Dendrite classification in rat hippocampal neurons according to signal propagation properties. Observation by multichannel optical recording in cultured neuronal networks

Two-dimensional neuronal networks were formed using a dissociated culture of rat hippocampal neurons on glass plates. Neural activity in response to pulse stimuli applied to the neurons by whole-cell clamp electrodes was observed with a 128-channel optical recording apparatus using a voltage-sensitive dye, RH482. Dendrites emerging from the somata of single neurons were classified according to two signal-transmission properties, those with lower conduction velocities (0.12+/-0.034 m/s, n=24) and those with very fast conduction velocity (faster than 1.0 m/s), by evaluating the conduction velocities of pulse responses. The distinction between these two properties seemed to be related to the morphological differences in input connectivity with the axons of neighboring neurons.     

Intraneuronal [Ca2+] changes induced by 2-deoxy-D-glucose in rat hippocampal slices

Temporary replacement of glucose by 2-deoxyglucose (2-DG; but not sucrose) is followed by long-term potentiation of CA1 synaptic transmission (2-DG LTP), which is Ca2+-dependent and is prevented by dantrolene or N-methyl--aspartate (NMDA) antagonists. To clarify the mechanism of action of 2-DG, we monitored [Ca2+]i while replacing glucose with 2-DG or sucrose. In slices (from Wistar rats) kept submerged at 30 degreesC, pyramidal neurons were loaded with [Ca2+]-sensitive fluo-3 or Fura Red. The fluorescence was measured with a confocal microscope. Bath applications of 10 mM 2-DG (replacing glucose for 15 +/- 0.38 min, means +/- SE) led to a rapid but reversible rise in fluo-3 fluorescence (or drop of Fura Red fluorescence); the peak increase of fluo-3 fluorescence (DeltaF/F0), measured near the end of 2-DG applications, was by 245 +/- 50% (n = 32). Isosmolar sucrose (for 15-40 min) had a smaller but significant effect (DeltaF/F0 = 94 +/- 14%, n = 10). The 2-DG-induced DeltaF/F0 was greatly reduced (to 35 +/- 15%, n = 16) by,-aminophosphono-valerate (50-100 microM) and abolished by 10 microM dantrolene (-4.0 +/- 2.9%, n = 11). A substantial, although smaller effect, of 2-DG persisted in Ca2+-free 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) medium. Two adenosine antagonists, which do not prevent 2-DG LTP, were also tested; 2-DG-induced DeltaF/F0 (fluo-3) was not affected by the A1 antagonist 8-cyclopentyl-3, 7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX 50 nM; 287 +/- 38%; n = 20), but it was abolished by the A1/A2 antagonist 8-SPT; 25 +/- 29%, n = 19). These observations suggest that 2-DG releases glutamate and adenosine and that the rise in [Ca2+] may be triggered by a synergistic action of glutamate (acting via NMDA receptors) and adenosine (acting via A2b receptors) resulting in Ca2+ release from a dantrolene-sensitive store. The discrepant effects of sucrose and 8-SPT on DeltaF/F0, on the one hand, and 2-DG LTP, on the other, support other evidence that increases in postsynaptic [Ca2+]i are not essential for 2-DG LTP.     

Epileptiform activity induced by low Mg2+ in cultured rat hippocampal slices

Organotypic cultured slices of the rat hippocampus undergo synaptic reorganization. Besides the establishment of reciprocal connections between area CA1 and the dentate gyrus (DG), collateral excitatory connections between granule cells are formed which are similar to those appearing in several epilepsy models and in the DG from patients with temporal lobe epilepsy. We studied the characteristics of epileptiform activity induced by low Mg2+ perfusion in cultured hippocampal slices using extra- and intracellular recordings. With low Mg2+ perfusion synchronous seizure like events (SLEs) were readily observed in the DG and areas CA3 and CA1. Also, the isolated DG was able to display seizure like activity. Intracellular recordings revealed long lasting depolarization shifts in granule cells of the DG and pyramidal cells of areas CA3 and CA1. The SLEs, lasting 2-3 s, could be recorded for at least 3 h in areas CA1 and CA3. However, approximately an hour after perfusion with low Mg2+, the epileptiform activity disappeared in the DG and responses to single pulse hilar stimulation progressively deteriorated. These responses returned to control values 1 week after reincubating the cultures. Interestingly, no deterioration of stimulus induced responses was observed in the isolated DG after exposure to low Mg2+.     

