Role In Long-term Potentiation Research Articles

Presynaptic Role of cGMP-Dependent Protein Kinase during Long-Lasting Potentiation

Previous research has suggested that cGMP-dependent protein kinases (cGKs) may play a role in long-term potentiation in hippocampus, but their site of action has been unknown. We examined this question at synapses between pairs of hippocampal neurons in dissociated cell culture. Injection of a specific peptide inhibitor of cGK into the presynaptic but not the postsynaptic neuron blocked long-lasting potentiation induced by tetanic stimulation of the presynaptic neuron. As controls, injection of a scrambled peptide or a peptide inhibitor of cAMP-dependent protein kinase into either neuron did not block potentiation. Conversely, injection of the alpha isozyme of cGK type I into the presynaptic but not the postsynaptic neuron produced activity-dependent potentiation that did not require NMDA receptor activation. Evidence from Western blots, reverse transcription-PCR, activity assays, and immunocytochemistry indicates that endogenous cGK type I is present in the neurons, including presynaptic terminals. These results support the idea that cGK plays an important presynaptic role during the induction of long-lasting potentiation in hippocampal neurons.

Dendritic potassium channels in hippocampal pyramidal neurons.

Potassium channels located in the dendrites of hippocampal CA1 pyramidal neurons control the shape and amplitude of back-propagating action potentials, the amplitude of excitatory postsynaptic potentials and dendritic excitability. Non-uniform gradients in the distribution of potassium channels in the dendrites make the dendritic electrical properties markedly different from those found in the soma. For example, the influence of a fast, calcium-dependent potassium current on action potential repolarization is progressively reduced in the first 150 micrometer of the apical dendrites, so that action potentials recorded farther than 200 micrometer from the soma have no fast after-hyperpolarization and are wider than those in the soma. The peak amplitude of back-propagating action potentials is also progressively reduced in the dendrites because of the increasing density of a transient potassium channel with distance from the soma. The activation of this channel can be reduced by the activity of a number of protein kinases as well as by prior depolarization. The depolarization from excitatory postsynaptic potentials (EPSPs) can inactivate these A-type K+ channels and thus lead to an increase in the amplitude of dendritic action potentials, provided the EPSP and the action potentials occur within the appropriate time window. This time window could be in the order of 15 ms and may play a role in long-term potentiation induced by pairing EPSPs and back-propagating action potentials.

ERK Plays a Regulatory Role in Induction of LTP by Theta Frequency Stimulation and Its Modulation by β-Adrenergic Receptors

MAP kinase (ERK) translates cell surface signals into alterations in transcription. We have found that ERK also regulates hippocampal neuronal excitability during 5 Hz stimulation and thereby regulates forms of long-term potentiation (LTP) that do not require macromolecular synthesis. Moreover, ERK-mediated changes in excitability are selectively required for some forms of LTP but not others. ERK is required for the early phase of LTP elicited by brief 5 Hz stimulation, as well as for LTP elicited by more prolonged 5 Hz stimulation when paired with β1-adrenergic receptor activation. By contrast, ERK plays no role in LTP elicited by a single 1 s 100 Hz train. Consistent with these results, we find that ERK is activated by β-adrenergic receptors in CA1 pyramidal cell somas and dendrites.

Activation of p42 mitogen-activated protein kinase by arachidonic acid and trans-1-amino-cyclopentyl-1,3-dicarboxylate impacts on long-term potentiation in the dentate gyrus in the rat: analysis of age-related changes

