beta-Hydroxybutyrate fuels synaptic function during development. Histological and physiological evidence in rat hippocampal slices.

To determine whether ketone bodies sustain neuronal function as energy substrates, we examined the effects of beta-hydroxybutyrate (betaHB) on synaptic transmission and morphological integrity during glucose deprivation in rat hippocampal slices. After the depression of excitatory postsynaptic potentials (EPSPs) by 60 min of glucose deprivation, administration of 0.5-10 mM D-betaHB restored EPSPs in slices from postnatal day (PND) 15 rats but not in slices from PND 30 or 120 rats. At PND 15, adding D-betaHB to the media allowed robust long-term potentiation of EPSPs triggered by high frequency stimulation, and prevented the EPSP-spike facilitation that suggests hyperexcitability of neurons. Even after PND 15,D-betaHB blocked morphological changes produced by either glucose deprivation or glycolytic inhibition. These results indicate that D-betaHB is not only able to substitute for glucose as an energy substrate but is also able to preserve neuronal integrity and stability, particularly during early development.

Oxygen deprivation produces delayed inhibition of long-term potentiation by activation of NMDA receptors and nitric oxide synthase.

The acute and delayed effects of anoxia on synaptic transmission and long-term potentiation (LTP) were examined in the CA1 region of rat hippocampal slices. Oxygen deprivation for 20 minutes completely but reversibly depressed excitatory postsynaptic potentials mediated by both N-methyl-D-aspartate receptors (NMDAR) and non-NMDAR. Although LTP was reliably produced by a single tetanus delivered 30 minutes after reoxygenation, LTP could not be induced when a tetanus was delivered 70 to 100 minutes after reoxygenation. A tetanus delivered 100 minutes after reoxygenation produced lasting synaptic enhancement when 100 mumol/L D,L-amino-phosphonovaleric acid (APV), a competitive NMDAR antagonist, was administered during the period of oxygen deprivation. The delayed effects of oxygen deprivation were not blocked when APV was administered after oxygen deprivation. Similarly, the delayed effects on LTP induction were overcome by inhibitors of nitric oxide synthase when the nitric oxide synthase inhibitors were administered during anoxia, but not when administered after oxygen deprivation. These results suggest that untimely activation of NMDAR and nitric oxide release during anoxia produce delayed inhibition of LTP induction and may be involved in the memory defects that occur subsequent to cerebral hypoxia.

Effects of fructose-1,6-bisphosphate on morphological and functional neuronal integrity in rat hippocampal slices during energy deprivation.

D-fructose-1,6-bisphosphate, a high energy glycolytic intermediate, attenuates ischemic damage in a variety of tissues, including brain. To determine whether D-fructose-1,6-bisphosphate serves as an alternate energy substrate in the CNS, rat hippocampal slices were treated with D-fructose-1,6-bisphosphate during glucose deprivation. Unlike pyruvate, an endproduct of glycolysis, 10 mM D-fructose-1,6-bisphosphate did not preserve synaptic transmission or morphological integrity of CA1 pyramidal neurons during glucose deprivation. Moreover, during glucose deprivation, 10-mM D-fructose-1,6-bisphosphate failed to maintain adenosine triphosphate levels in slices. D-fructose-1,6-bisphosphate, however, attenuated acute neuronal degeneration produced by 200 microM iodoacetate, an inhibitor of glycolysis downstream of D-fructose-1,6-bisphosphate. Because (5S, 10R)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine, an antagonist of N-methyl-D-aspartate receptors, exhibited similar protection against iodoacetate damage, we examined whether (5S, 10R)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine and D-fructose-1,6-bisphosphate share a common neuroprotective mechanism. Indeed, D-fructose-1,6-bisphosphate diminished N-methyl-D-aspartate receptor-mediated synaptic responses and partially attenuated neuronal degeneration induced by 100-microM N-methyl-D-aspartate. Taken together, these results indicate that D-fructose-1,6-bisphosphate is unlikely to serve as an energy substrate in the hippocampus, and that neuroprotective effects of D-fructose-1,6-bisphosphate are mediated by mechanisms other than anaerobic energy supply.

Effects of lactate and pyruvate on glucose deprivation in rat hippocampal slices.

Rat hippocampal slices were used to evaluate the effects of glucose deprivation and the ability of lactate or pyruvate to preserve histological integrity and synaptic function. Dark cell changes were observed during 180 min incubations in glucose-free solutions. These changes were blocked by substituting 10 mM lactate or pyruvate for glucose during the incubation. Excitatory postsynaptic potentials disappeared during 60 min of glucose deprivation but were restored by subsequent introduction of glucose, lactate or pyruvate. Incubation of slices with iodoacetate revealed a distinct pattern of damage that was blocked completely by pyruvate and partially by lactate. These results indicate that exogenous pyruvate and lactate can serve as energy substrates in the hippocampus when glucose is unavailable or glycolytic metabolism is impaired.

