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Epilepsy, a common neurological disorder, is characterized by the occurrence of spontaneous recurrent epileptiform discharges (SREDs). Acquired epilepsy is associated with long-term neuronal plasticity changes in the hippocampus resulting in the expression of spontaneous recurrent seizures. The purpose of this study is to evaluate and characterize endogenous epileptiform activity in hippocampal-entorhinal cortical (HEC) slices from epileptic animals. This study employed HEC slices isolated from a large series of control and epileptic animals to evaluate and compare the presence, degree and localization of endogenous SREDs using extracellular and whole cell current clamp recordings. Animals were made epileptic using the pilocarpine model of epilepsy. Extracellular field potentials were recorded simultaneously from areas CA1, CA3, dentate gyrus, and entorhinal cortex and whole cell current clamp recordings were obtained from CA3 neurons. All regions from epileptic HEC slices (n=53) expressed SREDs, with an average frequency of 1.3Hz. In contrast, control slices (n=24) did not manifest any SREDs. Epileptic HEC slices demonstrated slow and fast firing patterns of SREDs. Whole cell current clamp recordings from epileptic HEC slices showed that CA3 neurons exhibited paroxysmal depolarizing shifts associated with these SREDs. To our knowledge this is the first significant demonstration of endogenous SREDs in a large series of HEC slices from epileptic animals in comparison to controls. Epileptiform discharges were found to propagate around hippocampal circuits. HEC slices from epileptic animals that manifest SREDs provide a novel model to study in vitro seizure activity in tissue prepared from epileptic animals. [ABSTRACT FROM AUTHOR]

Cannabinoids have been shown to cause CB1-receptor-dependent anticonvulsant activity in both in vivo and in vitro models of status epilepticus (SE) and acquired epilepsy (AE). It has been further demonstrated in these models that the endocannabinoid system functions in a tonic manner to suppress seizure discharges through a CB1-receptor-dependent pathway. Although acute cannabinoid treatment has anticonvulsant activity, little is known concerning the effects of prolonged exposure to CB1 agonists and development of tolerance on the epileptic phenotype. This study was carried out to evaluate the effects of prolonged exposure to the CB1 agonist WIN55,212-2 on seizure activity in a hippocampal neuronal culture model of low-Mg(2+) induced spontaneous recurrent epileptiform discharges (SREDs). Following low-Mg(2+) induced SREDs, cultures were returned to maintenance media containing 10, 100 or 1000 nM WIN55,212-2 from 4 to 24 h. Whole-cell current-clamp analysis of WIN55,212-2 treated cultures revealed a concentration-dependent increase in SRED frequency. Immunocytochemical staining revealed that WIN55,212-2 treatment induced a concentration-dependent downregulation of the CB1 receptor in neuronal processes and at both glutamatergic and GABAergic presynaptic terminals. Prolonged exposure to the inactive enantiomer WIN55,212-3 in low-Mg(2+) treated cultures had no effect on the frequency of SREDs or CB1 receptor staining. The results from this study further substantiate a role for a tonic CB1-receptor-dependent endocannabinoid regulation of seizure discharge and suggest that prolonged exposure to cannabinoids results in the development of tolerance to the anticonvulsant effects of cannabinoids and an exacerbation of seizure activity in the epileptic phenotype.

Purpose: Although great advances have been made in the development of treatments for epilepsy, acquired epilepsy following brain injury still comprises approximately 50% of all the cases of epilepsy. Thus, development of drugs that would prevent or decrease the onset of epilepsy following brain injury represents an important area of research., Methods: Here, we investigated effects of carisbamate (RWJ 333369) on the development and expression of spontaneous recurrent epileptiform discharges (SREDs) and its neuroprotective potential in cultured hippocampal neurons. This model utilizes 3 h of low Mg(2+) treatment to mimic status epilepticus (SE-like) injury in vitro. Following the injury, networks of neurons manifest synchronized SREDs for their life in culture. Neuronal cultures were treated with carisbamate (200 microM) for 12 h immediately after the SE-like injury. The drug was then removed and neurons were patch clamped 24 h following drug washout., Results: Treatment with carisbamate after neuronal injury prevented the development and expression of epileptiform discharges. In the few neurons that displayed SREDs following carisbamate treatment, there was a significant reduction in SRED frequency and duration. In contrast, phenytoin and phenobarbital, when used in place of carisbamate, did not prevent the development and expression of SREDs. Carisbamate was also effective in preventing neuronal death when administered after SE-like injury., Conclusions: Carisbamate prevents the development and generation of epileptiform discharges and is neuroprotective when administered following SE-like injury in vitro and may offer a novel treatment to prevent the development of epileptiform discharges following brain injuries.

Traumatic brain injury (TBI) survivors often suffer from a post-traumatic syndrome with deficits in learning and memory. Calcium (Ca(2+)) has been implicated in the pathophysiology of TBI-induced neuronal death. However, the role of long-term changes in neuronal Ca(2+) function in surviving neurons and the potential impact on TBI-induced cognitive impairments are less understood. Here we evaluated neuronal death and basal free intracellular Ca(2+) ([Ca(2+)](i)) in acutely isolated rat CA3 hippocampal neurons using the Ca(2+) indicator, Fura-2, at seven and thirty days after moderate central fluid percussion injury. In moderate TBI, cognitive deficits as evaluated by the Morris Water Maze (MWM), occur after injury but resolve after several weeks. Using MWM paradigm we compared alterations in [Ca(2+)](i) and cognitive deficits. Moderate TBI did not cause significant hippocampal neuronal death. However, basal [Ca(2+)](i) was significantly elevated when measured seven days post-TBI. At the same time, these animals exhibited significant cognitive impairment (F(2,25)=3.43, p<0.05). When measured 30 days post-TBI, both basal [Ca(2+)](i) and cognitive functions had returned to normal. Pretreatment with MK-801 blocked this elevation in [Ca(2+)](i) and also prevented MWM deficits. These studies provide evidence for a link between elevated [Ca(2+)](i) and altered cognition. Since no significant neuronal death was observed, the alterations in Ca(2+) homeostasis in the traumatized, but surviving neurons may play a role in the pathophysiology of cognitive deficits that manifest in the acute setting after TBI and represent a novel target for therapeutic intervention following TBI.

Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM kinase II has not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long-lasting decrease in CaM kinase II activity in the hippocampal neuronal culture model of low Mg2+-induced spontaneous recurrent epileptiform discharges (SREDs). We show here that the decrease in CaM kinase II activity associated with SREDs in hippocampal cultures involves a Ca2+/N-methyl-d-aspartate (NMDA) receptor-dependent mechanism. Low Mg2+-induced SREDs result in a significant decrease in Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptide autocamtide-2. Reduction of extracellular Ca2+ levels (0.2 mM in treatment solution) or the addition of dl-2-amino-5-phosphonovaleric acid (APV) 25 microM blocked the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. Antagonists of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid receptor or L-type voltage sensitive Ca2+ channel had no effect on the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. The results of this study demonstrate that the decrease in CaM kinase II activity associated with this model of epileptogenesis involves a selective Ca2+/NMDA receptor-dependent mechanism and may contribute to the production and maintenance of SREDs in this model.

This study was initiated to investigate effects of the novel neuromodulator carisbamate (RWJ 333369) in the hippocampal neuronal culture model of status epilepticus and spontaneous epileptiform discharges. Whole-cell current clamp techniques were used to determine the effects of carisbamate on spontaneous recurrent epileptiform discharges (SREDs, in vitro epilepsy), depolarization-induced sustained repetitive firing (SRF) and low Mg(2+)-induced continuous high frequency spiking (in vitro status epilepticus). This in vitro model is an important tool to study the effects of anticonvulsant drugs (AEDs) on SREDs that occur for the life of the neurons in culture. Carisbamate dose dependently blocked the expression and reoccurrence of SREDs. The ED(50) value for its antiepileptic effect was 58.75+/-2.43 microM. Inhibition of SRF is considered a common attribute of many AEDs. Carisbamate (100 microM) significantly decreased SRF in hippocampal neurons. All these effects of carisbamate were reversed during a 5 to 30 min drug washout period. When exposed to low Mg(2+) medium cultured hippocampal neurons exhibit high frequency spiking. This form of in vitro status epilepticus is not effectively blocked by conventional AEDs that are known to be effective in treating status epilepticus in humans. Carisbamate, like phenytoin and phenobarbital, had little or no effect on low Mg(2+)-induced continuous high frequency spiking. These results characterize the effects of carisbamate in the hippocampal neuronal culture model of epileptiform discharges and suggest that the ability of carisbamate to inhibit depolarization-induced SRF may account in part for some of it's anticonvulsant effect.

Traumatic brain injury (TBI) survivors often suffer chronically from significant morbidity associated with cognitive deficits, behavioral difficulties and a post-traumatic syndrome and thus it is important to understand the pathophysiology of these long-term plasticity changes after TBI. Calcium (Ca2+) has been implicated in the pathophysiology of TBI-induced neuronal death and other forms of brain injury including stroke and status epilepticus. However, the potential role of long-term changes in neuronal Ca2+ dynamics after TBI has not been evaluated. In the present study, we measured basal free intracellular Ca2+ concentration ([Ca2+](i)) in acutely isolated CA3 hippocampal neurons from Sprague-Dawley rats at 1, 7 and 30 days after moderate central fluid percussion injury. Basal [Ca2+](i) was significantly elevated when measured 1 and 7 days post-TBI without evidence of neuronal death. Basal [Ca2+](i) returned to normal when measured 30 days post-TBI. In contrast, abnormalities in Ca2+ homeostasis were found for as long as 30 days after TBI. Studies evaluating the mechanisms underlying the altered Ca2+ homeostasis in TBI neurons indicated that necrotic or apoptotic cell death and abnormalities in Ca2+ influx and efflux mechanisms could not account for these changes and suggested that long-term changes in Ca2+ buffering or Ca2+ sequestration/release mechanisms underlie these changes in Ca2+ homeostasis after TBI. Further elucidation of the mechanisms of altered Ca2+ homeostasis in traumatized, surviving neurons in TBI may offer novel therapeutic interventions that may contribute to the treatment and relief of some of the morbidity associated with TBI.

The hippocampus is especially vulnerable to seizure-induced damage and excitotoxic neuronal injury. This study examined the time course of neuronal death in relationship to seizure duration and the pharmacological mechanisms underlying seizure-induced cell death using low magnesium (Mg2+) induced continuous high frequency epileptiform discharges (in vitro status epilepticus) in hippocampal neuronal cultures. Neuronal death was assessed using cell morphology and fluorescein diacetate-propidium iodide staining. Effects of low Mg2+ and various receptor antagonists on spike frequency were assessed using patch clamp electrophysiology. We observed a linear and time-dependent increase in neuronal death with increasing durations of status epilepticus. This cell death was dependent upon extracellular calcium (Ca2+) that entered primarily through the N-methyl-d-aspartate (NMDA) glutamate receptor channel subtype. Neuronal death was significantly decreased by co-incubation with the NMDA receptor antagonists and was also inhibited by reduction of extracellular (Ca2+) during status epilepticus. In contrast, neuronal death from in vitro status epilepticus was not significantly prevented by inhibition of other glutamate receptor subtypes or voltage-gated Ca2+ channels. Interestingly this NMDA-Ca2+ dependent neuronal death was much more gradual in onset compared to cell death from excitotoxic glutamate exposure. The results provide evidence that in vitro status epilepticus results in increased activation of the NMDA-Ca2+ transduction pathway leading to neuronal death in a time-dependent fashion. The results also indicate that there is a significant window of opportunity during the initial time of continuous seizure activity to be able to intervene, protect neurons and decrease the high morbidity and mortality associated with status epilepticus.

Protracted elevation in intracellular calcium caused by the activation of the N-methyl-d-aspartate receptor is the main cause of glutamate excitotoxic injury in stroke. However, upon excitotoxic injury, despite the presence of calcium entry antagonists, calcium unexpectedly continues to enter the neuron, causing extended neuronal depolarization and culminating in neuronal death. This phenomenon is known as the calcium paradox of neuronal death in stroke, and it represents a major problem in developing effective therapies for the treatment of stroke. To investigate this calcium paradox and to determine the source of this unexpected calcium entry after neuronal injury, we evaluated whether glutamate excitotoxicity activates an injury-induced calcium-permeable channel responsible for conducting a calcium current that underlies neuronal death. We used a combination of whole-cell and single-channel patch-clamp recordings, fluorescent calcium imaging, and neuronal cell death assays in a well characterized primary hippocampal neuronal culture model of glutamate excitotoxicity/stroke. Here, we report activation of a novel calcium-permeable channel upon excitotoxic glutamate injury that carries calcium current even in the presence of calcium entry inhibitors. Blocking this injury-induced calcium-permeable channel for a significant time period after the initial injury is still effective in preventing calcium entry, extended neuronal depolarization, and delayed neuronal death, thereby accounting for the calcium paradox. This injury-induced calcium-permeable channel represents a major source for the initial calcium entry following stroke, and it offers a new target for extending the therapeutic window for preventing neuronal death after the initial excitotoxic (stroke) injury.