Maintenance of long-term potentiation in perforant path–granule cell synapses is associated with an increase in glutamate release, which we have suggested relies on an interaction between arachidonic acid and the metabotropic glutamate receptor agonist, trans-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD). Evidence suggests that this interaction is dependent on stimulation of tyrosine kinase, which phosphorylates and activates phospholipase Cγ. In this study, we demonstrate that arachidonic acid and ACPD stimulate tyrosine phosphorylation of a protein of about 40,000 mol. wt and further analysis, using a specific antibody, suggested that this may be extracellular signal-regulated kinase, one member of the family of mitogen-activated protein kinases. Activity of extracellular signal-regulated kinase was increased by arachidonic acid and ACPD in vitro, but it was also increased by induction of long-term potentiation in perforant path–granule cell synapses. A role for extracellular signal-regulated kinase in long-term potentiation was supported by the observation that expression of long-term potentiation, as well as the associated increases in endogenous glutamate release and extracellular signal-regulated kinase activation, were inhibited by pretreatment with the mitogen-activated protein kinase inhibitor, PD98059, while PD98059 pretreatment inhibited the interaction between arachidonic acid and ACPD on glutamate release. An age-related decrease in extracellular signal-regulated kinase activity was observed in the dentate gyrus, and there was no evidence of increased extracellular signal-regulated kinase activity or endogenous glutamate release in tissue prepared from aged rats in which long-term potentiation was compromised. The evidence is consistent with the view that increased activation of extracellular signal-regulated kinase plays a role in long-term potentiation, and that activation of this kinase relies on the interaction between arachidonic acid and ACPD.

Brain-Derived Neurotrophic Factor Prevents Low-Frequency Inputs from Inducing Long-Term Depression in the Developing Visual Cortex

Brain-derived neurotrophic factor (BDNF) is reported to enhance synaptic transmission and to play a role in long-term potentiation in hippocampus and neocortex. If so, a shortage or blockade of BDNF might lead to another form of synaptic plasticity, long-term depression (LTD). To test this possibility and to elucidate mechanisms if it is the case, EPSCs evoked by test stimulation of layer IV were recorded from layer II/III neurons in visual cortical slices of young rats in the whole-cell voltage-clamp mode. LTD was induced by low-frequency stimulation (LFS) at 1 Hz for 10-15 min if each pulse of the LFS was paired with depolarization of neurons to -30 mV but was not induced if their membrane potentials were kept at -70 mV. Such an LTD was blocked by exogenously applied BDNF, probably through presynaptic mechanisms. Suppression of endogenous BDNF activity by the anti-BDNF antibody or an inhibitor for BDNF receptors made otherwise ineffective stimuli (LFS without postsynaptic depolarization) effective for LTD induction, suggesting that endogenous BDNF may prevent low-frequency inputs from inducing LTD in the developing visual cortex.

Reg1ulatory Role and Molecular Interactions of a Cell-Surface Heparan Sulfate Proteoglycan (N-syndecan) in Hippocampal Long-Term Potentiation

The cellular mechanisms responsible for synaptic plasticity involve interactions between neurons and the extracellular matrix. Heparan sulfates (HSs) constitute a group of glycosaminoglycans that accumulate in the beta-amyloid deposits in Alzheimer's disease and influence the development of neuron-target contacts by interacting with other cell surface and matrix molecules. However, the contribution of HSs to brain function is unknown. We found that HSs play a crucial role in long-term potentiation (LTP), a finding that is consistent with the idea that converging molecular mechanisms are used in the development of neuron-target contacts and in activity-induced synaptic plasticity in adults. Enzymatic cleavage of HS by heparitinase as well as addition of soluble heparin-type carbohydrates prevented expression of LTP in response to 100 Hz/1 sec stimulation of Schaffer collaterals in rat hippocampal slices. A prominent carrier protein for the type of glycans implicated in LTP regulation in the adult hippocampus was identified as N-syndecan (syndecan-3), a transmembrane proteoglycan that was expressed at the processes of the CA1 pyramidal neurons in an activity-dependent manner. Addition of soluble N-syndecan into the CA1 dendritic area prevented tetanus-induced LTP. A major substrate of src-type kinases, cortactin (p80/85), and the tyrosine kinase fyn copurified with N-syndecan from hippocampus. Moreover, association of both cortactin and fyn to N-syndecan was rapidly increased after induction of LTP. N-syndecan may thus act as an important regulator in the activity-dependent modulation of neuronal connectivity by transmitting signals between extracellular heparin-binding factors and the fyn signaling pathway.