Multiple actions of methohexital on hippocampal CA1 and cortical neurons of rat brain slices

To explore the mechanism by which methohexital (MTH) activates epileptiform activity in patients with epilepsy, we examined the effects of MTH on hippocampal CA1 and neocortical neurons via extracellular and whole-cell patch-clamp recordings in rat brain slices. Perfusion of slices with 10 to 100 microM MTH caused no significant change in glutamatergic transmission in the hippocampal CA1 region, but enhanced gamma-aminobutyric acid (GABA)A-mediated inhibitory postsynaptic currents and induced spontaneous inhibitory postsynaptic currents in neocortical and hippocampal CA1 neurons. In addition, MTH induced a tonic, bicuculline-sensitive hyperpolarization in association with increases in membrane conductance, suggesting a direct stimulation of GABAA receptors by MTH. Spontaneous epileptiform activity was not observed in the neocortex and hippocampus after exposure of slices to MTH, neither in the standard in vitro condition nor in the presence of 4-aminopyridine, which promotes rhythmic synaptic activities. We suggest that the activation of epileptiform activity in vivo by MTH may result from increased neuronal synchrony via the potentiation of GABAA-mediated synaptic inhibition.     

Age-dependent changes of presynaptic neuromodulation via A1-adenosine receptors in rat hippocampal slices

The presynaptic neuromodulation of stimulation-evoked release of [3H]-acetylcholine by endogenous adenosine, via A1-adenosine receptors, was studied in superfused hippocampal slices taken from 4-, 12- and 24-month-old rats. 8-Cyclopentyl-1,3-dimethylxanthine (0.25 microM), a selective A1-receptor antagonist, increased significantly the electrical field stimulation-induced release of [3H]-acetylcholine in slices prepared from 4- and 12-month-old rats, showing a tonic inhibitory action of endogenous adenosine via stimulation of presynaptic A1-adenosine receptors. In contrast, 8-cyclopentyl-1,3-dimethylxanthine had no effect in 24-month-old rats. 2-Chloroadenosine (10 microM), an adenosine receptor agonist decreased the release of [3H]-acetylcholine in slices taken from 4- and 12-month-old rats, and no significant change was observed in slices taken from 24-month-old rats. In order to show whether the number/or affinity of the A1-receptors was affected in aged rats, [3H]-8-cyclopentyl-1,3-dimethylxanthine binding was studied in hippocampal membranes prepared from rats of different ages. Whereas the Bmax value was significantly lower in 2-year-old rats than in younger counterparts, the dissociation constant (Kd) was not affected by aging, indicating that the density rather than the affinity of adenosine receptors was altered. Endogenous adenosine levels present in the extracellular space were also measured in the superfusate by high performance liquid chromatography (HPLC) coupled with ultraviolet detection, and an age-related increase in the adenosine level was found. In summary, our results indicate that during aging the level of adenosine in the extracellular fluid is increased in the hippocampus. There is a downregulation and reduced responsiveness of presynaptic adenosine A1-receptors, and it seems likely that these changes are due to the enhanced adenosine level in the extracellular space.     

NMDA antagonist displays anticonvulsant effect via NO synthesis inhibition in penicillin-treated rat hippocampal slices

The present study investigated the role of nitric oxide (NO) in epileptogenesis and whether this role correlated with ionotropic glutamate receptor (IGR). Using a self-constructed NO-sensitive microelectrode (SNM), we observed the effect of nitric oxide synthase (NOS) inhibitors, NMDA and non-NMDA selective antagonists on penicillin(PEN)-treated hippocampal slices by simultaneously recording evoked field potentials and nitric oxide release from CA1 pyramidal neurons. 7-nitroindazole (7-NI),Nomega-nitro-L-arginine (L-NNA) and DL-2-amino-phospho-novaleric acid (APV), but not 6,7-dinitroquinoxaline-2,3 (1h,4h)-dione(DNQX), depressed NO release and partly reversed PEN's epileptogenetic effect, while APV + 7-NI + L-NNA did not display a further inhibitory effect. These findings suggest both NOS inhibitor and NMDA antagonist involve as anticonvulsant factors in epileptogenesis, providing direct evidence for NO release in response to NMDA receptor activation. The anticonvulsant effect of NMDA antagonist may ascribe to its action on NO release.     

