Antagonism of the aconitine-induced inexcitability by the structurally related Aconitum alkaloids, lappaconitine and ajacine

Aconitine, lappaconitine and ajacine are structurally related alkaloids occurring in several species of the Aconitum genus. While aconitine is known to activate the voltage-dependent sodium channel, lappaconitine has been reported to block this channel. To investigate a possible antagonism of the aconitine action on neuronal activity by lappaconitine and the closely related alkaloid ajacine, we have performed extracellular recordings of stimulus evoked population spikes and field excitatory postsynaptic potential (EPSP) in rat hippocampal slices. Aconitine (10–100 nM) diminished the amplitude of the orthodromic population spike in a concentration-dependent manner. When aconitine was applied in presence of 10 μM lappaconitine, the concentration–response curve was shifted to the right. Furthermore, the complete suppression of the population spike evoked by 100 nM aconitine was reversed by 10 μM lappaconitine. The action of lappaconitine was mimicked by ajacine, however, the latter alkaloid was less potent. Both lappaconitine and ajacine shifted the input–output relationship of the presynaptic fiber spike as function of the stimulation intensity and of the field EPSP as function of the presynaptic fiber spike to the right. After pharmacological isolation, the presynaptic fiber spike was decreased by both compounds in a frequency-dependent manner indicative for a use-dependent action. Thus, electrophysiologically these alkaloids seem to inhibit predominantly the excitability of the afferent fibres and, in consequence, neurotransmission between Schaffer collaterals and the CA1 neurons, thereby suppressing the firing of the latter. Ajacine and lappaconitine inhibited stimulus-triggered epileptiform population bursts in area CA1 elicited by omission of Mg²⁺ as well as spontaneously occurring epileptiform discharges in area CA3 elicited by omission of Mg²⁺ and elevation of K⁺. It is concluded that the inhibitory and antiepileptiform effect of ajacine and lappaconitine is mediated by a frequency-dependent inhibition of the voltage-dependent sodium channel, thereby decreasing the excitability which might be important for filtering high frequency bursts of action potentials characteristic for epileptiform activity in the hippocampus. Moreover, these alkaloids are naturally occurring antagonists of the sodium channel activator aconitine.     

Heterosynaptic long-term depression is facilitated by blockade of inhibition in area CA1 of the hippocampus

Non-associative long-term depression (LTD) of the stratum radiatum input to area CA1 was studied in rat hippocampal slices. Tetanization of either the alveus or stratum oriens produced > 30min depression of the radiatum field EPSP and population spike, but generally only in the presence of picrotoxin. The spike depression was accounted for by the EPSP depression, and could be blocked by prior administration of anN-methyl-d-aspartate receptor antagonist. These data suggest that the induction of non-associative LTD is depolarization-dependent and involves theN-methyl-d-aspartate receptor/channel complex.     

Synchronization of rat hippocampal neurons in the absence of excitatory amino acid-mediated transmission

Extracellular and intracellular recordings and measurements of extracellular K⁺ concentration ([K⁺]_o) were performed in the adult rat hippocampus in an in vitro slice preparation. Excitatory amino acid receptor antagonists, as well as the K⁺-channel blockers 4-aminopyridine (4AP, 50 μM) and/or tetraethylammonium (TEA, 5 mM), were added to the bath. Synchronous, negative-going field potentials were recorded in the CA3 stratum radiatum during application of 4AP and excitatory amino acid receptor antagonists. Each of these events was associated with an intracellular long-lasting depolarization and a concomitant rise in [K⁺]_o that attained peak values of 4.3 + 0.1 mM (mean ± S.E.M., n = 6 slices) and lasted 29 ± 3 s. These field potentials were still recorded in CA3 stratum radiatum after addition of TEA. Under these conditions, prolonged field potentials (40.2 ± 4.5 s, n = 18) characterized by a prominent positive component; discharge of population spikes also occurred. [K⁺]_o, increases associated with these prolonged field-potential discharges had a considerable variability in magnitude (peak value = 3.8–14.1 mM, 6.1 ± 0.7 mM, n = 5) and duration (14–210 s; 48 ± 13 s, n = 5). In 8% of the cases spreading depression-like episodes were observed. [K⁺]_o increases during spreading depression-like episodes attained peak values of 11–27 mM (22.8 ± 0.2 mM, n = 2) and had a duration of 160–396 s (244 ± 29 s, n = 2). All types of synchronous activity were abolished by the GABAA receptor antagonist bicuculline methiodide (t0 μM) ( n= 11). A similar effect was obtained by applying Ca²⁺-free/high-Mg²⁺ medium ( n = 5). Simultaneous field-potential recordings in CA3, CAI, dentate area and subiculum demonstrated that negative-going potentials and prolonged field-potential discharges occurred in all areas in a synchronous fashion. Spreading depression-like episodes were more frequently recorded in the CAI than in the CA3 area and were not seen in the subiculum or dentate area. These experiments indicate that a glutamatergic-independent, synchronous GABA-mediated potential which is elicited by 4AP in the adult rat hippocampus continues to occur in the presence of TEA. In addition, concomitant application of these K⁺-channel blockers induces a novel type of prolonged field-potential discharge as well as spreading depression-like episodes. Since all synchronous potentials (including spreading depression-like episodes) were abolished by bicuculline methiodide, we conclude that their occurrence is presumably dependent upon the post-synaptic activation of GABA_A receptors located on neuronal and glial elements. As excitatory synaptic transmission was nominally blocked under our experimental conditions, we also propose that rises in [K⁺]_o and consequent redistribution processes are per se sufficient to make all types of synchronous activity propagate.     