Metabotropic glutamate receptor activation and blockade: their role in long-term potentiation, learning and neurotoxicity

Metabotropic glutamate receptors represent a fairly recent addition to the family of glutamate receptors. These receptors have the distinguishing feature of being coupled to G-proteins rather than ion channels and they appear to have a variety of functional characteristics. These receptors play a vital role, for example, in the induction and maintenance of long-term potentiation, the most popular current model of the biological correlates of learning and memory. Blockade of metabotropic glutamate receptors prevents long-term potentiation induction and learning in a variety of tasks in different species. Chronic metabotropic glutamate receptor activation is also associated with neurodegeneration and selective neuronal loss when agonists of these receptors are injected in high concentrations directly into the brain. Metabotropic glutamate receptors also play a role in the normal development of the nervous system and these sites within the central nervous system offer possible routes for drug therapies; selective receptor antagonists, for example, may prove to have the very desirable feature of endowing neuroprotection during ischaemic episodes whilst allowing normal excitatory neurotransmission to occur.

Proteolysis of cell adhesion molecules by serine proteases: a role in long term potentiation?

Tissue plasminogen activator (tPA), a serine protease endogenous to hippocampal neurons, is shown to recognize a highly conserved sequence in the extracellular domain of cell adhesion molecules (CAMs). When added to brain homogenates, tPA generated a CAM fragment similar in size to that produced in hippocampal slices by brief periods of NMDA receptor stimulation. The serine protease inhibitor 4-(2-Aminoethyl)-benzenesulfonyl fluoride blocked the effects of tPA with an approximately 50% suppression at 250 μM. The inhibitor at this concentration had no evident effect on synaptic responses but caused long term potentiation to decay back to baseline over a 1 h period. These results suggest that extracellular breakdown of cell adhesion molecules initiated by NMDA receptors and mediated by serine proteases contributes to the formation of stable potentiation.

Inhibition of group I metabotropic glutamate receptors blocks spatial learning in rats

Metabotropic glutamate receptors (mGluRs) are postulated to play a role in long-term potentiation and in learning and memory-formation. Previously, we found that the group I/II mGluR antagonist, (RS)- α-methyl-4-carboxyphenylglycine (MCPG), blocks Y-maze spatial alternation learning. In this study, we tested the group I mGluR antagonist (S)4-carboxyphenylglycine (4-CPG) in comparison with MCPG using the same behavioural paradigm. Male Wistar rats were intracerebroventricularly injected with either 29 μg 4-CPG or 209 μg MCPG, 30 min prior to learning. Neither 4-CPG nor MCPG had an effect on spatial alternation performance in the training session. In the memory-retention test 24 h later, however, both the 4-CPG- and the MCPG-treated animals were strongly impaired compared with NaCl-injected control rats. These results suggest a particular importance of group I mGluRs in spatial memory-formation and indicate that MCPG effects found in previous learning experiments were predominantly due to an action at group I mGluRs.

Evidence that nerve growth factor plays a role in long-term potentiation in the rat dentate gyrus