It is established that the majority but not all of the seizure-induced cell death is associated with status epilepticus while spontaneous recurrent seizures associated with epilepsy do not cause neuronal death. Extracellular effects and compensatory changes in brain physiology complicate assessment of neuronal death in vivo as the result of seizures. In this study we utilized a well-characterized in vitro hippocampal neuronal culture model of both continuous high-frequency epileptiform discharges (status epilepticus) and spontaneous recurrent epileptiform discharges (acquired epilepsy) to investigate the direct effects of continuous and episodic electrographic epileptiform discharges on cell death in a carefully controlled extracellular environment. The results from this study indicate that continuous high-frequency epileptiform discharges can cause neuronal death in a time-dependent manner. Episodic epileptiform seizure activity occurring for the life of the neurons in culture was not associated with increased neuronal cell death. Our data confirm observations from clinical and some animal studies that spontaneous recurrent seizures do not initiate cell death. The hippocampal neuronal culture model provides a powerful in vitro tool for carefully evaluating the effects of seizure activity alone on neuronal viability in the absence of various confounding factors and may provide new insights into the development of novel therapeutic agents to prevent neuronal injury during status epilepticus.

Aging is associated with increased vulnerability to neurodegenerative conditions such as Parkinson's and Alzheimer's disease and greater neuronal deficits after stroke and epilepsy. Emerging studies have implicated increased levels of intracellular calcium ([Ca(2+)](i)) for the neuronal loss associated with aging related disorders. Recent evidence demonstrates increased expression of voltage gated Ca(2+) channel proteins and associated Ca(2+) currents with aging. However, a direct comparison of [Ca(2+)](i) levels and Ca(2+) homeostatic mechanisms in hippocampal neurons acutely isolated from young and mid-age adult animals has not been performed. In this study, Fura-2 was used to determine [Ca(2+)](i) levels in CA1 hippocampal neurons acutely isolated from young (4-5 months) and mid-age (12-16 months) Sprague-Dawley rats. Our data provide the first direct demonstration that mid-age neurons in comparison to young neurons manifest significant elevations in basal [Ca(2+)](i) levels. Upon glutamate stimulation and a subsequent [Ca(2+)](i) load, mid-age neurons took longer to remove the excess [Ca(2+)](i) in comparison to young neurons, providing direct evidence that altered Ca(2+) homeostasis may be present in animals at significantly younger ages than those that are commonly considered aged (> or =24 months). These alterations in Ca(2+) dynamics may render aging neurons more vulnerable to neuronal death following stroke, seizures or head trauma. Elucidating the functionality of Ca(2+) homeostatic mechanisms may offer an understanding of the increased neuronal loss that occurs with aging, and allow for the development of novel therapeutic agents targeted towards decreasing [Ca(2+)](i) levels thereby restoring the systems that maintain normal Ca(2+) homeostasis in aged neurons.

Status epilepticus (SE) is a life-threatening neurological disorder associated with a significant morbidity and mortality. Benzodiazepines are the initial drugs of choice for the treatment of SE. Despite aggressive treatment, over 40% of SE cases are refractory to the initial treatment with two or more medications. It would be a major advance in the clinical management of SE to identify novel anticonvulsant agents that do not lose their ability to treat SE with increasing seizure duration. Cannabinoids have recently been demonstrated to regulate seizure activity in brain. However, it remains to be seen whether they develop pharmacoresistance upon prolonged SE. In this study, we used low Mg(2+) to induce SE in hippocampal neuronal cultures and in agreement with animal models and human SE confirm the development of resistance to benzodiazepine with increasing durations of SE. Thus, lorazepam (1 microM) was effective in blocking low Mg(2+) induced high-frequency spiking for up to 30 min into SE. However, by 1 h and 2 h of SE onset it was only 10-15% effective in suppressing SE. In contrast, the cannabinoid type-1 (CB1) receptor agonist, WIN 55,212-2 (1 microM) in a CB1 receptor-dependent manner completely abolished SE at all the time points tested even out to 2 h after SE onset, a condition where resistance developed to lorazepam. Thus, the use of cannabinoids in the treatment of SE may offer a unique approach to controlling SE without the development of pharmacoresistance observed with conventional treatments.

Status epilepticus is a serious neurological disorder associated with a significant morbidity and mortality. Antiepileptic drugs such as diazepam, phenobarbital and phenytoin are the mainstay of status epilepticus treatment. However, over 20% of status epilepticus cases are refractory to the initial treatment with two or more antiepileptic drugs. Endocannabinoids have been implicated as playing an important role in regulating seizure activity and seizure termination. This study evaluated the effects of the major endocannabinoids methanandamide and 2-arachidonylglycerol (2-AG) on status epilepticus in the low-Mg(2+) hippocampal neuronal culture model. Status epilepticus in this model was resistant to treatment with phenobarbital and phenytoin. Methanandamide and 2-AG inhibited status epilepticus in a dose-dependent manner with an EC(50) of 145+/-4.15 nM and 1.68+/-0.19 microM, respectively. In addition, the anti-status epilepticus effects of methanandamide and 2-AG were mediated by activation of the cannabinoid CB(1) receptor since they were blocked by the cannabinoid CB(1) receptor antagonist AM251. These results provide the first evidence that the endocannabinoids, methanandamide and 2-AG, are effective inhibitors of refractory status epilepticus in the hippocampal neuronal culture model and indicate that regulating the endocannabinoid system may provide a novel therapeutic approach for treating refractory status epilepticus.