Hyperexcitability in CA1 of the rat hippocampal slice following hypoxia or adenosine

Participation of adenosine receptors in the depression of synaptic transmission during hypoxia, and the production of multiple populations spikes in the pyramidal neurons following hypoxia, has been investigated in the CA₁ area of the rat hippocampal slice. A method is presented for analysing such hyperexcitability, using input/output curves of the second population spike. This method provides evidence that rebound hyperexcitability following hypoxia or prolonged adenosine-mediated inhibition results from an increase in excitability of the CA₁ pyramidal neurons rather than from an increase in excitatory neurotransmitter release. Hypoxia-induced depression of the synaptic components of evoked field potentials was blocked in a concentration dependent manner by the selective A₁ receptor antagonist 8-cyclopenthyltheophylline (8-CPT), demonstrating extracellular accumulation of adenosine during hypoxia. Upon reoxygenation of slices following 30 min hypoxia, multiple population spikes were evoked by a single orthodromic stimulus in slices that exhibited only a single population spike prior to hypoxia. Such post-hypoxic hyperexcitability was not prevented by superfusion of slices with 8-CPT during hypoxia. Depression of synaptic transmission by 30 min superfusion of slices with 50 μM adenosine was also followed, upon washout, by the appearance of multiple population spikes. However, such hyperexcitability could not be produced by superfusion with adenosine analogues selective for A₁ receptors, cyclopentyladenosine, selective for A_2a receptors, 2-p-(2-carboxyethyl)phenetheylamino-5′-ethylcarboxamidoadenosine (CGS 21680), or active at A_2a and A_2b receptors,N⁶-[2-(3,5-dimethyoxyphenyl)-2-(2-methyl-phenyl)ethyl]adenosine, suggesting that adenosine receptors other than the A₁, A_2a or A_2b subtypes are involved in its generation.     

Melatonin blocks the induction of long-term potentiation in an N-methyl-d-aspartate independent manner

Perfusion of 100 μM melatonin had no effect on low frequency synaptic transmission, but prevented the induction of tetanically induced long-term potentiation (LTP) when recorded in the dendritic region of the CA1 in rat hippocampal slices. Perfusion of 100 μM melatonin in this preparation had no effect on the multiple population spikes recorded in Mg²⁺-free medium, and, in grease-gap recordings from the CA1-subiculum slice, 100 μM melatonin had no effect on depolarisations evoked by N-methyl-d-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This suggests that melatonin has the ability to prevent the formation of LTP, and that this effect is not mediated by blockade of NMDA receptors.     

Evidence forN-methyl-d-aspartic acid receptor-mediated modulation of the commissural input to central vestibular neurons of the frog

We have investigated the role ofN-methyl-d-asparte (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg²⁺ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-appliedd-2-amino-5-phosphonovaleric acid (D-APV, 25–50 μM), a selective NMDA antagonist. In a low Mg²⁺ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and had a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of the latter was reduced by D-APV (25–50 μM) to about 65% of the control value in the presence of 1 mM Mg²⁺. During bath application of NMDA (100 μM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg²⁺ concentration. Bath application of NMDA (0.1–1 μM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were stimulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these stimulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.     