Lactate dehydrogenase release is facilitated by brief sonication of rat hippocampal slices and isolated retinas following acute neuronal damage.

Although useful for determining neuronal damage in cell cultures, the lactate dehydrogenase (LDH) assay is not suitable for acute brain preparations because LDH release is typically delayed relative to neuronal deterioration. The slow release suggests that LDH may remain trapped inside damaged cells until late in the degenerative process. To test this, we examined whether brief sonication facilitates LDH release from acutely damaged neurons. In rat isolated retinas and hippocampal slices, LDH release was minimal following acute administration of iodoacetate or kainate. However, these toxins promoted significant LDH release, when toxin exposure was followed by brief sonication. Increases in extracellular LDH correlated with changes in neuronal morphology. These findings suggest that sonication may facilitate the use of the LDH assay in acute brain preparations.

An ex vivo rat retinal preparation for excitotoxicity studies.

Although the isolated chicken embryo retina has been a very useful in vitro preparation for studying mechanisms of excitotoxicity, it is an avian rather than mammalian tissue and its embryonic age makes it unsuitable for a full range of developmental and aging studies. Therefore, we have explored the feasibility of using the rat retina at various ages for in vitro excitotoxicity studies. In this model, retinal segments were isolated in artificial cerebrospinal fluid (CSF) at 5 degrees C then incubated under various conditions at 30 degrees C and assessed histologically for signs of neurodegenerative changes. Retinal segments from 7-, 30-, 120- and 660-day-old rats incubated in CSF for 3 h and from 30-day-old rats incubated for 24 h retained a normal histological appearance. Thus, this preparation is suitable for in vitro studies pertaining to either acute or delayed excitotoxic phenomena in the mammalian CNS at any age from infancy to old age. Excitotoxin agonist experiments in the 30-day-old rat retina revealed the surprising result that the non-NMDA agonists, kainate and AMPA, at a low concentration (100 microM) damaged a much larger number of retinal neurons than NMDA did at a very high concentration (10 mM).

Nitric oxide inhibitors attenuate N-methyl-D-aspartate excitotoxicity in rat hippocampal slices.

To investigate whether nitric oxide (NO) plays a role in the neurotoxicity produced by N-methyl-D-aspartate (NMDA) we have examined the effects of NO inhibitors on NMDA-mediated neurodegeneration in the CA1 region of rat hippocampal slices. L-NG-Monomethylarginine, L-NG-nitroarginine and hemoglobin markedly diminished the toxicity produced by activation of NMDA receptors without interfering with NMDA receptor-mediated ion currents or synaptic responses. The neuroprotective effects are reversed by coapplication of L-arginine with the NO synthase inhibitors. These results suggest that activation of the NO system is an important component of the biochemical cascade leading to neurodegeneration produced by NMDA receptors.

Nitric oxide inhibitors attenuate ischemic degeneration in the CA1 region of rat hippocampal slices.

We examined the involvement of nitric oxide (NO) in ischemic brain damage using hippocampal slices prepared from 30 day old albino rats and exposed to 20 min of oxygen/glucose deprivation (ischemia) followed by 90 min postincubation in oxygen- and glucose-containing media. Damage in the CA1 region was rated on a 0 (intact) to 4 (severe neuronal damage) scale by a rater blind to the experimental condition. Control slices exposed to ischemia were rated as 2.8 +/- 0.4 (N = 12). L-NG-Monomethylarginine (100 microM) and L-NG-nitroarginine (100 microM), non-selective NO synthase (NOS) inhibitors, diminished ischemic damage (0.6 +/- 0.3, N = 8, and 1.0 +/- 0.5, N = 4, respectively). An inhibitor of brain NOS, 7-nitroindazole (30 microM), was also effective against ischemic degeneration (0.7 +/- 0.3, N = 5). These results suggest that activation of NOS is involved in ischemic degeneration in the CA1 region.

Endogenous monocarboxylates sustain hippocampal synaptic function and morphological integrity during energy deprivation.

