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To investigate the reversibility of neuronal functions during deep and mild hypothermia, we have examined changes in membrane properties of pyramidal neurons of the CA3 region of hippocampal slices during cooling and rewarming (8 approximately 37 degrees C) of the perfusion medium. Hypothermia reduced the excitatory postsynaptic potential (EPSP) slope in a temperature dependent manner, but the EPSP amplitude was enhanced transiently between 30 and 25 degrees C. In observing spikes generated by either orthodromic stimulation or by direct intracellular current injection, the critical threshold for spike generation was decreased transiently at a temperature of 30 degrees C. In addition, the numbers of spikes were increased transiently regardless of the progressive prolongation of spike duration and latency with cooling. The resting membrane potential was stable from 37 to 20 degrees C. However, this potential showed a depolarizing shift at 15 degrees C. The neuronal activities, including membrane properties, recovered fully when the temperature was raised to 35 degrees C even from a low of 15 degrees C. In addition, field population spikes (PS) recorded in the pyramidal cell layer showed a complete reversibility after long-term severe hypothermia (8 degrees C). These results suggest that synaptic function, neuronal excitability and membrane properties maintain reversibility during deep hypothermia, as well as in mild hypothermia.

The purpose of this present study is to clarify adenosine (ADO) metabolism in guinea pig brain slices during simulated ischemia. In slice preparations after decapitation, ADO levels were lowest in slices of the cerebellum (1.2 nmol/mg protein), followed by the superior colliculus (3.4) and highest in the hippocampus (6.4), and the combined concentrations of inosine (Ino) and hypoxanthine (HX) were highest in the cerebellum (5.5), followed by the superior colliculus (3.5) and the hippocampus (1.5). After preincubation with standard medium with oxygen and glucose for 30 min, total ADO levels (tissue ADO plus ADO lost into medium during incubation) decreased to 0.3 in the cerebellum, to 1.3 in the superior colliculus and to 2. 9 in the hippocampus. On the other hand, levels of total Ino and HX increased to 21.1 in the cerebellum, to 14.3 in the hippocampus and to 12.0 in the superior colliculus. To investigate the effect of simulated ischemia on ADO metabolism, preincubated slices were exposed for 10 min in medium deprived of oxygen and glucose. The increases of ADO content after 10 min ischemia were 0.2 in the cerebellum, 1.0 in the superior colliculus and 1.3 in the hippocampus. In contrast, the increases of both Ino and HX concentrations were 2.9 in the cerebellum, 2.2 in the superior colliculus and 1.4 in the hippocampus. The total amount of the increase in ADO, Ino and HX was approximately 3 in all three regions. These results indicate that there are significant differences in the metabolic rate to degrade ADO into Ino and HX in various areas of brain possibly due to differences in adenosine deaminase activity in those areas.

Changes in levels of adenosine, inosine, hypoxanthine and ATP during complete ischemia after decapitation were determined in various areas of the guinea pig and rat brain using an HPLC method. These results were compared with levels in brains fixed by microwave irradiation. The levels of adenosine during 60 min of complete ischemia were extremely high and unevenly distributed while levels in the microwaved brains were very low and evenly distributed. The ratios of inosine plus hypoxanthine levels to adenosine which indicate the rate of metabolic degradation from adenosine into inosine and hypoxanthine, were also unevenly distributed during complete ischemia in the cerebellum, superior colliculus, cerebral cortex and hippocampus of the guinea pig and rat, and the highest ratio was observed in the cerebellum of the guinea pig and the superior colliculus of the rat. The activities of adenosine deaminase (ADA), one of the enzymes involved in adenosine metabolism, were measured in the four regions of the guinea pig. The ADA activities were unevenly distributed and the highest ADA activity was found in the cerebellum. These regional differences in ADA activities are in good agreement with the regional differences in the ratio of inosine plus hypoxanthine levels to adenosine during complete ischemia. Furthermore, the administration of EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride] (10 mg/kg, i.p.), an ADA inhibitor, caused a significant increase of adenosine and decrease of inosine formation in all four regions and a drastic effect on the cerebellum with high ADA activity compared with the other regions in the guinea pig brain. These results indicate that the changes in concentrations of adenosine and its metabolites (inosine and hypoxanthine) during complete ischemia depend on ADA activity in each brain region.