An inbred strain of Wistar rat (GH), which is deficient in nerve growth factor (NGF), was used to assess the possible role of NGF in the generation of long-term potentiation in perforant path-granule cell synapses. The data show that NGF was significantly decreased in the dentate gyrus of GH rats, that this deficit was accompanied by an impairment in long-term potentiation (LTP) and that intraventricular injection of NGF substantially reversed this impairment. Analysis of depolarization-induced glutamate release in synaptosomes prepared from dentate gyrus of control rats revealed that NGF alone was without effect, but in combination with the metabotropic glutamate receptor agonist, aminocyclopentane-1,3-dicarboxylic acid (ACPD), NGF induced a significant increase in release. This effect was occluded by prior induction of LTP, suggesting that the interaction between these agents may be required to enhance transmitter release which accompanies LTP in dentate gyrus. In contrast to the effect of NGF and ACPD on glutamate release in control rats, the combination of these agents had no effect on release in synaptosomes prepared from GH rats, which might be explained by the marked decrease in trk receptors in dentate gyrus of GH rats. It was concluded that the impaired ability of GH rats to sustain LTP is associated with a reduction in NGF concentration, a reduction in stimulated release of NGF and a decrease in trk receptors in dentate gyrus. It is proposed that these data indicate a role for NGF in the generation of long-term potentiation in perforant path-granule cell synapses.

BDNF acutely increases tyrosine phosphorylation of the NMDA receptor subunit 2B in cortical and hippocampal postsynaptic densities

While neurotrophins are critical for neuronal survival and differentiation, recent work suggests that they acutely regulate synaptic transmission as well. Brain-derived neurotrophic factor (BDNF) enhances excitatory postsynaptic currents in cultured dissociated hippocampal neurons within 2–3 min through postsynaptic, phosphorylation-dependent mechanisms. Moreover, BDNF modulates hippocampal long-term potentiation, in which postsynaptic NMDA ( N-methyl- d-aspartate) receptors (NRs) play a key role. We now report that BDNF acutely increases tyrosine phosphorylation of the specific NMDA receptor subunit NR2B, which has recently been shown to play a role in long-term potentiation. Incubation of BDNF with cortical or hippocampal postsynaptic densities for 5 min increased tyrosine phosphorylation of the NR2B subunits in a dose-dependent manner. A maximal increase to 165% of control phosphorylation occurred at a BDNF concentration of 2 ng/ml. The BDNF action appeared to be specific, since nerve growth factor, another member of the neurotrophin gene family, had no effect on NR2B phosphorylation. Further, BDNF action was selective, since it did not alter tyrosine phosphorylation of NR2A subunits. Our results suggest that tyrosine phosphorylation of NR2B subunits of the NMDA receptor may contribute to neurotrophin modulation of postsynaptic responsiveness and long-term potentiation.

Adenosine A1 receptors mediate retinotectal presynaptic inhibition: uncoupling by C-kinase and role in LTP during regeneration.

Presynaptic adenosine receptors inhibit transmitter release at many synapses and are known to exist on retinotectal terminals. In this paper we show that adenosine decreases retinotectal field potentials by approximately 30% and investigate the mechanism. First, as judged by the effects of specific calcium channel blockers, retinotectal transmission was mediated almost exclusively by N-type calcium channels, which are known to be modulated by adenosine A1 receptors. Transmission was completely blocked by either omega-Conotoxin GVIA (-100%, N-type blocker) or omega-Conotoxin MVIIC (-99%, N-, P- and Q-type blocker) and was not significantly affected by omega-Agatoxin IVA [+1.7 +/- 9. 3% (SE), P-,Q-type blocker], but was augmented slightly by nifedipine(+9.3 +/- 2.1%, L-type blocker). Second, the adenosine inhibition was presynaptic, as indicated by a 43% increase in paired-pulse facilitation. Third, the selective A1 agonist cyclohexyl adenosine (CHA) at 50 nM caused a 21% decrease in amplitude and the selective A2 agonist N6-[2-(3, 5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) at 100 nM caused a 24% increase. Fourth, the selective A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) alone produced an increase in the field potential, suggesting a tonic inhibition mediated by endogenous adenosine. Fifth, pertussis toxin eliminated adenosine inhibition implicating Gi or Go protein coupling. Sixth, C-kinase activation eliminated the A1-mediated inhibition. In regenerating projections, adenosine also caused a decrease in transmission (-30 +/- 12%), but after induction of long-term potentiation (LTP) via trains of stimuli or via treatment with the phosphatase inhibitor okadaic acid, the adenosine response was converted to an augmentation. Because LTP is associated with C-kinase activation, this is consistent with C-kinase uncoupling the A1 receptor from inhibiting N-type Ca2+ channels. This uncovers the A2-mediated augmentation as demonstrated in normals with DPMA. Such an effect could account in part for the LTP of immature synapses and the change from rapidly fatiguing to robust synaptic transmission.

