Microelectrode array-based system for neuropharmacological applications with cortical neurons cultured in vitro

Microelectrode arrays (MEAs) provide a means to investigate the electrophysiological behavior of neuronal systems through the measurements from neuronal culture preparations. Changes in activity patterns of neuronal networks are usually detected by applying neural chemicals. Because of the difficulties of fabricating the arrays, and the delicate and less reliable properties of cortical neurons, MEA-based systems with cortical neuronal networks for neurophamacological applications are technically difficult, therefore restricting their utility. Here, we report a new approach to the development of such MEA-based system with sensitive and durable MEAs conveniently fabricated and the culture conditions optimized. Upon growth differentiation, cortical neurons, cultured directly on MEAs, reach a developmentally stable and reliable activity state. With this system, we monitored the global spontaneous activities of neuronal networks and demonstrated the fine discrimination for specific substances and unique property of cortical neurons, which validated both the applicability and necessity of such system in pharmacological bioassay.     

Networks of neurons coupled to microelectrode arrays: a neuronal sensory system for pharmacological applications

Two main features make microelectrode arrays (MEAs) a valuable tool for electrophysiological measurements under the perspective of pharmacological applications, namely: (i) they are non-invasive and permit, under appropriate conditions, to monitor the electrophysiological activity of neurons for a long period of time (i.e. from several hours up to months); (ii) they allow a multi-site recording (up to tens of channels). Thus, they should allow a high-throughput screening while reducing the need for animal experiments. In this paper, by taking advantages of these features, we analyze the changes in activity pattern induced by the treatment with specific substances, applied on dissociated neurons coming from the chick-embryo spinal cord. Following pioneering works by Gross and co-workers (see e.g. Gross and Kowalski, 1991. Neural Networks, Concepts, Application and Implementation, vol. 4. Prentice Hall, NJ, pp. 47-110; Gross et al., 1992. Sensors Actuators, 6, 1-8.), in this paper analysis of the drugs' effects (e.g. NBQX, CTZ, MK801) to the collective electrophysiological behavior of the neuronal network in terms of burst activity, will be presented. Data are simultaneously recorded from eight electrodes and besides variations induced by the drugs also the correlation between different channels (i.e. different area in the neural network) with respect to the chemical stimuli will be introduced (Bove et al., 1997. IEEE Trans. Biomed. Eng., 44, 964-977.). Cultured spinal neurons from the chick embryo were chosen as a neurobiological system for their relative simplicity and for their reproducible spontaneous electrophysiological behavior. It is well known that neuronal networks in the developing spinal cord are spontaneously active and that the presence of a significant and reproducible bursting activity is essential for the proper formation of muscles and joints (Chub and O'Donovan, 1998. J. Neurosci., 1, 294-306.). This fact, beside a natural variability among different biological preparations, allows a comparison also among different experimental session giving reliable results and envisaging a definition of a bioelectronic 'neuronal sensory system'.     

Network dynamics and synchronous activity in cultured cortical neurons

Neurons extracted from specific areas of the Central Nervous System (CNS), such as the hippocampus, the cortex and the spinal cord, can be cultured in vitro and coupled with a micro-electrode array (MEA) for months. After a few days, neurons connect each other with functionally active synapses, forming a random network and displaying spontaneous electrophysiological activity. In spite of their simplified level of organization, they represent an useful framework to study general information processing properties and specific basic learning mechanisms in the nervous system. These experimental preparations show patterns of collective rhythmic activity characterized by burst and spike firing. The patterns of electrophysiological activity may change as a consequence of external stimulation (i.e., chemical and/or electrical inputs) and by partly modifying the "randomness" of the network architecture (i.e., confining neuronal sub-populations in clusters with micro-machined barriers). In particular we investigated how the spontaneous rhythmic and synchronous activity can be modulated or drastically changed by focal electrical stimulation, pharmacological manipulation and network segregation. Our results show that burst firing and global synchronization can be enhanced or reduced; and that the degree of synchronous activity in the network can be characterized by simple parameters such as cross-correlation on burst events.     

Magnetic stimulation of one-dimensional neuronal cultures

Transcranial magnetic stimulation is a remarkable tool for neuroscience research, with a multitude of diagnostic and therapeutic applications. Surprisingly, application of the same magnetic stimulation directly to neurons that are dissected from the brain and grown in vitro was not reported to activate them to date. Here we report that central nervous system neurons patterned on large enough one-dimensional rings can be magnetically stimulated in vitro. In contrast, two-dimensional cultures with comparable size do not respond to excitation. This happens because the one-dimensional pattern enforces an ordering of the axons along the ring, which is designed to follow the lines of the magnetically induced electric field. A small group of sensitive (i.e., initiating) neurons respond even when the network is disconnected, and are presumed to excite the entire network when it is connected. This implies that morphological and electrophysiological properties of single neurons are crucial for magnetic stimulation. We conjecture that the existence of a select group of neurons with higher sensitivity may occur in the brain in vivo as well, with consequences for transcranial magnetic stimulation.     