The effects of chronic ethanol exposure on N-methyl-d-aspartate-stimulated overflow of [H]catecholamines from rat brain

The effects of acute and chronic ethanol administration on N-methyl-d-aspartate-stimulated catecholamine overflow were examined. Three groups of male Sprague-Dawley rats were used. The first group received a chronic liquid diet containing ethanol (37%) for 3 weeks. The second group was pair-fed a liquid diet with dextrin substituted for ethanol isocalorically. The third group received Purina rat lab chow and water ad libitum. N-methyl-d-aspartate-stimulated [³H]catecholamine overflow from brain tissue slices was determined. N-methyl-d-aspartate (50–2000 μM) produced a concentration-dependent increase in [³H]norepinephrine overflow from cortical and hippocampal slices with no significant alteration of the response following chronic ethanol treatment. [³H]Dopamine overflow from striatal slices of the chronic ethanol group was significantly different at 1000 μM N-methyl-d-aspartate. The response of the chronic ethanol-treated group at the 1000 μM N-methyl-d-aspartate concentration was 30% and 40% lower than the pair-fed and ad libitum controls, respectively. Ethanol when added in vitro (30–200 mM) produced a concentration-dependent inhibition of N-methyl-d-aspartate (150 μM) stimulated efflux in all brain regions, and chronic ethanol treatment did not alter the inhibitory response. These results indicate an apparant lack of adaptation in N-methyl-d-aspartate-stimulated transmitter release following chronic ethanol treatment in this particular paradigm.     

Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl-d-aspartate neurotoxicity

We examined the role of nitric oxide in N-methyl-d-aspartate (NMDA) receptor-mediated neurotoxicity in rat and mouse primary cortical cell cultures. In rat and mouse cultures, the NO synthase inhibitor, N^G-Nitro-l-arginine, blocked cGMP formation but not neuronal cell death following a 5–10 min exposure to 300–500 μM NMDA. N^G-Monomethyl-l-arginine was also unable to prevent neuronal death. In contrast, the non-competitive NMDA receptor antagonist, dextrophan, prevented both cGMP formation and cell death. While other data suggest that the synthesis of nitric oxide can mediate NMDA receptor-mediated neurotoxicity, present results suggest that such synthesis is not necessarily required.     

Long-term potentiation in the hippocampal slice: Possible involvement of pyruvate dehydrogenase

Tetanic stimulation of fibers in the hippocampal slice preparation produces long-term potentiation (LTP) and also decreases the in vitro incorporation of phosphate into the alpha subunit of pyruvate dehydrogenase (αPDH)¹². This paper describes 6 experiments that were undertaken to replicate this observation. Hippocampal slices were incubated in a specially designed chamber and stimulated with a tungsten wire electrode in the stratum radiatum for 1 s at 100 Hz. Two minutes after the tetanus, the stimulated slices were removed alternately with control (not tetanized) slices and each group was pooled for subcellular fractionation and labeling of the fractions with [³²P]ATP. Proteins were separated by electrophoresis and relative³²P contents of 41K and 50K protein bands were studied. Tetanic stimulation of the stratum radiatum did not affect subsequent phosphorylation of a 50K M_r protein that has been reported to be altered by perforant path activation². Stimulation also had no effect on pyruvate dehydrogenase enzyme activity or on the ratio of active (dephosphorylated) to inactive enzyme. In most cases tetanic stimulation produced no significant change in the in vitro phosphorylation of this enzyme. Only under one set of conditions, labeling with 250 μM [γ-³²P] ATP for 10 s, was a decrease in the in vivo labeling of αPDH shown to be statistically significant. These findings suggest that LTP is not necessarily accompanied by an initial change in PDH phosphorylation level or activity but may be associated with a decrease in the kinase activity directed toward this protein.     

The NMDA receptor contributes to anoxic aglcemic induced irreversible inhibition of synaptic transmission

Present recovery of CA1 field EPSP amplitude following various anoxic aglycemic (AA) periods was examined in rat hippocampal slices superfused with MK-801 (0.1 μM, 1 μM, 10 μM) or Mg²⁺-free artificial cerebrospinal fluid. Slices treated with 0.1 μM MK-801 showed greater percent recuperation of EPSP amplitude following 3 min 30 s of AA (36±12%vs6 ±4% in controls). Higher concentrations of MK-801 resulted in a greater recovery of EPSP amplitudes in more than one time period of AA, with 10 μM MK-801 providing protection in up to 4 min 30 s AA. Percent recuperation of EPSP amplitude was smaller in Mg²⁺-free slices following 2 min (34±15%vs81±11% in controls) and 2 min 30 (25±14%vs77±10% in controls) of AA. These results that the activation of theN-methyl-d-aspartate (NMDA) receptor channel by contribute to irreversible AA induced synaptic failure in CA1.     

