoxication, were effective. Membrane potential, membrane permeability, and amplitude of the action potential were relatively resistant to these low doses of alcohol; at the higher alcohol doses, no effect or only modest alterations of these characteristics were observed. The responses of the neurons to the putative excitatory neuro-transmitter glutamate, and inhibitory transmitters GABA and glycine were also relatively resistant to alcohol exposure. These data indicate that acute exposure to alcohol has a predominantly inhibitory action on the activity of the cultured mammalian CNS neurons, and that this inhibition is most likely due to an alteration in the process of synaptic transmission.     

Depression of cortical Na, K-ATPase acticvity in rats exposed to hyperbaric oxygen

Extracellular single unit recordings were used to study inhibitory synaptic responses evoked from preoptic-anterior hypothalamic neurones following arcuate-ventromedial stimulation. Intravenously administered methohexitone, pentobarbitone and thiopentone increased the duration of inhibitory synaptic responses by up to 400%. Submaximal responses to iontophoretically applied GABA but not glycine were also potentiated. Recovery from the actions of the short acting barbiturates was observed.     

The relationship between cortical electrical activity and regional cerebral glucose metabolic rate in rats exposed to 3 atmospheres absolute oxygen

The regional cerebral metabolic rate for glucose (rCMRgl) was autoradiographically measured in conscious rats during 180–210 min of exposure to 3 atmospheres absolute oxygen (ATA O₂), 3 ATA N₂-O₂ normoxia and air at 1 ATA. The exposure time and oxygen pressure in the present study were purposely matched to a parallel project in human subjects. The electrocorticogram (ECoG) was continuously recorded throughout the exposure. According to the ECoG responses, the oxygen-exposed rats fell into two categories: (1) ‘resistant’ ones, those without changes in ECoG throughout the exposure; and (2) ‘sensitive’ rats, those with changes in EcoG before or during the rCMRgl measurements. The observed ECoG changes were increased slow wave activity in the δ range, which was in some cases followed by paroxysmal electrical discharges. No changes in rCMRgl were observed in oxygen-exposed ‘resistant’ rats as compared to air breathing or N₂-O₂ normoxic rats at 3 ATA. However, in the ‘sensitive’ rats there were increases in rCMRgl in 8 of the 28 neuroanatomical structures examined as compared to the air breathing and 3 ATA normoxic controls. It is concluded that the increase in rCMRgl are related to the onset of the oxygen-induced preconvulsive changes in ECoG.     

Postsynaptic mechanism may contribute to inhibitory acetylcholine effect on GABAergic synaptic transmission in hippocampal cell cultures

The effect of acetylcholine (ACh) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections. Spontaneous IPSCs and IPSCs evoked by extracellular stimulation of a single presynaptic neuron were recorded. ACh inhibited the evoked IPSCs in most of the connections, although facilitation was also observed. Regardless of inhibitory or facilitatory effects on the evoked IPSCs, an enhanced spontaneous synaptic input to the postsynaptic neurons was usually observed. ACh-induced changes in the evoked IPSCs were usually accompanied by changes in paired pulse depression (PPD), which are thought to reflect presynaptic mechanisms of modulation. However, the time course of PPD changes did not always match that of the IPSC changes, suggesting a contribution of other, possibly postsynaptic, mechanism(s). To analyze this possibility, effects of ACh on responses to direct application of exogenous GABA were studied. In a proportion of the neurons (40%) ACh reversibly decreased GABA responses, indicating that postsynaptic mechanisms may also contribute to the inhibitory ACh effect on GABAergic transmission. We conclude that several different modulatory mechanisms of ACh action participate in the regulation of GABAergic transmission at the level of synaptic connection of a single GABAergic neuron.     

Co-release and interaction of two inhibitory co-transmitters in rat sacral dorsal commissural neurons

Conventional whole-cell recording configuration was used to characterize the interaction between two inhibitory neurotransmitters, GABA and glycine, in synaptic bouton preparation obtained from rat sacral dorsal commissural nucleus (SDCN). The co-release of GABA and glycine as well as the interaction between their corresponding receptors was demonstrated. Furthermore, taking advantage of pure glycinergic terminal preparation, the possible interaction between GABA and glycine at synaptic level was studied. The results revealed a novel cross-modulation between the two inhibitory cotransmitters systems. This interaction may contribute to sensory processing such as nociception in the mammalian deep dorsal horn under physiological and/or pathological conditions.     