Attenuation of hypoxic current by intracellular applications of ATP regenerating agents in hippocampal CA1 neurons of rat brain slices

Hypoxia-induced outward currents (hyperpolarization) were examined in hippocampal CA1 neurons of rat brain slices, using the whole-cell recording technique. Hypoxic episodes were induced by perfusing slices with an artificial cerebrospinal fluid aerated with 5% CO2/95% N2 rather than 5% CO2/95% O2, for about 3 min. The hypoxic current was consistently and reproducibly induced in CA1 neurons dialysed with an ATP-free patch pipette solution. This current manifested as an outward shift in the holding current in association with increased conductance, and it reversed at -78 +/- 2.5 mV, with a linear I-V relation in the range of -100 to -40 mV. To provide extra energy resources to individual neurons recorded, agents were added to the patch pipette solution, including MgATP alone, MgATP + phosphocreatine + creatine kinase, or MgATP + creatine. In CA1 neurons dialysed with patch solutions including these agents, hypoxia produced small outward currents in comparison with those observed in CA1 neurons dialysed with the ATP-free solution. Among the above agents examined, whole-cell dialysis with MgATP + creatine was the most effective at decreasing the hypoxic outward currents. We suggest that the hypoxic hyperpolarization is closely related to energy metabolism in individual CA1 neurons, and that the energy supply provided by phosphocreatine metabolism may play a critical role during transient metabolic stress.     

Frequency-dependent inhibition of neuronal activity by topiramate in rat hippocampal slices

1. Topiramate is a structurally novel anticonvulsant which was recently approved for adjunctive therapy in partial and secondarily generalized seizures. The present study was aimed at elucidating the mechanisms underlying the anticonvulsant efficacy of topiramate using intra- and extracellular recording techniques in the in vitro hippocampal slices. 2. When stimuli were delivered every 20 s, topiramate had no measurable effect on both field excitatory postsynaptic potentials (fEPSPs) and population spikes (PSs). However, increasing the stimulation frequency from 0.05-0.2 Hz, topiramate significantly decreased the slope of fEPSP and the amplitude of PS in a concentration-dependent manner. The amplitude of presynaptic fiber volley was also reduced. 3. Topiramate did not affect the magnitude of paired-pulse inhibition and monosynaptically evoked inhibitory postsynaptic potentials (IPSPs). 4. Sustained repetitive firing was elicited by injection of long duration (500 ms) depolarizing current pulses (500-800 pA). Superfusion with topiramate significantly reduced the number of action potentials evoked by a given current pulse. 5. After blockade of GABA receptors by bicuculline, burst firing which consisted of a train of several spikes riding on a large depolarizing wave termed paroxysmal depolarizing shift (PDS) was recorded. Application of topiramate reduced the duration of PDS and later spikes with less effect on the initial action potential. 6. These results suggest that frequency-dependent inhibition of neuronal activity due to blockade of Na+ channels may account largely for the anticonvulsant efficacy of topiramate.     

The effects of adenine dinucleotides on epileptiform activity in the CA3 region of rat hippocampal slices

Alpha, omega-adenine dinucleotides (Ap(n)A) consist of two adenosine molecules linked at the 5' position by phosphate groups, the number of which is denoted by n and can range from 2 to 6. The aim of this study was to investigate the effect of Ap4A and Ap5A on the rate of epileptiform activity. Hippocampal slices (450 microm), when perfused with a medium containing no added magnesium and 4-aminopyridine (50 microM), generate epileptiform activity of an interictal nature. Ap4A and Ap5A at 1 microM depressed the discharge rate to a significant extent. At this concentration adenosine (1 microM) did not produce any effect. However at 10 microM adenosine, Ap4A and Ap5A all decreased the burst frequency. Adenosine deaminase (0.2 U/ml) totally annulled the inhibition of epileptiform activity produced by 10 microM adenosine or 1 microM Ap4A and Ap5A. Adenosine deaminase did not significantly change the maximum depression of activity produced by 10 microM Ap4A and Ap5A. 8-cyclopentyl-1,3-dimethylxanthine, an A1, receptor antagonist, increased the basal rate of epileptiform activity and prevented the depression of burst discharges by Ap4A. 5'-adenylic acid deaminase converts AMP into IMP which is inactive. 5'-adenylic acid deaminase did not prevent the inhibitory effects of Ap4A. The results suggests that in the CA3 region of the hippocampus, Ap4A and Ap5A act partly by stimulating xanthine-sensitive receptors directly and partly through the formation of the metabolite, adenosine.     