The protective action of nefiracetam against electrophysiological and metabolic damage in the hippocampus after deprivation of glucose and oxygen

The present study examined the effect of nefiracetam on ischemic brain damage by monitoring population spikes (PSs) in the dentate gyrus of guinea pig hippocampal slices; assaying high-energy phosphates (ATP and CrP) in guinea pig hippocampal slices; and monitoring whole-cell membrane-currents and intracellular Ca²⁺ levels in cultured hippocampal neurons. Twenty-minute ischemic insult to slices, i.e., deprivation of glucose and oxygen from artificial cerebrospinal fluid, abolished PSs. As compared with only 35% recovery of the PS amplitude for control, PS amplitude reversed to 65% of basal levels 40 min after returning normal conditions by treatment with nefiracetam (0.01 μM). Ischemic insult reduced the levels of adenosine triphosphate (ATP) and creatine phosphate (CrP) in slices, and when returned to normal conditions, recovering to 70 and 85% of basal values, respectively, 30 min after returning normal conditions. Nefiracetam (0.01 μM) facilitated the recovery of ATP and CrP, reaching 110 and 140% of basal values, respectively. Nefiracetam inhibited N-methyl- -aspartate (NMDA)-evoked currents to 35% of basal amplitudes. Likewise, nefiracetam (0.01 μM) inhibited intracellular Ca²⁺ rise through NMDA receptor channels to 30% of basal levels. The results of the present study, thus, suggest that nefiracetam has the potential to protect against ischemic brain damage, possibly in part by preventing from accumulation of intracellular calcium through NMDA receptor channels.     

Impairment of the nitric oxide/cyclic GMP pathway in cerebellar slices prepared from thehph-1 mouse

In this study, the effect of tetrahydrobiopterin deficiency on the nitric oxide/cGMP pathway has been investigated in cerebellar slices derived from thehph-1 mouse. This animal displays a partial deficiency of tetrahydrobiopterin. Basal levels of cGMP were significantly reduced (−29.5%) in thehph-1 mouse cerebellum compared to controls. Following kainate stimulation (500 μM) cGMP levels increased in both control andhph-1 preparations but were again significantly lower (−29.1 %) in thehph-1 mouse. Exposure of slices to the nitric oxide donors,S-nitroso-N-acetylpenicillamine andS-nitroso-glutathione, revealed no difference in cGMP accumulation between the two groups. These findings suggest that the cerebellar nitric oxide/cGMP pathway may be impaired in partial tetrahydrobiopterin deficiency states due to diminished nitric oxide formation.     

Deramciclane inhibits N-methyl-D-aspartate receptor function

Effects of the novel anxiolytic drug deramciclane on excitatory amino acid release and transmembrane Ca²⁺ ion flux processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane (10 μM) significantly inhibited [³H]D-aspartate release and transmembrane Ca²⁺ flux to N-methyl-D-aspartate in the absence of Mg²⁺. By contrast, inhibition of [³H]D-aspartate release and transmembrane Ca²⁺ flux evoked by 0.1 mM (S)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the presence of Mg²⁺ and 10 μM cyclothiazide by 10 μM deramciclane was not significant. In the presence of N-methyl-D-aspartate receptor antagonists, deramciclane (10 μM) did not inhibit [³H]D-aspartate release to N-methyl-D-aspartate. These results suggest an involvement of the inhibition of a presynaptic N-methyl-D-aspartate receptor in the anxiolytic properties of deramciclane.     

Delayed neuronal injury induced by sub-lethal NMDA exposure in the hippocampal slice

Stroke produces neuronal death by two general processes which differ in their temporal course. Acute neuronal death occurs within minutes, while delayed neuronal death evolves within 24 h. To better examine mechanisms of delayed death, we developed a new in vitro model of delayed neuronal injury using extended electrophysiological recordings in paired hippocampal slices. We exposed one hippocampal slice of each pair to 10 μMN-methyl-d-aspartate (NMDA) until the orthodromic CA1 PS disappeared. Thereafter, NMDA-treated slices regained near full recovery of PS amplitude within one hour. However, 10 h later, NMDA-treated slices demonstrated a rapid decline in PS amplitude of 82% ± 15. CA1 orthodromic evoked PS was lost completely at an average 12.4 ± 1.6 h after NMDA exposure. This sudden loss of response contrasted with paired, untreated slices, where CA1 PS could be elicited for 22.6 ± 4.0 h (P < 0.05). Treatment with 10 mM MgCl₂ begun after NMDA exposure and continued for 35 min, prevented delayed loss of CA1 orthodromic PS, which then could be elicited for 20.3 ± 2.1 h. These results indicate that delayed injury can be evaluated using the hippocampal slice. They also suggest that activation of NMDA receptors can induce delayed neuronal injury in CA1 neurons, and that magnesium treatment after NMDA can prevent this injury.     