Cholecystokinin inhibits evoked inhibitory postsynaptic currents in the rat nucleus accumbens indirectly through gamma-aminobutyric acid and gamma-aminobutyric acid type B receptors

We recently reported that cholecystokinin (CCK) excited nucleus accumbens (NAc) cells and depressed excitatory synaptic transmission indirectly through gamma-aminobutyric acid (GABA), acting on presynaptic GABAB receptors (Kombian et al. [2004] J. Physiol. 555:71-84). The present study tested the hypothesis that CCK modulates inhibitory synaptic transmission in the NAc. Using in vitro forebrain slices containing the NAc and whole-cell patch recording, we examined the effects of CCK on evoked inhibitory postsynaptic currents (IPSCs) recorded at a holding potential of -80 mV throughout CCK-8S caused a reversible inward current accompanied by a concentration-dependent decrease in evoked IPSC amplitude. Maximum IPSC depression was approximately 25% at 10 microM, with an estimated EC50 of 0.1 microM. At 1 microM, CCK-8S induced an inward current of 28.3 +/- 4.8 pA (n=6) accompanied by an IPSC depression of -18.8% +/- 1.6% (n=6). This CCK-induced IPSC depression was blocked by pretreatment with proglumide (100 microM; -3.7% +/- 6.9%; n=4) and by LY225910 (100 nM), a selective CCKB receptor antagonist (4.4% +/- 2.6%; n=4). It was not blocked by SCH23390 (10 microM; -23.5% +/- 1.3%; P < 0.05; n=7) or sulpiride (10 microM; -21.8% +/- 5.1%; P <0.05; n=4), dopamine receptor antagonists. By contrast, it was blocked by CGP55845 (1 microM; -0.4% +/- 3.4%; n=5) a potent GABAB receptor antagonist, and by forskolin (50 microM; 9.9% +/- 5.2%; n=4), an adenylyl cyclase activator, and H-89 (1 microM; 6.9% +/- 3.9%; n=4), a protein kinase A (PKA) inhibitor. These results indicate that CCK acts on CCKB receptors to increase extracellular levels of GABA, which then acts on GABAB receptors to decrease IPSC amplitude.     

The effect of hyperbaric oxygen on regional brain and spinal cord levels of nitric oxide metabolites in rat

Hyperbaric oxygen (HBO(2)) therapy is reported to be beneficial in transient brain ischemia. The present study was conducted to determine the influence of HBO(2) on metabolites of nitric oxide (NO) in brain and spinal cord of rats. Rats were exposed to room air (RA), normobaric air (NBA), normobaric oxygen (NBO(2)), hyperbaric air (HBA) or HBO(2), the last two conditions at 2.5ATA (atmosphere absolute) for 60 min. The results demonstrate that, compared to the NBA control, oxygen alone generally reduced tissue levels of NO(x)(-) (nitrite plus nitrate). On the other hand, 2.5ATA alone tended to have a slight, if any, effect on tissue levels of NO(x)(-). The combination of oxygen and pressure (i.e., HBO(2)) generally led to an increase in tissue levels of NO(x)(-). Based on these findings, it is concluded that HBO(2) appears to markedly increase NO function most notably in the corpus striatum, brainstem, cerebellum and spinal cord.     

Oxygen tension in the globus pallidus and neostriatum of unanesthetized rats during exposure to hyperbaric oxygen

Unanesthetized rats were exposed 1 h to 100% oxygen at 60 psig (5 atmospheres absolute). Utilizing platinum semimicroelectrodes, oxygen tension was recorded from the globus pallidus and the neostriatum. Oxygen tension increased from control values (room air at ambient pressure) during the exposure period in both experimental groups. In the globus pallidus, oxygen tension reached the first stable peak in an average of 27.8 min, whereas in the neostriatum the first stable peak was reached in an average of 3.0 min. The results of this study suggest a correlation between oxygen tension and pathologic changes observed in the basal ganglia.     

Neuroprotection by oxygen in acute transient focal cerebral ischemia is dose dependent and shows superiority of hyperbaric oxygenation