Using hippocampal slices from guinea pigs, we investigated the effect of different concentrations of glucose and replacement of glucose with mannose, fructose and lactate on neural activity. As an index of neural activity, the population spikes (PS) were recorded in the granule cell layer of the dentate gyrus (DG) and the pyramidal cell layer of the CA3 area in the hippocampal slices. Lowering the concentration of glucose from 10 mM to 5, 3, 2, 1 and 0 mM caused a reduction in the PS amplitude. There were differences in the decay times of the PS evoked in these two regions. PS evoked in CA3 region decayed faster even at a concentration of 3 mM glucose at which PS in granule cell layer was well maintained. The decay time of the PS in the CA3 region in the presence of glucose up to a concentration of 3 mM was shorter than that evoked in the DG. After the replacement of glucose with mannose, fructose or lactate, the PS disappeared within 35 min and there were no significant differences between the decay times in the two regions of slices incubated in the same medium. ATP, creatine phosphate (CrP) and lactate levels in each slice were determined. To investigate whether mannose and fructose could be metabolized or not in the tissue slice, anaerobic production of lactate from glucose, mannose and fructose were measured during oxygen and glucose deprivation. Under anaerobic conditions for 60 min, the levels of high-energy phosphates decreased to 50% of the initial level and lactate was produced from glucose, mannose or fructose. However, there were significant differences in the rate of lactate production between the DG and CA3 areas during application of 3 mM glucose, 10 mM mannose and 10 mM fructose. These results indicate that mannose, fructose and lactate can be metabolized and are available for maintaining the levels of high-energy phosphates but not for neural activity in the tissue slices and that the presence of glucose is indispensable for the maintenance of neural activity.

The concentration of endogenous adenosine in the cerebrospinal fluid increased 2–3-fold of the original level in the area of rat superior colliculus after the intraperitoneal administration of an adenosine deaminase inhibitor, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenosine, 10 mg/kg). Potentials evoked in the superior colliculus by optic tract stimulation were also facilitated by 120–160% of their initial amplitudes. A selective A1 adenosine receptor antagonist, DPCPX (8-cyclopentyl-1,3-dipropylxanthine), failed to reduce such EHNA-induced facilitation. However, a selective A2A adenosine receptor antagonist, KF17837 (8(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine) completely eliminated the facilitatory effects of EHNA. Northern blot analysis demonstrated abundant expression of A1 adenosine receptor mRNA in the superior colliculus. RT-PCR analysis was able to detect the concomitant expression of A2A adenosine receptor mRNA, but at levels lower than one-tenth of the striatal expression. In the superior colliculus, A2A adenosine receptors function predominantly on the facilitatory effects of adenosine, irrespective of the ubiquitous expression of A1 adenosine receptors.

The block of synaptic transmission and neural activity during deprivation of oxygen or glucose has been simply attributed to the lack of energy due to the disorder of energy production. To clarify the interrelation between neural activity and energy metabolism during hopoxia or glucose deprivation, we studied the changes in ATP levels and electrical events of pyramidal neurons in the CA3 region and [Ca2+]i mobilization of the dendritic and cellular region of CA3 area, using guinea pig hippocampal slices. The studies of field potentials and intracellular recording from the pyramidal cell of CA3 area during hypoxia or glucose deprivation revealed that the cessation of synaptic activity and the depolarization of resting potential occurred earlier during hypoxia than during glucose deprivation while the increase of [Ca2+]i was slow during hypoxia but rapid during glucose deprivation although the ATP level of CA3 area was maintained at its original level for 20 min during both conditions. When glucose was replaced by lactate, ATP concentration was not reduced but the electrical activity decayed and [Ca2+]i increased with the similar time course as observed during lack of glucose, only. These results suggest that different mechanisms underlie the block of synaptic transmission in the CA3 pyramidal neurons during hypoxia and glucose deprivation and that lactate cannot substitute for glucose in the maintenance of neural activity.