Inhibition of an N-methyl-d-aspartate induced short-term potentiation in the rat hippocampal slice

The effects of the phorbol ester 4ß-phorbol-12,13 dibutyrate (PDBu) and the protein kinase (PK) inhibitors H-7 and sphingosine were investigated on the short-term potentiation (STP) of the population excitatory postsynaptic potential (EPSP) induced by perfusion of N-methyl-d-aspartate (NMDA) in the stratum radiatum of CA1 of the rat hippocampal slice. Bath perfusion of 130 μM NMDA for 10 s caused an initial depression of the population EPSP followed by a STP, which averaged 46% and lasted 16 min. PDBu (100 nM) perfused for 2 h completely inhibited the NMDA induced STP, suggesting that the stimulation of PKC inhibited an NMDA receptor activated process which induced the STP. The protein kinase inhibitors H-7 and sphingosine did not alter the NMDA induced STP.     

Presynaptic muscarinic modulation of nicotinic excitation in the rat neostriatum

In rat neostriatal slices, cholinergic agents were tested for their effects on endogenous ACh release and on electrical activity. ACh release was evoked by 25 mM K⁺ during two 5-min periods between which a slice was allowed to rest for 20 min; drugs were present during the second stimulation period. In the absence of a cholinesterase inhibitor, only Ch outflow was monitored. For the recording of electrical activity, intrastriatal stimulation evoked field potentials which were monitored in the absence and presence of drugs in the perfusate.Atropine (1–100 μM) increased endogeneous ACh release by 32–91% and effective doses were 10-fold lower in the presence of a cholinesterase inhibitor. Atropine also increased the amplitudes of synaptic population spikes in the field potentials.The muscarinic agonists muscarine (100 μM) and oxotremorine (25 and 100 μM) decreased endogenous ACh release. Atropine (10 μM) blocked the depressant effect of muscarine (100 μM). Muscarine (100 μM–1 mM) and oxotremorine (10–100 μM) decreased the electrically evoked excitation in the rat neostriatal slices, and their effects were reversed by atropine.Only higher concentrations of nicotine (1 and 5 mM) decreased the synaptic population spikes, but potassium-stimulated Ch outflow was not affected.It is concluded that in the neostriatum presynaptic muscarinic receptors modulate nicotinic excitation since potassium-stimulated ACh release and intrinsically evoked synaptic excitation are influenced by muscarinic drugs in the same way.     

Soman reversibly decreases the duration of Ca and Ba action potentials in bullfrog sympathetic neurons

Soman, (pinacoloxymethyl-phosphoryl fluoride) (0.1–10 μM) an irreversible cholinesterase inhibitor, reversibly reduced the duration of calcium (Ca²⁺)- and barium (Ba²⁺) spikes without significantly affecting spike amplitude in sympathetic postganglionic neurons of the adult bullfrog (Rana catesbeiana). The soman-induced shortening of the spike duration was not prevented by pretreatment with either (+)-tubocurarine (100 μM) or hexamethonium (100 μM) and atropine (10 μM) and was also recorded from acutely-dissociated sympathetic neurons. These results suggest that soman has a direct action to decrease calcium entry through voltage-dependent channels activated during a spike. This effect may contribute to both the decrease in the duration of the spike after-hyperpolarization (AHP) and the enhanced neuronal excitability produced by soman in these neurons.     