The neuroprotective effect of oxygen after acute stroke in rats has been shown previously. However, the question of optimal dosing still remains unanswered. Thus, we investigated the use of oxygen at different concentrations by either normobaric oxygenation (NBO) or hyperbaric oxygenation (HBO) at different pressures in a model of transient ischemia/reperfusion in rats. Animals underwent 90 min of middle cerebral artery occlusion (MCAO) followed by 90 min of reperfusion before oxygen treatment. Oxygen was applied either by NBO (100% O(2); 1.0 absolute atmosphere, ATA) or HBO (100% O(2); 1.5, 2.0, 2.5 or 3.0 ATA) for 1 h. Primary endpoints were infarct volume and clinical outcome measured 24 h and 7 days following the MCAO. A statistically significant and long-lasting reduction in infarct volume was seen in the HBO 2.5 ATA and 3.0 ATA groups over a period of 7 days. The reduced infarct volume was accompanied with a statistically significant improvement in clinical outcome in the high-dose oxygen-treated groups. The presented data indicate that oxygen is a highly neuroprotective molecule in transient focal cerebral ischemia in rats, when applied early and at high doses. The effect is dose dependent and shows a superiority of HBO over NBO, when the primary endpoints infarct volume reduction and clinical outcome are analyzed. These data are important for the development of new acute stroke treatment studies in humans.     

Attenuation of inhibitory synaptic transmission by glial dysfunction in rat thalamus

The thalamus serves as the obligatory gateway to the neocortex for sensory processing, and also serves as a pathway for corticocortical communication. In addition, the reciprocal synaptic connectivity between the thalamic reticular nucleus (TRN) and adjacent thalamic relay nuclei generates rhythmic activities similar to that observed during different arousal states and certain neurological conditions such as absence epilepsy. Epileptiform activity can arise from a variety of neural mechanisms, but in addition glia are thought to have an important role in such activities as well. Glia serve a central role in glutamine synthesis, a precursor for glutamate or GABA in nerve terminals. While alterations in glutamine shuttling from glia to neurons can influence GABA and glutamate neurotransmission; the consequences of such action on synaptic transmission and subsequent network activities within thalamic circuits is less understood. We investigated the consequences of altering glutamine transport on inhibitory transmission and intrathalamic activities using the in vitro thalamic slice preparation. Disruption of the glutamine shuttling by the neuronal glutamine transporter (system A transporter) antagonist, α-(methylamino)isobutyric acid (MeAIB), or the selective gliotoxic drug, fluorocitric acid (Fc) dramatically decreased intrathalamic rhythmic activities. At the single cell level, MeAIB and Fc significantly attenuated electrically evoked inhibitory postsynaptic currents (eIPSCs) in thalamic relay neurons; however, miniature IPSCs were unaffected. These data indicate that glutamate-glutamine shuttle is critical for sustaining thalamic synaptic transmission, and thereby alterations in this shuttle can influence intrathalamic rhythmic activities associated with absence epilepsy.     

The effect of hyperoxemia on cerebral blood flow in normal humans

The aim of this study was to evaluate the effect of various degrees of hyperoxemia on cerebral blood flow (CBF), including the hyperbaric oxygenation (HBO) environment. Study subjects were 28 healthy volunteers (17 males and 9 females) from 26 to 60 (average: 42 +/- 11) years old. CBF measurements were done by 19 mCi 133Xe intravenous injection method using rCBF analyzer BI-1400 (Valmet). Two-compartmental analysis was used for the calculation of Fast, Slow Flow and initial slope index (ISI). The three CBF study series included: Rest (before HBO 1 ATA.air)-1 ATA.O2-2 ATA.O2 series in 8 cases; Rest-1 ATA.O2 50% N2 50%-1.5 ATA.O2 series in 10 cases; and Rest-2.5 ATA.O2-after HBO (1 ATA.air) series in 8 cases. CBF measurements commenced 5 to 10 minutes after fixing a mask for oxygen inhalation. Arterial blood gas analyses using IL-813 (IL) and blood pressure measurements were done immediately after CBF measurements. CBF changes evaluated by ISI, estimating resting flow as 100% (PaO2: 93 +/- 8 mmHg), were 91% at 1 ATA.O2 50% (PaO2: 201 +/- 50 mmHg), 79% at 1 ATA.O2 (PaO2: 432 +/- 44 mmHg), 77% at 1.5 ATA.O2 (PaO2: 693 +/-79 mmHg) and 71% at 2 ATA.O2 (PaO2: 838 +/- 95 mmHg). CBF gradually decreased to the level shown for 2 ATA.O2, but CBF showed a tendency to increase somewhat at 2.5 ATA.O2 (81%, PaO2: 1103 +/- 111 mmHg). CBF decreases were statistically significant at 1 ATA.O2, 1.5 ATA.O2, 2 ATA.O2 and also 2.5 ATA.O2 compared with Rest (P less than 0.05). Arterial blood gas analyses clearly showed the stepwise increase in PaO2 to the level of 2.5 ATA.O2 (P less than 0.01). Changes in PaCO2 and blood pressure were slight and not significant statistically in each series. Since the data showed no significant change in the PaCO2 level in each series, it was concluded that the CBF decrease was due to vasoconstriction caused by the elevated PaO2. The mechanism of cerebral vasoconstriction caused by hyperoxemia is not yet clearly understood, but the direct vasoconstrictive effect of oxygen, neurogenic control and the metabolic effect of an elevated cerebral tissue oxygen level may contribute to the CBF decrease. CBF decrease during elevated PaO2 may be a protective physiological response to maintain normal brain metabolism and function against the excessive oxygen supply. Disturbance of this regulatory mechanism may result in oxygen poisoning of the central nervous system.(ABSTRACT TRUNCATED AT 400 WORDS)     