To investigate the involvement of N-methyl-D-aspartate (NMDA) receptor, protein kinase C (PKC) and calmodulin on long-term potentiation (LTP) formation in the superior colliculus (SC), the effects of an NMDA receptor antagonist (D-APV), PKC inhibitors (H-7, K-252a, K-252b, polymyxin B), a protein kinase A (PKA) inhibitor (H-8) and a calcium/calmodulin-dependent kinase inhibitor (calmidazolium) on LTP formation were studied in guinea pig SC slices. APV (100 microM) masked the expression of LTP by tetanic stimulation, but the LTP once formed was not influenced by application of APV. LTP was blocked by application of H-7 (100 microM), but LTP reappeared 20 min after removal of H-7 from the perfusion medium without further tetanic stimulation. On the other hand, established LTP was also inhibited by application of H-7 even 90 min after the tetanic stimulation. Application of K-252a (500 nM) inhibited LTP formation, but K-252b (500 nM) had no inhibitory effect on LTP formation since K-252b, unlike K-252a, cannot permeate the cell membrane. Tetanic stimulation was applied 20 min after application of polymyxin B (1 microM) to the medium but it could not induce LTP, while established LTP was not influenced by the drug. Application of calmidazolium (50 microM) inhibited LTP formation, but had no inhibitory effect on LTP once formed. These results suggest that both the NMDA receptor and calmodulin system are involved in the induction of LTP after tetanic stimulation. This leads to PKC activation which maintains the LTP.

Superior colliculus (SC) slices were prepared from guinea pig. Electrical stimulation was applied to the optic layer (OL) of the SC slices and the postsynaptic potential (PSP) was evoked in the superficial grey layer (SGL) of the SC. Tetanic stimulation to the OL evoked long-term potentiation (LTP) in the PSP. To investigate the involvement of the protein kinase C (PKC) system on the LTP formation in the SC, the effects of PKC activator (phorbol ester) and PKC inhibitors (polymyxin B, melittin and H-7) on the LTP formation have been studied. Application of phorbol ester to the medium at concentrations between 10 −8 and 10 −10 M increased the amplitude of the PSP. On the other hand, the presence of phorbol ester at a concentration of 10 −6 M increased the glutamate release from the SGL slices. Tetanic stimulation which could not induce LTP by itself could elicit LTP during application of phorbol esters at the low concentration (3 × 10 −12 M). PKC inhibitors such as polymyxin B (10 −7 M), melittin (10 −8 M) and H-7 (10 −4 M) prevented LTP formation. When H-7 was applied once after LTP was formed, the enhanced PSP reduced to the original level. These results strongly indicate the involvement of PKC system on the LTP formation in the SC slices.

Postsynaptic potentials (PSPs) were recorded from the granular cell layer of guinea pig hippocampal slices. Application of adenosine at a concentration of more than 10 microM in the incubation medium depressed the amplitude of the PSP, but adenosine at a concentration of 10 nM to 1 microM enhanced the amplitude of the PSP. The dose-response curve of the effect of adenosine showed an excitatory and inhibitory biphasic pattern. Adenosine analogues such as adenosine triphosphate (ATP), adenosine diphosphate (ADP) and nicotinamide adenine dinucleotide (NAD) showed similar biphasic effects, however beta-gamma-methyleneadenosinetriphosphate (beta-gamma-ATP), 5'-N-ethylcarboxamidoadenosine (NECA). N6-cyclohexyladenosine (CHA) and N6-R-phenylisopropyladenosine (RPIA) revealed only inhibitory effects.