Status epilepticus (SE) is a major medical emergency associated with a significant morbidity and mortality. Little is known about the mechanisms that terminate seizure activity and prevent the development of status epilepticus. Cannabinoids possess anticonvulsant properties and the endocannabinoid system has been implicated in regulating seizure duration and frequency. Endocannabinoids regulate synaptic transmission and dampen seizure activity via activation of the presynaptic cannabinoid receptor 1 (CB1). This study was initiated to evaluate the role of CB1 receptor-dependent endocannabinoid synaptic transmission towards preventing the development of status epilepticus-like activity in the well-characterized hippocampal neuronal culture model of acquired epilepsy using patch clamp electrophysiology. Application of the CB1 receptor antagonists SR141716A (1 microM) or AM251 (1 microM) to "epileptic" neurons caused the development of continuous epileptiform activity, resembling electrographic status epilepticus. The induction of status epilepticus-like activity by CB1 receptor antagonists was reversible and could be overcome by maximal concentrations of CB1 agonists. Similar treatment of control neurons with CB1 receptor antagonists did not produce status epilepticus or hyperexcitability. These findings suggest that CB1 receptor-dependent endocannabinoid endogenous tone plays an important role in modulating seizure frequency and duration and preventing the development of status epilepticus-like activity in populations of epileptic neurons. The regulation of seizure activity and prevention of status epilepticus by the endocannabinoid system offers an important insight into understanding the basic mechanisms that control the development of continuous epileptiform discharges.

Stroke is a major risk factor for developing acquired epilepsy (AE). Although the underlying mechanisms of ischemia-induced epileptogenesis are not well understood, glutamate has been found to be associated with both epileptogenesis and ischemia-induced injury in several research models. This chapter discusses the development of an in vitro model of epileptogenesis induced by glutamate injury in hippocampal neurons, as found in a clinical stroke, and the implementation of this model of stroke-induced AE to evaluate calcium's role in the induction and maintenance of epileptogenesis. To monitor the acute effects of glutamate on neurons and chronic alterations in neuronal excitability up to 8 days after glutamate exposure, whole-cell current-clamp electrophysiology was employed. Various durations and concentrations of glutamate were applied to primary hippocampal cultures. A single 30-min, 5-microM glutamate exposure produced a subset of neurons that died or had a stroke-like injury, and a larger population of injured neurons that survived. Neurons that survived the injury manifested spontaneous, recurrent, epileptiform discharges (SREDs) in neural networks characterized by paroxysmal depolarizing shifts (PDSs) and high-frequency spike firing that persisted for the life of the culture. The neuronal injury produced in this model was evaluated by determining the magnitude of the prolonged, reversible membrane depolarization, loss of synaptic activity, and neuronal swelling. The permanent epileptiform phenotype expressed as SREDs that resulted from glutamate injury was found to be dependent on the presence of extracellular calcium. The "epileptic" neurons manifested elevated intracellular calcium levels when compared to control neurons, independent of neuronal activity and seizure discharge, demonstrating that alterations in calcium homeostatic mechanisms occur in association with stroke-induced epilepsy. Findings from this investigation present the first in vitro model of glutamate injury-induced epileptogenesis that may help elucidate some of the mechanisms that underlie stroke-induced epilepsy.

Background: Bilirubin encephalopathy or kernicterus is a potentially serious complication of neonatal hyperbilirubinemia. The mechanism of bilirubin-induced neurotoxicity is not known. Many neurological insults are mediated through NMDA receptor activation., Objective: We assessed the effect of the NMDA channel antagonist, MK-801 on bilirubin neurotoxicity in vivo and in vitro., Methods: Bilirubin toxicity in vitro was assessed using trypan blue staining. Sulfadimethoxine injected (i.p.) jaundiced Gunn rat pups exhibit many neurological sequelae observed in human hyperbilirubinemia. Brainstem auditory-evoked potentials (BAEPs), a noninvasive sensitive tool to assess auditory dysfunction due to bilirubin neurotoxicity, were used to assess neuroprotection with MK-801 (i.p.) in vivo., Results: In primary cultures of hippocampal neurons, 20 min exposure to 64:32 microM bilirubin:human serum albumin reduced the cell viability by approximately 50% ten hours later. MK-801 treatment did not protect the cells. MK-801 pretreatment doses ranging from 0.1-4.0 mg/kg did not protect against BAEP abnormalities in Gunn rat pups 6 h after sulfadimethoxine injection., Conclusion: Our findings suggest that bilirubin neurotoxicity is not mediated through NMDA receptor activation., ((c) 2007 S. Karger AG, Basel.)

Epilepsy is characterized by the occurrence of spontaneous recurrent epileptiform discharges (SREDs) in neurons. A decrease in calcium/calmodulin-dependent protein kinase II (CaMK-II) activity has been shown to occur with the development of SREDs in a hippocampal neuronal culture model of acquired epilepsy, and altered calcium (Ca(2+)) homeostasis has been implicated in the development of SREDs. Using antisense oligonucleotides, this study was conducted to determine whether selective suppression of CaMK-II activity, with subsequent induction of SREDs, was associated with altered Ca(2+) homeostasis in hippocampal neurons in culture. Antisense knockdown resulted in the development of SREDs and a decrease in both immunocytochemical staining and enzyme activity of CaMK-II. Evaluation of [Ca(2+)](i) using Fura indicators revealed that antisense-treated neurons manifested increased basal [Ca(2+)](i), whereas missense-treated neurons showed no change in basal [Ca(2+)](i). Antisense suppression of CaMK-II was also associated with an inability of neurons to restore a Ca(2+) load. Upon removal of oligonucleotide treatment, CaMK-II suppression and Ca(2+) homeostasis recovered to control levels and SREDs were abolished. To our knowledge, the results demonstrate the first evidence that selective suppression of CaMK-II activity results in alterations in Ca(2+) homeostasis and the development of SREDs in hippocampal neurons and suggest that CaMK-II suppression may be causing epileptogenesis by altering Ca(2+) homeostatic mechanisms.