A depolarizing inhibitory response to GABA in brainstem auditory neurons of the chick

Neurons in the brainstem auditory nuclei, n. magnocellularis and n. laminaris, of the chick are contacted by terminals containing the inhibitory neurotransmitter γ-aminobutyric acid (GABA). In this report we describe the physiological response of these neurons to GABA using an in vitro slice preparation. In brainstem auditory neurons, GABA produced a depolarization of up to 20 mV and an associated decrease in input resistance. This depolarization was inhibitory; action potentials generated by orthodromic synaptic drive, antidromic stimulation and intracellular current injection were prevented by GABA application. The GABA response still occurred when synaptic transmission was prevented by perfusing the slice with a medium containing low Ca²⁺ and high Mg²⁺ concentrations. Thus, the effects of GABA were directly on the postsynaptic neuron and not via an interneuron. Whole-cell voltage clamp of neurons revealed that the reversal potential of the inward current was approximately −45 mV, suggesting that the channel responsible for this response is not selective for Cl⁻ or K⁺. Pharmacological analyses suggest that this GABA receptor has properties distinct from those typical of either GABA_a or GABA_b receptors. Although a similar response was observed with the GABA_a agonist, muscimol, it was not blocked by the GABA_a antagonist, bicuculline. The response was not evoked by the GABA_b agonist, baclofen, and was not blocked by the GABA_b antagonist phaclofen. This unusual depolarizing response is not a common feature of all brainstem neurons. Neurons located in the neighboring medial vestibular nucleus show a more traditional response to GABA application. At resting potential, these neurons show a hyperpolarizing or biphasic response associated with a decrease in input resistance and inhibition of their spontaneous activity. GABA-induced responses in the medial vestibular nucleus are blocked by bicuculline. These results suggest that an unusual form of the GABA receptor is present in the brainstem auditory system of the chick. It is possible that this form of GABA receptor provides an efficient mechanism for inhibiting the relatively powerful EPSPs received by brainstem auditory neurons, or it may play a trophic role in the afferent regulation of neuronal integrity in this system.     

The effect of breathing 100 per cent oxygen at 1 and 3 atmospheres absolute on the accumulation of 3 H-lysine into different brain regions, plasma, muscle and liver of ethanol-anesthetized and unanesthetized rats

The effect of breathing 100 % oxygen at 1 and 3 atmospheres absolute was studied in relation to the accumulation of ³H-lysine into different brain regions, plasma, muscle and liver of ethanol-anesthetized and unanesthetized rats, as well as into the TCA-precipitable protein (PCF) of brain, plasma, muscle and liver. It was shown that hyperoxia at 1 and 3 ATA caused a depression of the ³H-lysine accumulation into the hypothalamus and cerebellum of unanesthetized rats two hours after the injection of the amino acid. Hyperoxia at 3 ATA also caused a depression of lysine accumulation into white matter 2 hours after injection. No changes were noted in the ethanol-anesthetized rats. By group analysis, there was also a depression of ³H-lysine accumulation in the cerebral grey matter in the 1 ATA animals at both levels of anesthesia. Hyperoxia at 1 and 3 ATA caused a depression in the ³H-lysine accumulation into plasma protein at two and three hours in unanesthetized rats and had somewhat more variable effects in the anesthetized animals. In the skeletal muscle, hyperoxia caused an increase in amino acid accumulation in animals breathing 100 % 0₂ at 3 ATA as compared to those breathing air or 100 % oxygen at 1 ATA when compared by group analysis of whole tissue. Analysis of the muscle PCF revealed a depression in lysine uptake associated with anesthesia (group analysis), and changes in all groups related to time elapsed since injection of labelled amino acid. Finally, 0₂ at 1 ATA was observed to depress amino acid accumulation while 0₂ at 3 ATA had no effect in the unanesthetized rats. In the liver, there was a significant depression of ³H-lysine accumulation in those animals breathing 100 % oxygen at 1 ATA as compared to controls in both the ethanol-anesthetized and unanesthetized rats. ³H-lysine accumulation     