Drug evaluations using neuronal networks cultured on microelectrode arrays

We used spontaneously active neuronal networks derived from dissociated embryonic murine spinal cord and auditory cortex and grown on substrate-integrated thin-film microelectrodes to determine characteristic responses to the cannabinoid agonists anandamide (AN) and methanandamide (MA). AN and MA reversibly inhibited spike and burst production in both tissue types. Responses of 21 cultures ranging in age from 23 to 111 days in vitro (d.i.v.) showed high intra- and inter-culture reproducibility at all ages. However, responses were tissue and substance-dependent. AN and MA were equipotent in cortical cultures and terminated bursting and spiking at 2.5 +/- 0.9 microM (n = 10). Spinal cultures were shut-off by 1.3 +/- 0.7 microM (n = 15) AN, but required 5.8 +/- 1.2 microM MA for activity cessation. MA, but not AN, demonstrated a biphasic influence: excitation at 0.25-3.5 microM and suppression at 4-7.1 microM. Palmitoylethanolamide, a related lipophilic molecule with no reported binding to the CBI receptor (to which AN and MA bind in the central nervous system), did not affect network activity at concentrations up to 6.5 microM. Irreversible cessation of activity was observed after 30 min applications of AN or MA at > 7 microM.     

Effect of lamotrigine on a novel model of epilepsy

The effect of lamotrigine (LTG) on evoked and spontaneous seizure-like activity induced by veratridine, was investigated. Rat brain slices were examined using conventional electrophysiological intracellular techniques. Alteration of sodium channel function by veratridine (0.3 microM) induced spontaneous seizure-like activity in the hippocampal CA1 pyramidal neurons. Therapeutic concentrations of LTG (5-10 microM) inhibited both evoked and spontaneous bursting induced by veratridine. This inhibition was voltage-dependent indicating possible interaction between the drug and the inactivated state of sodium channels. There was an increase in the firing threshold of the bursting but no change in the resting membrane potential (RMP) and membrane input resistance. Results from this work suggest that the veratridine model of epilepsy is very sensitive to drugs which act on sodium channels. These data make the veratridine model a suitable tool for screening potential sodium channel-dependent antiepileptic drugs.     

Effect of moxonidine on putative sympathetic neurons in the rostral ventrolateral medulla of the rat

We used an intracellular recording technique in vitro to investigate the effects of moxonidine on neurons in the rostral ventrolateral medulla (RVLM) with electrophysiological properties similar to premotor sympathetic neurons in vivo. These neurons were classified as firing regularly and irregularly, according to previous reports. Moxonidine is a sympathoinhibitory and antihypertensive agent that is thought to be a ligand of alpha(2)-adrenergic receptors and imidazoline type-1 receptors in the RVLM. Moxonidine (2-10 microM) was applied to the perfusate on 4 irregularly firing neurons, and 2 regularly firing neurons. Moxonidine (2 microM) produced only minor depolarization in 2 of these neurons. However, on 4 tested neurons, moxonidine (10 microM) elicited a profound inhibitory effect with hyperpolarization (near -20 mV); these neurons practically ceased firing. These changes were partially reversible. The results would indicate that neurons in the RVLM, recorded in vitro and with similar electrophysiological characteristics to a group of neurons previously identified in vivo in the same bulbar region as barosensitive premotor sympathetic neurons, can be modulated by imidazoline-derivative adrenergic agonists. These results could help to understand the hypotensive effects of moxonidine.     

Resolution, configurational assignment, and enantiopharmacology at glutamate receptors of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and demethyl-ACPA

We have previously described (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) as a potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of (S)-glutamic acid (Glu) receptors. We now report the chromatographic resolution of ACPA and (RS)-2-amino-3-(3-carboxy-4-isoxazolyl)propionic acid (demethyl-ACPA) using a Sumichiral OA-5000 column. The configuration of the enantiomers of both compounds have been assigned based on X-ray crystallographic analyses, supported by circular dichroism spectra and elution orders on chiral HPLC columns. Furthermore, the enantiopharmacology of ACPA and demethyl-ACPA was investigated using radioligand binding and cortical wedge electrophysiological assay systems and cloned metabotropic Glu receptors. (S)-ACPA showed high affinity in AMPA binding (IC(50) = 0.025 microM), low affinity in kainic acid binding (IC(50) = 3.6 microM), and potent AMPA receptor agonist activity on cortical neurons (EC(50) = 0.25 microM), whereas (R)-ACPA was essentially inactive. Like (S)-ACPA, (S)-demethyl-ACPA displayed high AMPA receptor affinity (IC(50) = 0.039 microM), but was found to be a relatively weak AMPA receptor agonist (EC(50) = 12 microM). The stereoselectivity observed for demethyl-ACPA was high when based on AMPA receptor affinity (eudismic ratio = 250), but low when based on electrophysiological activity (eudismic ratio = 10). (R)-Demethyl-ACPA also possessed a weak NMDA receptor antagonist activity (IC(50) = 220 microM). Among the enantiomers tested, only (S)-demethyl-ACPA showed activity at metabotropic receptors, being a weak antagonist at the mGlu(2) receptor subtype (K(B) = 148 microM).     