Cannabinoids have been shown to have anticonvulsant properties, but no studies have evaluated the effects of cannabinoids in the hippocampal neuronal culture models of acquired epilepsy (AE) and status epilepticus (SE). This study investigated the anticonvulsant properties of the cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol[1,2,3 de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2) in primary hippocampal neuronal culture models of both AE and SE. WIN 55,212-2 produced dose-dependent anticonvulsant effects against both spontaneous recurrent epileptiform discharges (SRED) (EC50 = 0.85 microM) and SE (EC50 = 1.51 microM), with total suppression of seizure activity at 3 microM and of SE activity at 5 microM. The anticonvulsant properties of WIN 55,212-2 in these preparations were both stereospecific and blocked by the cannabinoid type-1 (CB1) receptor antagonist N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A; 1 microM), showing a CB1 receptor-dependent pathway. The inhibitory effect of WIN 55,212-2 against low Mg2+-induced SE is the first observation in this model of total suppression of SE by a selective pharmacological agent. The clinically used anticonvulsants phenytoin and phenobarbital were not able to abolish low Mg2+-induced SE at concentrations up to 150 microM. The results from this study show CB1 receptor-mediated anticonvulsant effects of the cannabimimetic WIN 55,212-2 against both SRED and low Mg2+-induced SE in primary hippocampal neuronal cultures and show that these in vitro models of AE and SE may represent powerful tools to investigate the molecular mechanisms mediating the effects of cannabinoids on neuronal excitability.

Alterations in hippocampal neuronal Ca(2+) and Ca(2+)-dependent systems have been implicated in mediating some of the long-term neuroplasticity changes associated with acquired epilepsy (AE). However, there are no studies in an animal model of AE that directly evaluate alterations in intracellular calcium concentration ([Ca(2+)](i)) and Ca(2+) homeostatic mechanisms (Ca(2+) dynamics) during the development of AE. In this study, Ca(2+) dynamics were evaluated in acutely isolated rat CA1 hippocampal, frontal, and occipital neurons in the pilocarpine model by using [Ca(2+)](i) imaging fluorescence microscopy during the injury (acute), epileptogenesis (latency), and chronic-epilepsy phases of the development of AE. Immediately after status epilepticus (SE), hippocampal neurons, but not frontal and occipital neurons, had significantly elevated [Ca(2+)](i) compared with saline-injected control animals. Hippocampal neuronal [Ca(2+)](i) remained markedly elevated during epileptogenesis and was still elevated indefinitely in the chronic-epilepsy phase but was not elevated in SE animals that did not develop AE. Inhibiting the increase in [Ca(2+)](i) during SE with the NMDA channel inhibitor MK801 was associated in all three phases of AE with inhibition of the changes in Ca(2+) dynamics and the development of AE. Ca(2+) homeostatic mechanisms in hippocampal neurons also were altered in the brain-injury, epileptogenesis, and chronic-epilepsy phases of AE. These results provide evidence that [Ca(2+)](i) and Ca(2+)-homeostatic mechanisms are significantly altered during the development of AE and suggest that altered Ca(2+) dynamics may play a role in the induction and maintenance of AE and underlie some of the neuroplasticity changes associated with the epileptic phenotype.

Altered GABAergic inhibitory tone has been observed in association with a number of both acute and chronic models of epilepsy and is believed to be the result, in part, of a decrease in function of the postsynaptic GABAA receptor (GABAAR). This study was carried out to investigate if alterations in receptor internalization contribute to the decrease in GABAAR function observed with epilepsy, utilizing the hippocampal neuronal culture model of low-Mg2+-induced spontaneous recurrent epileptiform discharges (SREDs). Analysis of GABAAR function in "epileptic" cultures showed a 62% reduction in [3H]flunitrazepam binding to the GABAA alpha receptor subunit and a 50% decrease in GABA currents when compared with controls. Confocal microscopy analysis of immunohistochemical staining of GABAAR beta2/beta3 subunit expression revealed approximately a 30% decrease of membrane staining in hippocampal cultures displaying SREDs immediately after low-Mg2+ treatment and in the chronic epileptic state. Low-Mg2+-treated cultures internalized antibody labeled GABAA receptor with an increase in rate of 68% from control. Inhibition of GABAAR endocytosis in epileptic cultures resulted in both a recovery to control levels of membrane GABAA beta2/beta3 immunostaining and a total blockade of SREDs. These results indicate that altered GABAAR endocytosis contributes to the decrease in GABAAR expression and function observed in this in vitro model of epilepsy and plays a role in causing and maintaining SREDs. Understanding the mechanisms underlying altered GABAA R recycling may offer new insights into the pathophysiology of epilepsy and provide novel therapeutic strategies to treat this major neurological condition.

In this report we investigated the effects of the aqueous fraction (AF) isolated from Delphinium denudatum on sustained repetitive firing in cultured neonatal rat hippocampal pyramidal neurons. Blockade of SRF is one of the basic mechanisms of antiepileptic drugs (AED) at the cellular level. The effects of aqueous fraction (0.2-0.6 mg/ml) were compared with the prototype antiepileptic drug, phenytoin (PHT). Using the whole cell current-clamp technique, sustained repetitive firing was elicited in neurons by a depolarizing pulse of 500 ms duration, 0.3 Hz and 0.1-0.6 nA current strength. Similar to phenytoin, aqueous fraction reduced the number of action potentials (AP) per pulse in a concentration-dependent manner until no action potentials were elicited for the remainder of the pulse. There was a corresponding use-dependent reduction in amplitude and Vmax (velocity of upstroke) of action potentials. The Vmax and amplitude of the first action potential was not affected by phenytoin, while aqueous fraction exhibited concentration-dependent reduction. At 0.6 mg/ml aqueous fraction reduced Vmax to 58-63% and amplitude to 16-20% of the control values. The blockade of sustained repetitive firing by aqueous fraction was reversed with hyperpolarization of membrane potential (-65 to -75 mV) while depolarization of membrane potential (-53 to -48 mV) potentiated the block. The results suggest that aqueous fraction blocks sustained repetitive firing in hippocampal neurons in a use-dependent and voltage-dependent manner similar to phenytoin. However, unlike phenytoin, which interacts preferably with the inactive state of the Na+ channel, the compounds present in aqueous fraction apparently also interact with the resting state of the Na+ channels as suggested by dose-dependent reduction of Vmax and amplitude of first AP. We conclude that aqueous fraction contains potent anticonvulsant compounds.

Altered calcium homeostatic mechanisms have been implicated in the development of acquired epilepsy in numerous models. Stroke is one of the leading brain injuries that cause acquired epilepsy, yet little is known concerning the molecular mechanisms underlying stroke-induced epileptogenesis. Recently an in vitro model of stroke-induced epilepsy was developed and characterized as a powerful tool to study the pathophysiology of injury and stroke-induced epileptogenesis. Using this glutamate injury-induced epileptogenesis model, we have investigated the role of altered calcium homeostatic mechanisms in the development and maintenance of stroke-induced epilepsy. Epileptic neurons manifested elevated intracellular calcium levels compared to control neurons independent of neuronal activity and seizure discharge for the remainder of the life of the neurons in culture. In addition, epileptic neurons were found to have alterations in the ability to reduce intracellular calcium levels following a calcium load. These long-term epileptic changes in calcium homeostasis were dependent on calcium during the initial glutamate injury. The data demonstrate that significant alterations in calcium homeostatic mechanisms occur in association with stroke-induced epilepsy and suggest that these changes may play a role in both the induction and maintenance of the epileptic phenotype in this model.