The mechanisms of the strong inhibitory modulation of long-term potentiation in the rat dentate gyrus

The hippocampus is essential for the formation of certain types of memory, and synaptic plasticity such as long-term potentiation (LTP) is widely accepted as a cellular basis of hippocampus-dependent memory. Although LTP in both perforant path-dentate gyrus (DG) granule cell and CA3-CA1 pyramidal cell synapses is similarly dependent on activation of postsynaptic N-methyl-D-aspartate receptors, several reports suggest that modulation of LTP by γ-aminobutyric acid (GABA) receptor-mediated inhibitory inputs is stronger in perforant path-DG granule cell synapses. However, little is known about how different the mechanism and physiological relevance of the GABAergic modulation of LTP induction are among different brain regions. We confirmed that the action of GABA(A) receptor antagonists on LTP was more prominent in the DG, and explored the mechanism introducing such difference by examining two types of GABA(A) receptor-mediated inhibition, i.e. synaptic and tonic inhibition. As synaptic inhibition, we compared inhibitory vs. excitatory monosynaptic responses and their summation during an LTP-inducing stimulus, and found that the balance of the summated postsynaptic currents was biased toward inhibition in the DG. As tonic inhibition, or sustained activation of extrasynaptic GABA(A) receptors by ambient GABA, we measured the change in holding currents of the postsynaptic cells induced by GABA(A) receptor antagonists, and found that the tonic inhibition was significantly stronger in the DG. Furthermore, we found that tonic inhibition was associated with LTP modulation. Our results suggest that both the larger tonic inhibition and the larger inhibitory/excitatory summation balance during conditioning are involved in the stronger inhibitory modulation of LTP in the DG.     

Monitoring synaptic transmission in primary neuronal cultures using local extracellular stimulation

Various techniques have been applied for the functional analysis of synaptic transmission in cultured neurons. Here, we describe a method of studying synaptic transmission in neurons cultured at high-density from different brain regions such as the cortex, striatum and spinal cord. We use postsynaptic whole-cell recordings to monitor synaptic currents triggered by presynaptic action potentials that are induced by brief stimulations with a nearby extracellular bipolar electrode. Pharmacologically isolated excitatory or inhibitory postsynaptic currents can be reliably induced, with amplitudes, synaptic charge transfers, and short-term plasticity properties that are reproducible from culture to culture. We show that the size and kinetics of pharmacologically isolated inhibitory postsynaptic currents triggered by single action potentials or stimulus trains depend on the Ca2+ concentration, temperature and stimulation frequency. This method can be applied to study synaptic transmission in wildtype neurons infected with lentiviruses encoding various components of presynaptic release machinery, or in neurons from genetically modified mice, for example neurons carrying floxed genes in which gene expression can be acutely ablated by expression of Cre recombinase. The preparation described in this paper should be useful for analysis of synaptic transmission in inter-neuronal synapses formed by different types of neurons.     

Attenuation of malonate toxicity in primary mesencephalic cultures using the GABA transport blocker, NO-711

Cultured rat mesencephalic neurons were used to assess the effects of gamma-aminobutyric acid (GABA) transport blockers on toxicity caused by malonate, a reversible, competitive inhibitor of succinate dehydrogenase. Previous studies utilizing an ex vivo chick retinal preparation have shown that GABA release and cell swelling are early consequences of acute energy impairment and that GABA transport blockers attenuate this toxicity. The present results demonstrate that the nonsubstrate GABA transport blocker, NO-711 (1 nM-1 microM), dose-dependently protected cultured mesencephalic dopamine (DA) and GABA neurons from malonate-induced toxicity. Similar protection was demonstrated with nipecotic acid (1 mM) and SKF89976A (100 nM), substrate and nonsubstrate GABA transport blockers, respectively. These compounds by themselves produced no signs of toxicity, although nipecotic acid caused a long-term decrease in GABA uptake not associated with toxicity. Compounds which decrease intracellular reactive oxygen species (ROS) are protective in this model, but NO-711 did not prevent the rise in intracellular ROS induced by malonate, indicating its protective effects were downstream of ROS production. Supplementation of malonate treated cultures with the GABA(A) agonist, muscimol (10 microM), increased the toxicity toward the DA and GABA neuron populations. Antagonists at the GABA(A) and glycine receptors provided partial protection to both the GABA and DA neurons. These findings suggest that the GABA transporter, GABA(A), and/or glycine channels contribute to cell damage associated with energy impairment in this model.