To investigate the correlation between neural activity and intracellular Ca2+ ([Ca2+]i) mobilization in immature and adult brain during ischemia (hypoxia and glucose deprivation) and deprivation of glucose, hippocampal slices were prepared from 7-, 10-day-old and adult rats. Population spikes (PS) and antidromic responses (AR) were recorded in the pyramidal cell layer of the CA1 area as an index of neural function. [Ca2+]i mobilization of the stratum radiatum in the CA1 area was measured using the fluorescent dye fura-2 AM. The rise in [Ca2+]i occurred earlier in the adult animal and the decay times for the orthodromic PS and antidromic responses were shorter in the adult during ischemia. The field potentials and antidromic responses decreased substantially prior to the elevation of [Ca2+]i in both developing and adult brains. Furthermore, ATP levels decreased substantially before the elevation of [Ca2+]i during ischemia. These results suggest that neural activity and intracellular Ca2+ homeostasis in the immature rats brain are more resistant to energy failure than adult rats and that neuronal activity in the developing and adult brain is impaired initially by energy depletion during ischemia. In the immature animal, during glucose deprivation, the antidromic responses were slowly decayed or even failed to extinguish and [Ca2+]i levels were maintained for a longer period or even failed to rise in spite of the rapid loss of PS. Furthermore, ATP levels were well preserved at the time of PS loss. These results agree well with our previous reports showing that glucose plays an important role in the preservation of synaptic transmission in addition to its major function as an energy substrate.

The purpose of the present experiments was to clarify the possible mechanism for the depression of long term potentiation (LTP) induction in the superficial gray layer of the rat superior colliculus after optic nerve stimulation. A postsynaptic field potential was recorded in vitro in the superficial gray layer of superior colliculus slices after stimulation of the optic layer. Tetanic optic layer stimulation (50 Hz, 20 s) induced LTP of the postsynaptic field potential elicited in the superficial gray layer. The postsynaptic field potential, with unitary discharges, produced in the superficial gray layer by optic nerve stimulation in vivo was depressed by a conditioning stimulus to the visual cortex. Identical inhibition of the cortical response of the superficial gray layer was produced by optic nerve stimulation. The application of picrotoxin (2.5 mg/kg, i.p.), a GABAA antagonist or methoxypyridoxine (100 mg/kg, i.v.), an anti-glutamate decarboxylase agent which reduces GABA levels, blocked the inhibitory interaction between the optic nerve-superficial gray layer and visual cortex-superficial gray layer. Tetanic optic nerve stimulation (50 Hz, 20 s) failed to induce LTP in the superficial gray layer of the intact rat. LTP was only elicited by tetanic optic nerve stimulation when picrotoxin or methoxypyridoxine was administered prior to the tetanic stimulation and when the ipsilateral visual cortex was removed. These results indicate that GABAergic interneurons in the superficial gray layer activated by corticotectal input, may stop the formation of LTP in the superficial gray layer.

The effects of afferent lesions on the levels of glutamate, aspartate and gamma-aminobutyric acid (GABA) in the laminae of the superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN) of the rat were studied, using microassay methods for these amino acids. The analysis was performed 12-14 days after left eye enucleation, or ablation of right visual cortical area, or both left eye enucleation and ablation of right visual cortex. Superficial gray layer (SGL) and deep layers in the SC were dissected out from the thin-sectioned, freeze-dried sample. In the dLGN, the outer and inner laminae were separately dissected. The glutamate contents in the upper half of SGL and outer lamina of dLGN contralateral to eye enucleation decreased significantly (15%). Combination of eye enucleation and visual cortical ablation further decreased the glutamate content in the upper half of the right SGL (29.3%). On the other hand, aspartate and GABA concentrations in the SC and dLGN exhibited no significant reduction after deafferentations. These results indicate that the retino-tectal and retino-geniculate pathway of the rat may be glutamatergic in nature.

Postsynaptic potentials (PSPs) were recorded on the surface of the rat in the superior colliculus (SC) after electrical stimulation of the optic nerve. Long-term potentiation (LTP) was elicited in the PSP by tetanic stimulation of the optic nerve either when the ipsilateral visual cortex was removed or when a GABA antagonist, picrotoxin, was administered before the tetanic stimulation. These results suggest that the corticotectal tract may inhibit the appearance of LTP via the GABAergic interneurons within the superficial gray layer (SGL).