Learning by neurones: role of attention, reinforcement and behaviour

The importance of the behavioural situation, attentional demands of the task, and stimulus-reinforcement contingencies in promoting or permitting experience-dependent neuronal plasticity is argued. Evidence is provided for the specific activation of the locus coeruleus noradrenergic system of the rat by novel stimuli encountered while investigating the environment, as well as during a formal learning situation. Noradrenergic neurons are particularly concerned with changes in the predictive value of the stimulus, when new learning should occur. Noradrenaline, released at LC terminals in target sensory systems, could facilitate shifts in attention, information processing and memory through its well-documented gating and tuning effects and its permissive role in long-term potentiation. Dopamine neurons, which fire persistently to reward during learning, could be involved in maintaining the behavioural response.

Inhibitory avoidance learning inhibits ectonucleotidases activities in hippocampal synaptosomes of adult rats.

Several lines of evidence indicate that ATP may play an important role in Long-Term Potentiation. In this investigation we evaluated the effect of a memory task (step-down inhibitory avoidance) on the synaptosomal ecto-enzymes (ATP diphosphohydrolase and 5'-nucleotidase) involved in the degradation of ATP to adenosine. After the training session, a decrease in the ATPase (40%) and ADPase (29%) activities of ATP diphosphohydrolase as well as was a decrease in 5'-nucleotidase activity (31%) was observed in hippocampal synaptosomes of rats trained and killed immediately after training. In synaptosomes of rats killed 30 minutes after training, a decrease in ATPase activity (28%) was observed. In the test session, no significant changes were observed in the enzyme activities studied. These results provide new information about the activity of ecto-enzymes involved in nucleotide degradation and their possible participation in mechanisms of acquisition and modulation of memory processing.

Extrasynaptic glutamate spillover in the hippocampus: evidence and implications

In the mammalian brain most excitatory transmission is mediated by glutamate binding to AMPA and NMDA receptors. These receptors have markedly different biophysical properties, and at synapses in the CA1 region of the hippocampus they play complementary roles in long-term potentiation (LTP): while postsynaptic NMDA receptor activation is necessary for the induction of this form of plasticity, AMPA receptors play a larger role in its expression. Recent studies in hippocampal slices have revealed a further striking difference in the behaviour of the two receptor types: NMDA receptors consistently sense a larger number of quanta of glutamate released from presynaptic terminals than do AMPA receptors. Two alternative explanations for this are either that AMPA receptors are functionally silent at a proportion of synapses (although they can be uncovered by LTP), or that glutamate can spill over from neighbouring synapses and selectively activate NMDA (but not AMPA) receptors. Both of these competing hypotheses have extensive implications for the mechanisms of expression of LTP. Extrasynaptic glutamate diffusion appears to depend critically on the recording temperature, but if excitatory synapses are sufficiently close for cross-talk to occur under physiological conditions, it could have profound implications for the specificity of synaptic communication in the brain.