The ability to fuel neurons via glycogenolysis is believed to be an important function of glia. Indeed, the slow, rather than immediate, depression of synaptic transmission in hippocampal slices during exogenous glucose deprivation suggests that intrinsic energy reservoirs help to sustain neurotransmission. It is believed that glia fuel neighboring neurons via diffusible monocarboxylates such as pyruvate and lactate, although a role for glucose has been proposed also. Using alpha-cyano-4-hydroxycinnamate (4-CIN) to inhibit monocarboxylate transport and cytochalasin B (CCB) to inhibit glucose transport, we examined the role of glucose and monocarboxylates in supporting the functional and morphological integrity of hippocampal neurons during glucose deprivation. Although 200 microM 4-CIN failed to depress EPSPs supported by 10 mM glucose, pretreatment with 4-CIN accelerated the depression of EPSPs during glucose deprivation. 4-CIN also accelerated the decline in glucose-supported EPSPs after administration of 50 microM CCB, whereas CCB failed to alter the slow decay of pyruvate-supported EPSPs during pyruvate deprivation. 4-CIN did not alter the morphology of pyramidal neurons in the presence of 10 mM glucose but produced significant damage during glucose deprivation or CCB administration. These results suggest that endogenous monocarboxylates rather than glucose maintain neuronal integrity during energy deprivation. Furthermore, EPSPs supported by 2-3.3 mM glucose were sensitive to 4-CIN, suggesting that endogenous monocarboxylates are involved in maintaining neuronal function even under conditions of mild glucose deprivation.

Müller cell swelling, glutamate uptake, and excitotoxic neurodegeneration in the isolated rat retina.

We characterized morphological effects of the endogenous excitotoxin, glutamate in ex vivo retinal segments prepared from 30-day-old rats. Initial changes induced by glutamate consisted of reversible, sodium-dependent Müller cell swelling. This glial swelling was mimicked by glutamate transport substrates but not by ionotropic glutamate receptor agonists. Only very high concentrations of exogenous glutamate (3,000 microM) produced excitotoxic neuronal damage. The neuronal damage was accompanied by severe glial swelling and was blocked by an antagonist of non-N-methyl-D-aspartate (NMDA) receptors but not by an NMDA receptor antagonist. Because glutamate uptake can be influenced by changes in cellular energy levels, we studied the effects of oxidative and glycolytic energy depletion on glutamate-mediated Müller cell swelling. Oxygen deprivation produced little morphological change and did not alter either glutamate-mediated Müller cell swelling or glutamate-induced excitotoxicity. In contrast, inhibition of glycolysis by iodoacetate produced severe neuronal damage without Müller cell swelling. In the presence of iodoacetate, exogenous glutamate failed to cause glial swelling. The neuronal damage produced by iodoacetate was inhibited by pyruvate, a substrate that sustains oxidative energy pathways. In the presence of iodoacetate plus pyruvate, glutamate failed to cause Müller cell swelling but became neurotoxic at low concentrations through activation of non-NMDA receptors. These results indicate that glycolytic energy metabolism plays a critical role in sustaining ionic balances required for Müller cell glutamate uptake and glial uptake helps to prevent glutamate-mediated excitotoxicity.

Neurotoxic effects of sodium nitroprusside in rat hippocampal slices.

To investigate the possible role of nitric oxide (NO) in n-methyl-D-aspartate (NMDA)-induced neurotoxicity we examined the effects of sodium nitroprusside (SNP) in rat hippocampal slices. Incubation with 3 mM SNP for up to 2 h produced neuronal damage characterized by "nuclear ballooning" in CA3 and CA1 pyramidal neurons and interneurons. The nuclear ballooning was not blocked by lowering the external calcium concentration or coincubation with MK-801 and/or 6,7-dinitro-quinoxaline-2,3-dione. Two lines of evidence suggest that the SNP neurotoxicity is not mediated by NO. First, inactivation of SNP by UV light failed to prevent the nuclear ballooning. Second, the toxicity was not attenuated by coadministration of hemoglobin. Although cyanide can be released from SNP, potassium cyanide did not mimic the nuclear ballooning. In electrophysiological experiments, 100 microM SNP irreversibly diminished the CA1 population spike amplitude while having less effect on the field excitatory postsynaptic potential. The effect of SNP on population spikes was inhibited by hemoglobin and was not mimicked by light-inactivated SNP. These results suggest that active SNP and light-treated SNP have different effects on hippocampal neurons and that SNP induces damage that differs from that produced by NMDA or cyanide.

Ketamine, phencyclidine, and MK-801 protect against kainic acid-induced seizure-related brain damage.

Recent evidence implicates the endogenous excitotoxin, glutamate (Glu), in several neurologic disorders, including seizure-related brain damage. Ketamine, phencyclidine, and MK-801, which are noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) subtype of Glu receptor (but do not antagonize kainic acid receptors) were tested in the present study for their effects on behavioral and/or electrographic seizures and seizure-related brain damage induced by kainic acid. Behavioral seizure activity was reduced by these agents, as was spread of electrographic seizures to neocortex, but seizures recorded from deep brain regions such as hippocampus, piriform cortex, and amygdala were not significantly diminished. All three agents prevented seizure-related brain damage in the amygdala, piriform cortex, thalamus, and CA1 region of the hippocampus but conferred little or no protection in the lateral septum and CA3 region of the hippocampus. The regional selectivity of the neuroprotective effect suggests that NMDA receptors may play a more dominant role in seizure-related brain damage in some brain regions than in others. The ability of NMDA antagonists to prevent seizure-related damage in several brain regions without suppressing seizure activity suggests that in these brain regions persistent seizure activity can be maintained by other transmitter systems, with or without NMDA receptor participation, but that seizure-related brain damage is critically dependent on NMDA receptor participation.