Protein tyrosine kinase inhibitors reduce high-voltage activating calcium currents in CA1 pyramidal neurones from rat hippocampal slices

We have investigated the effects of protein tyrosine kinases (PTKs) inhibitors on high-threshold voltage activating (HVA) calcium currents in CA1 pyramidal neurones, whole-cell patch-clamp recorded from rat hippocampal slices. Genistein (100 microM) and tyrphostin B42 (100 microM), two PTKs inhibitors, reduced the steady-state barium current (IBa). On the other hand, daidzein and genistin (100 microM), two inactive analogues of genistein, had no effect on IBa amplitude. The inhibition induced by genistein was more pronounced at negative potentials. In order to characterize the calcium channels subtypes inhibited by PTKs inhibitors, we examined the effect of genistein in the presence of different calcium channel blockers. When L-type calcium channels were blocked by nifedipine, genistein induced a strong inhibition of the nifedipine-resistant IBa, suggesting an effect on non-L-type channels. Genistein did not antagonize the depressant effect of omega-Conotoxin-GVIA, a selective N-type calcium channel blocker, suggesting that N-type channels were not blocked by genistein. omega-Conotoxin-MVIIC (3-10 microM), a selective P/Q-type calcium channel blocker, greatly antagonized the depressant effect of genistein. Our results suggest that PTKs inhibitors reduce P-/Q-type, but not L- or N-types calcium currents in neurones of the CNS. The possible modulation of calcium channels by endogenous PTKs is discussed.     

Comparison of effects induced by toxic applications of kainate and glutamate by glucose deprivation on area CA1 of rat hippocampal slices

This study focuses on the fortuitous discovery of an atypical atherosclerotic lesion in four of 49 male adult cynomolgus monkeys (macacus fascicularis) which were maintained for a long time at a high level of hypercholesterolemia, and in seven of 19 female cynomolgus monkeys examined from the second to the 24th week of hypercholesterolemic diet: this lesion was in formation or already mature during this period of diet. This atypical lesion was formed by a collagen and elastic network surrounding synthetic smooth muscle cells without fibrofatty or fibrous plaques. Lipids were occasionally seen in the inner intima. The lesion appeared early (from the third week of diet). Once established, its morphology did not change. It became more extensive, but was not complicated by lipid overload in spite of prolonged, permanent hypercholesterolemia. This response to hypercholesterolemia is interesting because the activity of the smooth muscle cells differs from that observed in the classic lesion: they intervene earlier, their replication is very marked and rapid, their elastin secretion is greater and remains constant over time, and their phagocytic properties are reduced. This experimental study examines the installation and the maintenance of this lesion and raises the problem of its origin.     

Examination of persistent effects of repeated administration of pentylenetetrazol on rat hippocampal CA1: evidence from in vitro study on hippocampal slices

The early and long-lasting effects of pentylenetetrazol-kindling on hippocampal CA1 synaptic transmission were investigated. Experiments were carried out in the hippocampal slices from control and kindled rats at two post-kindling periods, i.e. 48-144 h (early phase) and 30-33 days (long-lasting phase). Field potentials, i.e. population excitatory postsynaptic potential (pEPSP) and population spike (PS) were recorded at the stratum pyramidale following stimulation of the stratum radiatum. Kindling-induced changes in synaptic transmission were assessed by stimulus-response functions and paired-pulse responses. The results showed that 48-144 h after kindling, the PS amplitude in the CA1 of kindled slices enhanced, and a second PS appeared compared to control slices. But at 30-33 days after kindling, the pEPSP slope in the CA1 of kindled slices enhanced without any change in the PS compared with those in the control slices. Evaluation of paired-pulse responses showed a significant reduction in paired-pulse inhibition for PS 48-144 h after kindling and a significant increase in paired-pulse inhibition for pEPSP 30-33 days after kindling. Our results suggest that pentylenetetrazol-kindling is accompanied by enhanced excitability and a reduction of paired-pulse inhibition in hippocampal CA1. The increased paired-pulse inhibition one month after kindling, may be interpreted as an adaptive process to cope with subsequent seizures.     