LTP: Memory, arousal, neither, both

The neurophysiological phenomenon of LTP (long term potentiation) is considered by many to represent an adequate mechanism for acquiring or storing memories in the mammalian brain. In our target article, we reviewed the various arguments put forth in support of the LTP/memory hypothesis. We concluded that these arguments were inconsistent with the purported data base and proposed an alternative interpretation that we suggested was at least as compatible with the available data as the more widely held view. In doing so, we attempted to illustrate that the inadequacy of present experimental designs did not permit us to distinguish between equally viable hypotheses. In the four years since we wrote the first draft of our target article, hundreds of additional studies on LTP have been published and their results have been incorporated into current theories about memory. A diverse group of commentators responded to our target article with their own theories of how memories might be stored in the brain, some of which rely on LTP. Some commentators doubted whether memories can be stored through modifications of synaptic strength. Some assert that it will never be possible to understand the neural mechanisms of memory; still others remain hopeful that we will accomplish some semblance of a resolution, provided we appreciate LTP's role in a subset of seemingly amorphous memory systems. In summary, although it is commonly written that “LTP is a memory storage device,” the divergence of views among the commentators suggests, at least as strongly as our target article, that such conviction is unwarranted and fails to acknowledge both the lack of consensus regarding the role of LTP in memory and the complexity of the phenomenon of memory itself.

The role of long-term potentiation in persistent epileptiform burst-induced hyperexcitability following GABA A receptor blockade

Persistent hyperexcitability follows synchronized bursting induced in the CA3 region of hippocampal slices by perfusion with high concentrations (2 000 IU/ml) of the GABA A antagonist, penicillin. This hyperexcitable state is characterized by: i) slow recovery from bursting following penicillin washout; ii) persistent “post-burst” field potential oscillations and iii) increased probability of spontaneous bursting with ordinarily sub-convulsant doses of GABA A antagonists. An N-methyl- d-aspartate-independent type of long-term potentiation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate excitatory postsynaptic potentials occurred following bursting. However, similar increases in excitatory postsynaptic potential magnitude also occurred after a subconvulsant dose of penicillin (500 IU/ml) which did not produce the other features of persistent hyperexcitability. Furthermore, long-term potentiation either increased or remained stable after bursting stopped, whereas, post-burst oscillations gradually diminished with time. Low doses of the AMPA/kainate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, which restored the potentiated excitatory postsynaptic potentials to control levels, reduced but did not eliminate the post-burst oscillation. Tetanus-induced long-term potentiation did not reproduce the hyperexcitable state seen after bursting. These findings indicate that the epileptiform bursting caused by blocking GABA A-mediated inhibition induces long-term potentiation which is partially responsible for persistent burst-induced hyperexcitability but is not sufficient to entirely explain it. The hippocampus which is critical for normal memory is also frequently the generator of intractable epileptic seizures. Seizure-like discharges in the hippocampus induced long-lasting increases in synaptic efficacy similar to those thought to underlie normal memory. This form of long-term potentiation contributed to the network oscillations characteristic of the hyperexcitable state persisting after epileptiform activity but was not sufficient to entirely explain them. Epileptic seizures may engage normal memory mechanisms which increase neuronal excitability and predispose the hippocampal network to further seizures. This may, in part, account for the propensity for hippocampal seizure foci to become intractable.

Brain-Derived Neurotrophic Factor Enhances Long-Term Potentiation in Rat Visual Cortex

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the nerve growth factor (NGF) gene family, have been suggested to play a role in experience-dependent modification of neural networks in the developing nervous system. In this study we addressed the question of whether these neurotrophins are involved in long-term potentiation (LTP) in developing visual cortex. We recorded layer II/III field potentials and whole-cell currents evoked by test stimulation of layer IV at 0.1 Hz in visual cortical slices prepared from young rats (postnatal day 15-25) and observed effects of BDNF, NT-3, and NGF on these responses. Then we analyzed the effects of these neurotrophins on LTP induced by tetanic (Theta-burst type) stimulation of layer IV. We found that BDNF at 200 ng/ml potentiated field potentials and EPSCs in most cases and that this potentiation lasted after cessation of the BDNF application. At the concentration of 20 ng/ml, BDNF did not show such an effect, but it enhanced the magnitude of expressed LTP. On the other hand, NT-3 and NGF had none of these effects. Immunohistochemical staining of slices with antibody against BDNF showed that exogenous BDNF penetrated into the whole slice within approximately 5 min of its application. The actions of BDNF were blocked by preincubation of slices with TrkB-IgG fusion protein, a BDNF scavenger, or coapplication of K252a, an inhibitor for receptor tyrosine kinases. TrkB-IgG or K252a itself completely blocked LTP, suggesting that endogenous BDNF or another TrkB ligand plays a role in LTP in the developing visual cortex.