Inhibition of NMDA-induced protein kinase C translocation by a Zn chelator: Implication of intracellular Zn

The role of intracellular Zn²⁺ in the translocation of protein kinase C from cytosol to membrane fractions was examined by the [³H]phorbol 12,13-dibutyrate (PDBu) binding method in guinea pig cerebral synaptoneurosomes. N-methyl-d-aspartate (NMDA, 100 μM) and calcium ionophore A23187 (0.3–30 μM) decreased the binding activity in the cytosol with a concomitant increase in the membrane fractions. Pretreatment of synaptoneurosomes with a heavy metal chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), inhibited the NMDA- and A23187-induced changes of the distribution of [³H]PDBu binding sites in cytosol and membrane fractions. The inhibitory effect of TPEN was negated by a preincubation of TPEN with equimolar Zn²⁺ but not by that with Ca²⁺. The addition of 500 μM Zn²⁺ to the lysate of synaptoneurosomes induced an increase of [³H]PDBu binding activity in the membrane fraction with a concomitant decrease in the cytosol fraction, as did 100 μM Ca²⁺. Low concentrations of Zn²⁺ (10 μM), which alone had no effect on the distribution of the binding, significantly enhanced the effect of 10 μM Ca²⁺ in the lysate. Under those conditions TPEN inhibited the Zn²⁺-potentiated Ca²⁺-dependent changes in the binding. These results suggest that intracellular Zn²⁺ is essential for the agonist-induced translocation of protein kinase C in guinea pig synaptoneurosomes.     

Involvement of peripheral NMDA and non-NMDA receptors in development of persistent firing of spinal wide-dynamic-range neurons induced by subcutaneous bee venom injection in the cat

To study the roles of peripheral excitatory amino acids receptor subtypes N-methyl- -aspartate (NMDA) and non-NMDA receptors in persistent nociception, extracellular single unit recording technique was used to assess the effects of a single dose NMDA and non-NMDA receptor antagonists, AP₅ (5-aminophosphonovaleric acid) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) or DNQX (6,7-dinitroquinoxaline-2,3-dione), on s.c. bee venom-induced increase in firing of wide-dynamic-range (WDR) neurons in the spinal dorsal horn of the urethane–chloralose anesthetized cats. Subcutaneous bee venom injection into the cutaneous receptive field resulted in a single phase of increased firing of WDR neurons over the background activity for more than 1 h. Local pre-administration of AP₅ (200 μg/100 μl) or CNQX (8.3 μg/100 μl) into the bee venom injection site produced 94% (1.01±0.96 spikes/s, n=5) or 76% (2.97±0.58 spikes/s, n=4) suppression of the increased neuronal firing when compared with local saline (16.32±4.55 spikes/s, n=10) or dimethyl sulfoxide (DMSO) (12.37±6.36 spikes/s, n=4) pre-treated group, respectively. Local post-administration of the same dose of AP₅ produced a similar result to the pre-treatment group with a 67% inhibition of the mean firing rate, however, the same treatment with CNQX and even a higher dose of DNQX (100 μg/100 μl) did not produce any inhibition of the neuronal firing induced by s.c. bee venom injection (DNQX vs. DMSO: 23.91±0.25 vs. 22.14±0.04 spikes/s, P=0.0298, n=5). In the control experiments, local pre-administration of the same dose of AP₅ or CNQX into a region on the contralateral hindpaw symmetrical to the bee venom injection site produced no significant influence on the increased firing of the WDR neurons [contralateral AP₅ vs. saline: 14.17±6.27 spikes/s (n=5) vs. 16.32±4.55 spikes/s (n=10), P>0.05; contralateral CNQX vs. DMSO: 12.85±6.38 spikes/s (n=4) vs. 12.37±6.36 spikes/s (n=4), P>0.05], implicating that the suppressive action of local AP₅ or CNQX was not the result of systemic effects. The present results suggest that activation of the peripheral NMDA receptors is involved in both induction and maintenance, while activation of non-NMDA receptors is only involved in induction, but not in the maintenance of persistent firing of the dorsal horn WDR neurons induced by s.c. bee venom injection.     

Dynorphin A (1–13) Neurotoxicity in Vitro: Opioid and Non-Opioid Mechanisms in Mouse Spinal Cord Neurons

Dynorphin A is an endogenous opioid peptide that preferentially activates κ-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1–13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both κ-opioid and N-methyl- -aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through κ-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing κ-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both κ-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1–13) on intracellular calcium concentration ([Ca²⁺]_i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1–13) elevated [Ca²⁺]_i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 μM), 2-amino-5-phosphopentanoic acid (100 μM), or 7-chlorokynurenic acid (100 μM)—suggesting that dynorphin A (1–13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (−)-naloxone (3 μM), or the more selective κ-opioid receptor antagonist nor-binaltorphimine (3 μM), exacerbated dynorphin A (1–13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 μM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 μM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates κ-opioid receptors and suggests that κ receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1–13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.