After electrical stimulation of the optic layer (OL) of superior colliculus (SC) slices, the postsynaptic potential (PSP) was recorded in the superficial gray layer (SGL) of the SC. The degeneration studies of retinotectal or corticotectal inputs to the SGL of the SC indicated that this PSP evoked in the SGL of the SC slices was retinotectal in origin. Neurotransmission in this pathway may be mediated by glutamate, because the PSP amplitude was reduced and blocked by application of kynurenate or quinoxaline dione (DNQX) to the medium. Furthermore, the concentration of glutamate in the right SGL was significantly reduced by 32% after left optic denervation and by 30% after ablation of the right visual cortex, compared with that in the left SGL. Long-term potentiation (LTP) in the SGL was induced by tetanic stimulation (50 Hz, 20 s) to the OL. The LTP formation was facilitated by the removal of Mg2+ from the medium. The effects of glutamate antagonistsd-amino-5-phosphonovalerate (D-APV), γ-d-glutamylglycine (γ-DGG), and (+)-5-methyl-10,11-dihydro-5H-dibenzo, a,d-cycloheptene-5,10-imine maleate (MK-801) on the induction of LTP were investigated. D-APV (100 μM) or γ-DGG (1 mM) masked the expression of LTP by tetanic stimulation, however LTP was induced after removal of the agents. LTP formation was observed without further tetanic stimulation following the removal of D-APV from the medium even 80 min after the tetanic stimulation. LTP once formed was not influenced by application of D-APV. The effects of D-APV on the LTP formation suggest the possibility that theN-methyl-d-aspartate (NMDA) receptor is involved in the expression of LTP but it does not contribute to the initiation and the maintenance of LTP.

Using guinea pig hippocampal slice preparations, the effect of temperature on the electrical activity and the protective effect of hypothermia against deprivation of both oxygen and glucose were studied by recording field potentials of pyramidal cell layer (CA 3–4 area) and by measuring the content of adenosine triphosphate (ATP), phosphocreatine (PCr) and lactate of each slice. Cooling the perfusion medium from 37 to 21 °C caused a decrease in the amplitude of field potentials, although the amplitude increased (120%) transiently at around 33 °C. The electrical activity ceased at around 22 °C. When the temperature was raised from 21 to 37 °C, the activity recovered reversibly. However, when the temperature was raised to above 38 °C, the amplitude decreased and disappeared irreversibly at 42 °C. During deprivation, energy consumption (total ∼P used;2 × ΔATP+ ΔPCr+ 1.3 × Δlactate) was suppressed by lowering the tissue temperature and the initial (0–2.5 min deprivation) energy use rate was calculated to be 42.2 at 37°C, 22.8 at 28 °C and 7.0 at 21 °C (∼P m mol/kg protein/min), respectively. From these values, Q10 was estimated to be 2.05. With regard to the protective effect of hypothermia, the critical survival time (period of deprivation of oxygen and glucose for the complete recovery in neural activity and the level of high energy phosphates) was 10 min at 37 °C, 15 min at 28 °C, and 45 min at 21 °C, respectively. These results indicate that moderate hypothermia (28 °C) is not very effective in protecting the brain tissue against deprivation of oxygen and glucose, and that deep hypothermia (21 °C) has a much more potent protective effect against deprivation from the functional and metabolic viewpoint.

The effects of excitatory amino acids such as glutamate (Glu) and aspartate (Asp), and their receptor agonists, kainate (Ka), N-methyl-D-aspartate (NMDA), and quisqualate (Quis) on the neuronal activity and the oxygen consumption were investigated using hippocampal slices of the guinea pig. Bath application of these excitants elevated the amplitude of the postsynaptic field potential (PSP) to approximately 120% of the original level at low concentrations, although effective doses varied for the different excitants (Ka greater than NMDA greater than Quis greater than Glu greater than Asp). At concentrations over each effective dose the PSP was diminished and subsequently abolished. The application of Ka (1 x 10(-8) to 1 x 10(-6) M), NMDA (1 x 10(-8) to 1 x 10(-4) M), Quis (1 x 10(-7) to 1 x 10(-4) M), Glu (1 x 10(-5) to 5 x 10(-4) M), and Asp (1 x 10(-5) to 5 x 10(-3) M) enhanced the oxygen consumption dose-dependently, to a maximum of 120-146% of the resting level (8.43 mumol/g protein/min). It was notable that the initial increase in the oxygen consumption was associated with neuronal excitation and the doses of the excitants producing maximal oxygen consumption was in good agreement with those demonstrating disappearance of the PSP, probably because of massive depolarization of the neurones. The increase of the oxygen consumption and the neuronal activity induced by the excitants were specifically inhibited by their antagonists, such as glutamic acid diethylester (GDEE), DL-2-amino-5-phosphonovaleric acid (APV), and Joro spider toxin (JSTX). The present results strongly suggest that the enhancement of oxygen consumption due to the excitatory amino acids and agonists must reflect the neuronal activation induced by them.