Comparison of rat retinal fixation techniques: chemical fixation and microwave irradiation.

In histological studies using retinas, eyes are commonly fixed with aldehyde derivatives administered by immersion or perfusion. However, the histology of rat retinas chemically fixed as a whole eye is typically inferior to the histology of retinas that are immediately fixed after acute dissection from the rest of the eye. Chemical fixation without dissection often results in neuronal swelling resembling excitotoxic damage induced by ischemia because the retina is protected by the sclera and is thus poorly accessible to immersion or perfusion fixation techniques. In order for the acute dissection technique to work properly, it must be completed in a timely manner, which may be difficult under some circumstances. Microwave irradiation is an alternative method for fixing tissues that are inaccessable to chemicals. We examined the effectiveness of microwave irradiation of the whole eye as a substitute for acute retinal dissection. To study the feasibility of microwave methods, we compared retinal morphology using microwave irradiation to morphology using conventional immersion fixation methods. Eyes were removed from rats, placed in a container with 2 or 20 ml artificial cerebrospinal fluid (aCSF) and irradiated with a household microwave oven. For morphological comparison, control eyes were immersed in a chemical fixative containing 1% paraformaldehyde and 1.5% glutaraldehyde. All eyes were embedded in araldite for evaluation by light microscopy. Retinal segments acutely isolated before immersion fixation revealed intact histology whereas retinal segments exposed to 60 min of simulated ischemia showed severe neuronal degeneration. Using an immersion technique, the retinas of chemically fixed whole eyes showed neuronal swelling similar to excitotoxic ischemic damage, suggesting that conventional immersion methods provide poor whole eye fixation. The neuronal degeneration observed with conventional immersion fixation was not found in retinas of whole eyes fixed with 20 sec of microwave irradiation. During microwave irradiation the temperature in the bathing aCSF rose to 55-72 degrees C. In some eyes, overcooking produced chromatin clumping and a small loss of contrast in staining. Although nuclear clumping and diminished staining occasionally result from overcooking, ischemic damage is well controlled with microwave fixation of enucleated eyes. When the optimal conditions are defined, microwave fixation may be preferable for retinal histology if chemical fixation following acute dissection is not feasible.

Swelling of Müller cells induced by AP3 and glutamate transport substrates in rat retina.

Previous studies have shown that a single systemic injection of 2-amino-3-phosphonopropionate (AP3), an agonist/antagonist at metabotropic glutamate receptors, produces marked swelling of rodent Müller cells. To investigate the effects of AP3 on Müller cells, we used in vitro retinal segments prepared from 30 day old rats. Incubation with AP3 for 1 h or more caused severe swelling of Müller cells with the appearance of mitotic cellular profiles in the outer nuclear layer. The Müller cell swelling was mimicked by substrates for glutamate transporters, suggesting that AP3 may produce its effects via transport into glial cells. To determine whether AP3 is a substrate for glutamate transporters, we studied cultured rat hippocampal astrocytes using whole-cell patch clamp recordings. In hippocampal astrocytes, AP3 activated currents via an Na(+)-dependent glutamate transporter. Consistent with this, substitution of extracellular sodium with choline blocked Müller cell swelling in the rat retina. These results indicate that the acute glial swelling produced by AP3 results primarily from a fluid shift that accompanies the transport of AP3 and sodium into Müller cells.

Age dependent sensitivity of the rat retina to the excitotoxic action of N-methyl-D-aspartate.

We have found that the rat retina can be isolated atraumatically and incubated ex vivo for up to 24 h without showing signs of histological deterioration, and that retinas from adult or aged rats can be isolated as successfully as those from immature rats. In the present study we used this preparation to show that rat retinal neurones at postnatal day zero (PND 0) are relatively insensitive to the excitotoxic action of the glutamate agonist, N-methyl-D-aspartate (NMDA), then gradually show increasing sensitivity that peaks at about PND 9 and declines from PND 15-30 after which it remains at a low level up to the last time point studied (10 months of age). This is consistent with other developmental NMDA receptor data and underscores the need for caution in using immature in vitro central; nervous system (CNS) tissue preparations as a basis for interpreting the role of NMDA receptors in adult neurological diseases.