Neurotoxic effects of sodium nitroprusside in rat hippocampal slices

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were 1) down-regulated to basal levels (P0 and MBP mRNAs), 2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), 3) upregulated (mRNAs encoding 2'3'-cyclic nucleotide phosphodiesterase and beta-actin), or 4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons.     

Activity-induced enhancement of HB-GAM expression in rat hippocampal slices

Heparin-binding growth-associated molecule (HB-GAM) is a developmentally regulated secretory protein with neurite outgrowth-promoting activity. High-frequency stimulation leading to induction of long-term potentiation (LTP) resulted in increased expression of HB-GAM in rat hippocampal area CA1. When tetanization was given in the presence of antagonists of the N-methyl-D-aspartate (NMDA) receptor and postsynaptic voltage-gated calcium channels, the mRNA level was comparable to control levels. The results indicate that high frequency stimulation inducing LTP results in calcium-dependent enhancement in HB-GAM expression, and imply a role for this extracellular protein in the modulation of synaptic function in the hippocampus.     

Thapsigargin inhibits bicuculline-induced epileptiform excitability in rat hippocampal slices

Evoked field potentials were recorded in the CA3 region of rat hippocampal slices to detect whether intracellular Ca2+ stores are involved in the epileptiform effects of the two prototypic GABA(A) antagonists, bicuculline methiodide (BMI) and gabazine (SR-95531; GBZ). Field population spikes gradually increased and became repetitive (epileptiform bursting) in the presence of either BMI (5 microM), or GBZ (5 microM). Thapsigargin (2 microM), a depletor of intracellular Ca2+ stores, reduced the epileptiform effect of BMI, but had no significant effect on the GBZ-induced hyperexcitability. These data suggest that Ca2+ release from intracellular stores participates in the epileptiform response of hippocampal CA3 neurons to BMI, but not in the response to GBZ.     

Kindling-like state occurring on periodic increases in the extracellular K+ concentration in field CA1 in rat hippocampal slices

Transient periodic increases in the extracellular K+ concentration (20 mM, 30 sec, 3-6 episodes) led to the appearance of a kindling-like state in local neuronal networks of field CA1 of rat hippocampal slices. A criterion for the appearance of this state was a reduction in the threshold for the generation of multiple population discharges and an increase in the total number of population spikes within discharges (epileptiform activity). This state correlated with potentiation of excitatory postsynaptic potentials (EPSP) (long-term increases in pyramidal neuron excitability), but not with potentiation of glutamatergic synaptic transmission in field CA1 of hippocampal slices. The role of the various Ca2+ channels in inducing and maintaining the kindling-like state in rat hippocampal sections, evoked by periodic increases in the extracellular K+ concentration, is discussed.     

Evidence from simultaneous intracellular recordings in rat hippocampal slices that area CA3 pyramidal cells innervate dentate hilar mossy cells

1. Simultaneous intracellular recordings of area CA3 pyramidal cells and dentate hilar "mossy" cells were made in rat hippocampal slices to test the hypothesis that area CA3 pyramidal cells excite mossy cells monosynaptically. Mossy cells and pyramidal cells were differentiated by location and electrophysiological characteristics. When cells were impaled near the border of area CA3 and the hilus, their identity was confirmed morphologically after injection of the marker Neurobiotin. 2. Evidence for monosynaptic excitation of a mossy cell by a pyramidal cell was obtained in 7 of 481 (1.4%) paired recordings. In these cases, a pyramidal cell action potential was followed immediately by a 0.40 to 6.75 (mean, 2.26) mV depolarization in the simultaneously recorded mossy cell (mossy cell membrane potentials, -60 to -70 mV). Given that pyramidal cells used an excitatory amino acid as a neurotransmitter (Cotman and Nadler 1987; Ottersen and Storm-Mathisen 1987) and recordings were made in the presence of the GABAA receptor antagonist bicuculline (25 microM), it is likely that the depolarizations were unitary excitatory postsynaptic potentials (EPSPs). 3. Unitary EPSPs of mossy cells were prone to apparent "failure." The probability of failure was extremely high (up to 0.72; mean = 0.48) if the effects of all presynaptic action potentials were examined, including action potentials triggered inadvertently during other spontaneous EPSPs of the mossy cell. Probability of failure was relatively low (as low as 0; mean = 0.24) if action potentials that occurred during spontaneous activity of the mossy cell were excluded. These data suggest that unitary EPSPs produced by pyramidal cells are strongly affected by concurrent synaptic inputs to the mossy cell. 4. Unitary EPSPs were not clearly affected by manipulation of the mossy cell's membrane potential. This is consistent with the recent report that area CA3 pyramidal cells innervate distal dendrites of mossy cells (Kunkel et al. 1993). Such a distal location also may contribute to the high incidence of apparent failures. 5. Characteristics of unitary EPSPs generated by pyramidal cells were compared with the properties of the unitary EPSPs produced by granule cells. In two slices, pyramidal cell and granule cell inputs to the same mossy cell were compared. In other slices, inputs to different mossy cells were compared. In all experiments, unitary EPSPs produced by granule cells were larger in amplitude but similar in time course to unitary EPSPs produced by pyramidal cells. Probability of failure was lower and paired-pulse facilitation more common among EPSPs triggered by granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three-dimensional structure and composition of CA3-->CA1 axons in rat hippocampal slices: implications for presynaptic connectivity and compartmentalization