The effects were investigated of a partially purified subfraction (FS-1) isolated from Delphinium denudatum on sustained repetitive firing (SRF) of cultured neonatal rat hippocampal pyramidal neurons. The blockade of sustained repetitive firing is one of the basic mechanisms of antiepileptic drugs at the cellular level. Using the whole cell current-clamp technique, sustained repetitive firing was elicited in pyramidal neurons under study by a depolarizing pulse of 500 ms duration, 0.3 Hz and 0.1-0.6 nA current strength. FS-1 (0.01-0.06 mg/mL) reduced the number of action potentials per pulse in a dose-dependent manner until no action potentials were elicited for the remainder of the pulse. There was a corresponding use-dependent reduction in amplitude and Vmax of action potentials. The Vmax of action potential 1 exhibited a dose-dependent reduction. At a dose of 0.06 mg/mL FS-1 reduced Vmax to 29%-38% and amplitude to 16%-20 % of the control values. The blockade of sustained repetitive firing by FS-1 was reversed by hyperpolarization of the membrane potential (-65 to -75 mV) while depolarization of the membrane potential (-53 mV to -48 mV) potentiated the block. The results suggest that FS-1 blocks sustained repetitive firing in hippocampal neurons in a use-dependent and voltage-dependent manner similar to the prototype anticonvulsant drug, phenytoin. However, unlike phenytoin, which binds preferably to the inactive state, the compounds present in FS-1 also interacted with the resting state of the Na+ channels by reducing Vmax of action potential 1. The results indicate that the partially purified FS-1 subfraction of Delphinium denudatum contains a potent anticonvulsant compound., (Copyright 2003 John Wiley & Sons, Ltd.)

Roots of Delphinium denudatum W. are used for the treatment of epilepsy by traditional healers in subcontinent. Aqueous fraction (AF) isolated from D. denudatum has previously shown significant anticonvulsant activity in in vivo and in vitro models of seizures. We investigated anticonvulsant effects of AF on pentylenetetrazole (PTZ) and bicuculline (BIC)-induced epileptiform activity in primary hippocampal neuronal cultures. Electrophysiological studies on single pyramidal neurons were carried out by using whole-cell current clamp technique. Introduction of AF (0.6 mg/ml) in perfusate blocked PTZ (10 mM) and BIC (100 micro M)-induced epileptiform activity comprising of paroxysmal depolarization shifts (PDS). The PDS were elicited again when AF was removed from perfusate. We conclude that AF contains anticonvulsant compounds that possibly interact with GABA(A) receptor to produce blockade of epileptiform activity. Further studies on isolation of compounds from AF may lead to discovery of new class of anticonvulsants.

Purpose: Stroke is the most common cause of acquired epilepsy. The purpose of this investigation was to characterize the role of calcium in the in vitro, glutamate injury-induced epileptogenesis model of stoke-induced epilepsy., Methods: Fura-2 calcium imaging and whole-cell current clamp electrophysiology techniques were used to measure short-term changes in neuronal free intracellular calcium concentration and long-term alterations in neuronal excitability in response to epileptogenic glutamate injury (20 microM, 10 min) under various extracellular calcium conditions and in the presence of different glutamate-receptor antagonists., Results: Glutamate injury-induced epileptogenesis was associated with prolonged, reversible elevations of free intracellular calcium concentration during and immediately after injury and chronic hyperexcitability manifested as spontaneous recurrent epileptiform discharges for the remaining life of the cultures. Epileptogenic glutamate exposure performed in solutions containing low extracellular calcium, barium substituted for calcium, or N-methyl-d-aspartate (NMDA)-receptor antagonists reduced the duration of intracellular calcium elevation and inhibited epileptogenesis. Antagonism of non-NMDA-receptor subtypes had no effect on glutamate injury-induced calcium changes or the induction epileptogenesis. The duration of the calcium elevation and the total calcium load statistically correlated with the development of epileptogenesis. Comparable elevations in neuronal calcium induced by non-glutamate receptor-mediated pathways did not cause epileptogenesis., Conclusions: This investigation indicates that the glutamate injury-induced epileptogenesis model of stroke-induced epilepsy is calcium dependent and requires NMDA-receptor activation. Further, these experiments suggest that prolonged, reversible elevations in neuronal free intracellular calcium initiate the long-term plasticity changes that underlie the development of injury-induced epilepsy.

Several models that develop epileptiform discharges and epilepsy have been associated with a decrease in the activity of calmodulin-dependent kinase II. However, none of these studies has demonstrated a causal relationship between a decrease in calcium/calmodulin kinase II activity and the development of seizure activity. The present study was conducted to determine the effect of directly reducing calcium/calmodulin-dependent kinase activity on the development of epileptiform discharges in hippocampal neurons in culture. Complimentary oligonucleotides specific for the alpha subunit of the calcium/calmodulin kinase were used to decrease the expression of the enzyme. Reduction in kinase expression was confirmed by Western analysis, immunocytochemistry, and exogenous substrate phosphorylation. Increased neuronal excitability and frank epileptiform discharges were observed after a significant reduction in calmodulin kinase II expression. The epileptiform activity was a synchronous event and was not caused by random neuronal firing. Furthermore, the magnitude of decreased kinase expression correlated with the increased neuronal excitability. The data suggest that decreased calmodulin kinase II activity may play a role in epileptogenesis and the long-term plasticity changes associated with the development of pathological seizure activity and epilepsy.