Chronic exposure to U18666A induces apoptosis in cultured murine cortical neurons

Niemann-Pick disease type C (NPC) is a juvenile neurodegenerative disorder characterized by premature neuronal loss and altered cholesterol metabolism. Previous reports applying an 8-h exposure of U18666A, a cholesterol transport-inhibiting agent, demonstrated a dose-dependent reduction in beta-amyloid (Abeta) deposition and secretion in cortical neurons, with no significant cell injury. In the current study, we examined the chronic effect of 24-72h of U18666A treatment on primary cortical neurons and several cell lines. Our results showed caspase-3 activation and cellular injury in U18666A-treated cortical neurons but not in the cell lines, suggesting cell death by apoptosis only occurred in cortical neurons after chronic exposure to U18666A. We also demonstrated through filipin staining the accumulation of intracellular cholesterol in cortical neurons treated with U18666A, indicating the phenotypic mimic of NPC by U18666A. However, additions of 10 and 25microM pravastatin with 0.5microg/ml U18666A significantly attenuated toxicity. Taken together, these data showed for the first time that U18666A induces cell death by apoptosis and suggested an important in vitro model system to study NPC.     

Role of nucleotide- and base-excision repair in genotoxin-induced neuronal cell death

Base-excision (BER) and nucleotide-excision (NER) repair play pivotal roles in protecting the genomes of dividing cells from damage by endogenous and exogenous agents (i.e. environmental genotoxins). However, their role in protecting the genome of post-mitotic neuronal cells from genotoxin-induced damage is less clear. The present study examines the role of the BER enzyme 3-alkyladenine DNA glycosylase (AAG) and the NER protein xeroderma pigmentosum group A (XPA) in protecting cerebellar neurons and astrocytes from chloroacetaldehyde (CAA) or the alkylating agent 3-methyllexitropsin (Me-Lex), which produce ethenobases or 3-methyladenine (3-MeA), respectively. Neuronal and astrocyte cell cultures prepared from the cerebellum of wild type (C57BL/6) mice or Aag(-/-) or Xpa(-/-) mice were treated with 0.1-50 microM CAA for 24h to 7 days and examined for cell viability, DNA fragmentation (TUNEL labeling), nuclear changes, and glutathione levels. Aag(-/-) neurons were more sensitive to the acute (>20 microM) and long-term (>5 microM) effects of CAA than comparably treated wild type neurons and this sensitivity correlated with the extent of DNA fragmentation and nuclear changes. Aag(-/-) neurons were also sensitive to Me-Lex at comparable concentrations of CAA. In contrast, Xpa(-/-) neurons were more sensitive than either wild type or Aag(-/-) neurons to CAA (>10 microM), but less sensitive than Aag(-/-) neurons to Me-Lex. Astrocytes from the cerebellum of wild type, Aag(-/-) or Xpa(-/-) mice were essentially insensitive to CAA at the concentrations tested. These studies demonstrate that BER and NER are required to protect neurons from genotoxin-induced cell death.     

Effects of neuromuscular blocking agents on central respiratory chemosensitivity in newborn rats

Neuromuscular blocking agents suppress central respiratory activity through their inhibitory effects on preinspiratory neurons and the synaptic drive from preinspiratory neurons to inspiratory neurons. Central CO2-chemosensitive areas, which partly consist of CO2-excited neurons, in the rostral ventrolateral medulla are thought to provide tonic drive to the central respiratory network and involve cholinergic mechanisms, which led us to hypothesize that neuromuscular blocking agents can inhibit CO2-excited neurons and attenuate respiratory CO2 responsiveness. To test this hypothesis, we used isolated brainstem-spinal cord preparations from newborn rats. The increase of C4 burst frequency induced by a hypercapnic superfusate, i.e. respiratory CO2 responsiveness, was suppressed by the application of neuromuscular blocking agents, either d-tubocurarine (10, 100 microM) or vecuronium (100 microM). These agents (40 microM) also induced hyperpolarization and decreases in firing frequency of CO2-excited neurons in the rostral ventrolateral medulla. Our results demonstrate that neuromuscular blocking agents inhibit CO2-excited tonic firing neurons and attenuate respiratory CO2 responsiveness.     