Ethanol's inhibition of LTP may not be mediated solely via direct effects on the NMDA receptor.

Acute abuse of alcohol is well known to have deleterious effects on memory. However, the molecular and cellular bases of this effect are not well understood. Ethanol is known to inhibit long-term potentiation (LTP), a putative cellular substrate of memory. However, there is controversy concerning the doses of ethanol required for inhibition of LTP. We examined the doses of ethanol required to inhibit LTP in the CA1 region of the hippocampus. We used two different LTP-inducing paradigms in these studies and found that only doses of ethanol associated with profound intoxication (50-100 mM) can produce significant inhibition of LTP. We also investigated the molecular mechanisms of ethanol's effect on LTP. Activation of the N-methyl-D-aspartate receptor plays a critical role in LTP, and ethanol has been shown to partially inhibit N-methyl-D-aspartate receptor function. We tested directly whether the level of N-methyl-D-aspartate inhibition produced by 100 mM ethanol is sufficient to account for the complete inhibition of LTP produced by 100 mM ethanol. Our data suggest that ethanol's effects on the N-methyl-D-aspartate receptor can account for most, but not all of ethanol's inhibition of LTP.

Characterization of ionotropic glutamate receptor-mediated nitric oxide production in vivo in rats.

Glutamate receptor activation can stimulate nitric oxide (NO) production and possibly play a role in long-term potentiation and excitotoxic-mediated injury. We studied the differential effect of agonist-induced activation of ion channel-linked N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtypes on NO production in vivo in rat hippocampus. We also studied whether dantrolene, a ryanodine calcium channel inhibitor previously shown to attenuate metabotropic glutamate receptor stimulation of NO production, also attenuated ionotropic glutamate receptor-mediated stimulation of NO production. Microdialysis probes were placed bilaterally into the CA3 region of the hippocampus of pentobarbital-anesthetized adult Sprague-Dawley rats and were perfused for 5 hours with artificial cerebrospinal fluid (CSF) containing 3 mumol/L [14C]L-arginine. Recovery of [14C]L-citrulline in the effluent was used as a marker of NO production. In 13 groups of rats, increases in [14C]L-citrulline recovery were compared between right- and left-sided probes perfused with no additional drugs versus combinations of NMDA, AMPA, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), the non-competitive glutamate receptor blocker MK-801, the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and dantrolene. Recovery of [14C]L-citrulline during perfusion with artificial CSF progressively increased to 272 +/- 73 fmol/min (+/-SEM) over 5 hours. Contralateral perfusion with 1 mmol/L L-NAME inhibited [14C]L-citrulline recovery. Perfusion with 1 mmol/L MK-801 or 1 mmol/L CNQX reduced [14C]L-citrulline recovery compared with contralateral perfusion with CSF alone. Perfusion with 1 mmol/L NMDA enhanced [14C]L-citrulline recovery, and this enhancement was attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. Perfusion with 1 mmol/L AMPA enhanced [14C]L-citrulline recovery, and this enhancement was also attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. Through an indirect method of assessing NO production in vivo, results with MK-801 and CNQX indicate that NMDA and AMPA receptor activation contribute to basal NO production in the rat hippocampus. Enhanced NO production with NMDA and AMPA agonists appears to involve a complex neuronal interaction because the effect of NMDA was attenuated by both MK-801 and CNQX and because the effect of AMPA was attenuated by both CNQX and MK-801. In contrast to metabotropic glutamate receptor activation, release of calcium from intracellular ryanodine calcium channels does not appear to be a prominent mediator of ionotropic glutamate receptor stimulation of NO production.