Purpose: This study was undertaken to evaluate the effects of topiramate (TPM) on excitatory amino acid-evoked currents., Methods: Kainate and N-methyl-D-aspartate (NMDA) were applied to cultured rat hippocampal neurons by using a concentration-clamp apparatus to selectively activate the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)/kainate and NMDA receptor subtypes, respectively. The evoked membrane currents were recorded by using perforated-patch whole-cell voltage-clamp techniques., Results: TPM partially blocked kainate-evoked currents with an early-onset reversible phase (phase I) and a late-onset phase (phase II) that occurred after a 10- to 20-min delay and did not reverse during a 2-h washout period. Application of dibutyryl cyclic adenosine monophosphate (cAMP; 2 mM) during washout after phase II block enhanced reversal, with the kainate current amplitude being restored by approximately 50%. Phase II but not phase I block was prevented by prior application of okadaic acid (1 microM), a broad-spectrum phosphatase inhibitor, suggesting that phase II block may be mediated through interactions with intracellular intermediaries that alter the phosphorylation state of kainate-activated channels. Topiramate at 100 microM blocked kainate-evoked currents by 90% during phase II, but had no effect on NMDA-evoked currents. The median inhibitory concentration (IC50) values for phase I and II block of kainate currents were 1.6 and 4.8 microM, respectively, which are within the range of free serum levels of TPM in patients., Conclusions: The specific blockade of the kainate-induced excitatory conductance is consistent with the ability of TPM to reduce neuronal excitability and could contribute to the anticonvulsant efficacy of this drug.

Purpose: In this study, we examined the effects of topiramate (TPM) on the electrophysiologic properties of cultured rat hippocampal pyramidal neurons., Methods: Whole-cell current-clamp recording techniques were used to determine the effects of TPM on sustained repetitive firing (SRF), spontaneous epileptiform-burst firing, and spontaneous recurrent seizures (SRS)., Results: Topiramate at therapeutic concentrations (10-100 microM) significantly decreased or abolished SRF in a dose-dependent and partially reversible manner. When transiently exposed to a medium in which Mg2+ is omitted, hippocampal neurons in culture develop SRS ("epilepsy") and epileptiform discharges. Application of TPM at concentrations ranging from 10 to 100 microM to cells displaying seizure activity caused a concentration-dependent decrease in the number of action potentials within a burst and in the average duration of epileptiform activity. Both effects were partially reversed during a 5- to 30-min drug washout period., Conclusions: These effects on the electrophysiologic properties of cultured neurons are consistent with the concept that TPM exerts modulatory effects on voltage-dependent Na+ and/or Ca2+ conductances responsible for the generation and propagation of action potentials. Topiramate also may inhibit synaptic conductances responsible for transmission of epileptiform discharges.

The molecular basis for developing symptomatic epilepsy (epileptogenesis) remains ill defined. We show here in a well characterized hippocampal culture model of epilepsy that the induction of epileptogenesis is Ca2+-dependent. The concentration of intracellular free Ca2+ ([Ca2+]i) was monitored during the induction of epileptogenesis by prolonged electrographic seizure activity induced through low-Mg2+ treatment by confocal laser-scanning fluorescent microscopy to directly correlate changes in [Ca2+]i with alterations in membrane excitability measured by intracellular recording using whole-cell current-clamp techniques. The induction of long-lasting spontaneous recurrent epileptiform discharges, but not the Mg2+-induced spike discharges, was prevented in low-Ca2+ solutions and was dependent on activation of the N-methyl-D-aspartate (NMDA) receptor. The results provide direct evidence that prolonged activation of the NMDA-Ca2+ transduction pathway causes a long-lasting plasticity change in hippocampal neurons causing increased excitability leading to the occurrence of spontaneous, recurrent epileptiform discharges.

Cultured rat hippocampal neurons previously exposed to a media containing no added Mg2+ for 3 h begin to spontaneously trigger recurrent epileptiform discharges following return to normal medium, and this altered population epileptiform activity persisted for the life of the neurons in culture (> 2 wk). Neurons in "epileptic" cultures appeared similar in somatic and dendritic morphology and cellular density to control, untreated cultures. In patch-clamp recordings from hippocampal pyramidal cells from "epileptic," low Mg2+ pretreated hippocampal cultures, a rapid (within 2 h of treatment), permanent (lasting > or = 8 days) and statistically significant 50-65% reduction in the current density of functional gamma-aminobutyric acid-A (GABA(A)) receptors was evident when the GABA responses of these cells were compared with control neurons. Functional GABA receptor current density was calculated by determining the maximal response of a cell to GABA 1 mM application and normalizing this response to cellular capacitance. Despite the marked GABA efficacy differences noted above, the potency of GABA in activating chloride currents was not significantly different when the responses to control and "epileptic" pyramidal cells to multiple concentrations of GABA were compared. The EC50 for GABA was 4.5 +/- 0.2 (mean +/- SE) for control neurons and 3.5 +/- 0.4 microM, 5.2 +/- 0.5 microM, 3.7 +/- 0.3 microM, and 4.6 +/- 0.3 microM for epileptic neurons 2 h, 2 days, 3 days, and 8 days after low Mg2+ pretreatment, respectively. Modulation of GABA responses by the benzodiazepine, clonazepam, was significantly reduced in epileptic neurons compared with controls. The kinetically determined clonazepam 100 nM GABA augmentation efficacy decreased from 44.1% in control neurons to 9.3% augmentation in neurons recorded from cultures 10 days posttreatment. The kinetics of GABA current block by the noncompetitive antagonist picrotoxin were determined in hippocampal cultured neurons, and an IC50 of 14 microM determined. Bath application of picrotoxin at half of the IC50 concentration (7 microM) induced epileptiform activity in control cultures and this activity appeared very similar to the epileptiform activity induced by prior low Mg2+ treatment. This concentration of picrotoxin was determined experimentally to block 30% of the GABA(A)-mediated receptor responses in these cultures, and this level of block was sufficient to trigger spontaneous epileptiform activity. The 50% reduction of GABA responses induced as a permanent consequence of low Mg2+ treatment therefore was determined to be sufficient in and of itself to induce the spontaneous epileptiform activity, which was also a consequence of this treatment.