It is well known that during generalized seizures induced by antivitamin B6 agents, such as hydrazides, as well as by direct antagonists of pyridoxine, such as desoxypyridoxine, the GABA concentration is reduced in the whole brain2, 23. The same is true in the vicinity of the human epileptic focus z2. However, previous studies have not established if the GABA reduction is the primary cause of the seizure, i.e. whether the decrease in GABA content occurs prior to the onset of the seizure or in the course of the seizure. Comparing the changes taking place during the preictal period of drug induced seizures with those of the ictal period may answer this problem. A thorough study of the preictal alterations necessitates a regional investigation for two reasons. First, the GABA level differs among brain regions 6,16 in this respect the substantia nigra with its high GABA content is very interesting and second, the susceptibility towards seizure discharges differs among brain regions in this respect the hippocampus, one of the most seizure prone regions of the brain 1°,2°, earns special regard. This led us to determine the GABA content in 11 different regions of the rabbit brain during the preictal and ictal period of generalized seizure discharges. For this purpose it is necessary to use a convulsant which induces seizures after a constant latency, so that the moment at which the seizure starts can be predicted. This requirement is fulfilled by the pyridoxine-analog methoxypiridoxine (MP), a convulsant agent described by Purpura and Gonzales-Monteagudo in 195919, which reduces GABA content in cat cortex after systemic application 18. An intravenous administration of 100 mg/kg MP to immobilized rabbits induced generalized seizure discharges starting constantly after 22-28 min as checked by EEG recordings 7. This made it possible to analyze precisely changes occurring during the preictal period. The MP-induced sustained epileptic discharges lasted throughout the experiment for 2 h and longer. To avoid side-effects such as dyspnoea and anoxia, which may also alter the GABA content, all rabbits were artificially respirated and immobili

The laminar distribution of gamma-aminobutyric acid (GABA) was studied in certain structures of the visual system of the adult cat. A microassay method to measure GABA (10(-12) mol) was established using enzymatic cycling of NADP-NADPH. In the dorsal lateral geniculate nucleus, GABA concentration was highest in lamina A (average concentration 23 mmol/kg dry weight) and lowest in lamina C. In the visual cortex (Areas 17 and 18), the concentration of GABA was 10-12 mmol/kg dry weight in layers I-IV and 5-8 mmol/kg dry weight in layers V and VI. No significant difference was found in the GABA distribution in Areas 17 and 18. In the superior colliculus, the highest level of GABA was found in the upper part of the superficial gray layer (40 mmol/kg dry weight), whereas the deep layers contained GABA at a concentration of 23-28 mmol/kg dry weight. The results of the GABA distribution measurements revealed an orderly, layer-specific disposition of the neurotransmitter in the cat visual system. GABA may play an important role in the function of the visual system.

The effects of intravenous injection of cholinergic or anticholinergic agents in inducing the PS state were studied in acute cats with the midbrain transected. Preparations were made by precollicular decerebrations (mesencephalic cat) and by transsection at the prepontine level (prepontine preparation). The intravenous administration of physostigmine sulfate (0.05–0.1 mg/kg) induced PS state in the mesencephalic cat and prepontine preparation. The PS state appeared with a delay of 1.5–7 min after injection, and continued for about 20 min. TEPP (0.05 mg/kg), DDVP (0.5 mg/kg) and pilocarpine hydrochloride (0.2 mg/kg) were also effective for induction of PS state in these preparations. On the other hand, acetylcholine chloride, neostigmine methylsulfate and bethanechol chloride had no effect. Atropine sulfate suppressed the effect of physostigmine, TEPP, DDVP and pilocarpine in inducing PS state, while atropine methylnitrate had no such suppressive effect. Intravenous injection of physostigmine or TEPP provoked EEG arousal in the forebrain of prepontine preparation. The mechanisms of PS state induction are discussed.