Physiological studies of CA3-->CA1 synaptic transmission and plasticity have revealed both pre- and postsynaptic effects. Understanding the extent to which individual presynaptic axonal boutons could provide local compartments for control of synaptic efficacy and microconnectivity requires knowledge of their three-dimensional morphology and composition. In hippocampal slices, serial electron microscopy was used to examine a nearly homogeneous population of CA3-->CA1 axons in the middle of stratum radiatum of area CA1. The locations of postsynaptic densities (PSDs), vesicles, and mitochondria were determined along 75 axon segments (9.1 +/- 2.0 micrometer in length). Synapses, defined by the colocalization of PSDs and vesicles, occurred on average at 2.7 micrometer intervals along the axons. Most varicosities (68%) had one PSD, 19% had 2-4 PSDs, and 13% had none. Synaptic vesicles occurred in 90% of the varicosities. One-half (53%) of the varicosities lacked mitochondria, raising questions about their regulation of ATP and Ca2+, and 8% of varicosities contained only mitochondria. Eleven axons were reconstructed fully. The varicosities were oblong and varied greatly in both length (1.1 +/- 0.7 micrometer) and volume (0.13 +/- 0.14 micrometer 3), whereas the intervaricosity shafts were narrow, tubular, and similar in diameter (0.17 +/- 0.04 micrometer) but variable in length (1.4 +/- 1.2 micrometer). The narrow axonal shafts resemble dendritic spine necks and thus could promote biochemical compartmentalization of individual axonal varicosities. The findings raise the intriguing possibility of localized differences in metabolism and connectivity among different axons, varicosities, and synapses.     

The neurotoxicity of sulfur-containing amino acids in energy-deprived rat hippocampal slices

The rat hippocampal slice preparation and its electrophysiology were used to assess the toxicity of two sulfur-containing amino acids, L-cysteate (CA) and L-cysteine (CYS). Both compounds were innocuous under normal conditions but became toxic in energy-deprived (lack of oxygen or glucose) slices. CA and CYS toxicity was apparent as both reduced the number of slices that normally recover their neuronal function (evoked CA1 population spike) after a standardized period of hypoxia or glucose deprivation (GD). The competitive N-methyl-D-aspartate (NMDA) antagonist DL-2-amino-5-phosphonovalerate blocked the toxicity of both CA and CYS in hypoxic slices, but it was effective only against CYS toxicity in glucose-deprived slices. The glycine antagonist 7-chlorokynurenate blocked CA and CYS toxicity in hypoxic slices but was unable to block their toxicity in glucose-deprived tissue. Perfusing slices with medium containing a high magnesium concentration blocked the toxicity of CA in both hypoxic and glucose-deprived slices, a treatment that was ineffective against CYS toxicity under either condition. Calcium depletion from the perfusion medium completely blocked the damaging effect of both amino acids in hypoxic slices, but it only partially blocked the toxicity of CA and did not block that of CYS in glucose-deprived slices. These results suggest that CA and CYS activate different NMDA receptor subsets and other glutamate receptor subtypes. Moreover, the results indicate a possible difference between the mechanism that lead to hypoxic neuronal damage and the one that lead to hypoglycemic neuronal damage.