The hippocampus is especially vulnerable to excitotoxicity and delayed neuronal cell death. Chronic elevations in free intracellular calcium concentration ([Ca2+]i) following glutamate-induced excitotoxicity have been implicated in contributing to delayed neuronal cell death. However, no direct correlation between delayed cell death and prolonged increases in [Ca2+]i has been determined in mature hippocampal neurons in culture. This investigation was initiated to determine the statistical relationship between delayed neuronal cell death and prolonged increases in [Ca2+]i in mature hippocampal neurons in culture. Using indo-1 confocal fluorescence microscopy, we observed that glutamate induced a rapid increase in [Ca2+]i that persisted after the removal of glutamate. Following excitotoxic glutamate exposure, neurons exhibited prolonged increases in [Ca2+]i, and significant delayed neuronal cell death was observed. The N-methyl-D-aspartate (NMDA) channel antagonist MK-801 blocked the prolonged increases in [Ca2+]i and cell death. Depolarization of neurons with potassium chloride (KCl) resulted in increases in [Ca2+]i, but these increases were buffered immediately upon removal of the KCl, and no cell death occurred. Linear regression analysis revealed a strong correlation (R = 0.973) between glutamate-induced prolonged increases in [Ca2+]i and delayed cell death. These data suggest that excitotoxic glutamate exposure results in an NMDA-induced inability to restore resting [Ca2+]i (IRRC) that is a statistically significant indicator of delayed neuronal cell death.

Activation of excitatory amino acid (EAA) receptors in cultured hippocampal neurons causes down-regulation of the protein ligatin, a receptor for phosphoglycoproteins and a marker protein for membrane-vesicle transport systems. This reduction occurs at both physiologic and excitotoxic levels of glutamate stimulation and is accompanied by a significant decrease in steady state levels of ligatin mRNA. Reduction in ligatin mRNA occurs within 60 min and persists 24 h later. Steady state levels of mRNAs encoding cyclophilin, an ubiquitous cytosolic protein, and neuron specific-enolase (N-SE) are not diminished by glutamate receptor activation, demonstrating that down-regulation of ligatin mRNA was not a result of general catabolism. Further, this reduction in ligatin mRNA occurred without induction of HSP 70. Pharmacological studies using selective antagonists and agonists indicate that this down-regulation of ligatin gene expression is predominantly mediated by the N-methyl-D-aspartate (NMDA) subclass of EAA receptors and that Ca2+ is required. This is the first report that EAA receptor activation in hippocampal neurons can pretranslationally down-regulate gene expression in a rapid and long-lasting manner under physiologic, as well as cytotoxic conditions. The data support the hypothesis that modulation of neuronal gene expression may represent a molecular mechanism mediating some of the long-lasting functional and pathophysiological effects of EAA on cell function.

Intracellular recording revealed that cytotoxic activation of excitatory amino acid receptors by glutamate or N-methyl-D-aspartate (NMDA) elicited an extended neuronal depolarization (END) of at least 5 h duration following washout of glutamate in hippocampal neurons in culture. During END, cells were still responsive to glutamate, and still able to fire sodium spikes. END induction could be blocked by concurrent application of D-2-amino-5-phosphonovalerate (APV) or MK-801, but not 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), during the glutamate exposure. The induction of END by excitotoxic glutamate receptor activation may play a role in the pathophysiology of glutamate toxicity.

Habituation of excitatory synaptic inputs onto identified motor neurons of the locust metathoracic ganglion, driven electrically and by natural stimuli, was examined using intracellular recording. Rapid progressive reduction in amplitude of EPSPs from a variety of inputs onto fast-type motor neurons occurred. The habituated EPSPs were quickly dishabituated by iontophoretic release of octopamine from a microelectrode into the neuropilar region of presumed synaptic action. The zone within which release was effective for a given neuron was narrowly-defined. With larger amounts of octopamine applied at a sensitive site the EPSP became larger than normal, and in many instances action potentials were initiated by the sensitized response. Very small EPSPs onto a motor neuron, which were associated with proprioceptive feedback, and which were originally too small to be detected above the noise, were potentiated to a level of several mV by the iontophoresed octopamine. A DUM neuron (presumed to be octopaminergic) was found, whose direct stimulation was followed by a strong dishabituating and sensitizing action leading to spikes, of inputs to an identified flexor tibiae motor neuron. The action and its time course were closely similar to those evoked by octopamine iontophoresed into the neuropil in the region of synaptic inputs to the motor neuron. It is concluded that DUM (octopaminergic) neurons exert large potentiating actions on central neuronal excitatory synaptic transmission in locusts.

The natural insect neuromodulator octopamine (OCT) was released iontophoretically into regions of neuropil in locust metathoracic ganglia. A narrowly-defined site was found on one side of the ganglion at which release caused a prolonged bout of repetitive flex-extend-flex movements of the tibia on the injected side, at a frequency of from 2-3.5 Hz. When a bout had terminated, repetition of the OCT release caused an extremely similar bout to occur, and again with further treatments, indefinitely. OCT iontophoresis at the equivalent site on the contralateral side caused the contralateral flexor to make stepping movements. Two sites were found, in each half of the ganglion, at which similar OCT release evoked a bout of flight motor activity at 10 Hz. The flight bout involved both sides synchronously and nearly equally, except for a slightly greater motor output on the injected side. Evoked bouts lasted from 20 sec to 25 min depending on the preparation and amount of OCT released. At a site in the 6th abdominal ganglion of mature female locusts OCT release suppressed ongoing rhythmic oviposition digging evoked by severing the ventral nerve cord. A number of previously undescribed DUM neurons was encountered and their dendritic patterns, which are distinctive, determined following dye injection. A hypothesis, termed the Orchestration Hypothesis is presented, which considers how modulator neurons such as locust octopaminergic neurons, might be involved in the generation of specific behaviors.

It is generally believed that neural transmission in the central nervous systems of insects is cholinergic, on the basis of secondary evidence: the presence of cholinesterase, and sensitivity of a nonsynaptic region of the neuron, its cell body, to iontophoresed acetylcholine. In the present work a preparation has been developed which takes advantage of the availability of identified motor neurons in the locust metathoracic ganglion with known 3-dimensional geometry of dendritic fields. These neurons transmit at their peripheral neuromuscular junctions with glutamate. The fast extensor tibiae motor neuron also makes excitatory central connections onto its functional antagonists the flexor tibiae motor neurons. Unless Dale's principle is contravened, transmission at these central synapses should also be glutamatergic. This transmission onto flexor motor neurons was found to be attenuated 70% by a glutamatergic blocker. Glutamate iontophoresed into selected areas of neuropil into which the motor neurons have dendritic branches caused the neurons to be depolarized, in a dose-dependent manner. Individual motor neurons were directly excited to spike with suprathreshold iontophoretic current. With long durations of release they were desensitized, but recovered quickly with rest. The data provide evidence that central transmission onto motor neurons in the locust metathoracic ganglion is glutamatergic.