Neurotrophic activity of honokiol on the cultures of fetal rat cortical neurons

Honokiol, a main biphenyl neolignan of the traditional crude medicine, Magnoliae cortex, was found to show neurotrophic activity on the cultures of rat cortical neurons at concentration from 0.1 to 10 microM. In the cortical neurons cultured in serum-free medium supplemented with B27, honokiol could promote neurite outgrowth. In addition, the survival and growth of neurons were significantly enhanced by adding honokiol to the primary cultures in serum-free medium supplemented with N2. Its neurotrophic activity was comparable to 40 ng mL(-1) of bFGF at concentration of 10 microM.     

Alpha2A-adrenoceptors strengthen working memory networks by inhibiting cAMP-HCN channel signaling in prefrontal cortex

Spatial working memory (WM; i.e., "scratchpad" memory) is constantly updated to guide behavior based on representational knowledge of spatial position. It is maintained by spatially tuned, recurrent excitation within networks of prefrontal cortical (PFC) neurons, evident during delay periods in WM tasks. Stimulation of postsynaptic alpha2A adrenoceptors (alpha2A-ARs) is critical for WM. We report that alpha2A-AR stimulation strengthens WM through inhibition of cAMP, closing Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels and strengthening the functional connectivity of PFC networks. Ultrastructurally, HCN channels and alpha2A-ARs were colocalized in dendritic spines in PFC. In electrophysiological studies, either alpha2A-AR stimulation, cAMP inhibition or HCN channel blockade enhanced spatially tuned delay-related firing of PFC neurons. Conversely, delay-related network firing collapsed under conditions of excessive cAMP. In behavioral studies, either blockade or knockdown of HCN1 channels in PFC improved WM performance. These data reveal a powerful mechanism for rapidly altering the strength of WM networks in PFC.     

Effects of docosahexaenoic acid on the survival and neurite outgrowth of rat cortical neurons in primary cultures

Effects of docosahexaenoic acid (DHA) on survival and neurite outgrowth were investigated in primary cultures of rat cortical neurons. Cell cultures were prepared from cortex on embryonic day 18 (E-18) for treatment with a series of DHA concentrations (12.5, 25, 50, 75, 100 and 200 microM). Docosahexaenoic acid (25-50 microM) significantly enhanced neuronal viability, but lower concentration of DHA (12.5 microM) did not show an obvious effect. In contrast, higher concentrations of DHA (100-200 microM) exerted the significant opposite effects by decreasing neuronal viability. Furthermore, treatment with 25 microM DHA significantly prevented the neurons from death after different culture days in vitro (DIV). Moreover, measurements from the cultures exposed to 25 microM DHA immediately after plating showed significant increases in the percentage of cells with neurites, the mean number of neurite branches, the total neuritic length per cell and the length of the longest neurite in each cell after 24 and 48 h in vitro (HIV). The DHA-treated neurons had greater growth-associated protein-43 (GAP-43) immunoactivity and higher phosphatidylserine (PS) and phosphatidylethanolamine (PE) contents, but lower phosphatidylcholine (PC) content than control neurons. The significant increased DHA contents were also observed in both PE and PS in the treated neurons. These findings suggest that optimal DHA (25 microM) may have positive effects on the survival and the neurite outgrowth of the cultured fetal rat cortical neurons, and the effects probably are related to DHA-stimulating neuron-specific protein synthesis and its enhancing the discrete phospholipid (PL) content through enrichment of DHA in the PL species.     

Platelet-activating factor in the enteric nervous system of the guinea pig small intestine

Platelet-activating factor (PAF) is a proinflammatory mediator that may influence neuronal activity in the enteric nervous system (ENS). Electrophysiology, immunofluorescence, Western blot analysis, and RT-PCR were used to study the action of PAF and the expression of PAF receptor (PAFR) in the ENS. PAFR immunoreactivity (IR) was expressed by 6.9% of the neurons in the myenteric plexus and 14.5% of the neurons in the submucosal plexus in all segments of the guinea pig intestinal tract as determined by double staining with anti-human neuronal protein antibody. PAFR IR was found in 6.1% of the neurons with IR for calbindin, 35.8% of the neurons with IR for neuropeptide Y (NPY), 30.6% of the neurons with IR for choline acetyltransferase (ChAT), and 1.96% of the neurons with IR for vasoactive intestinal peptide (VIP) in the submucosal plexus. PAFR IR was also found in 1.5% of the neurons with IR for calbindin, 51.1% of the neurons with IR for NPY, and 32.9% of the neurons with IR for ChAT in the myenteric plexus. In the submucosal plexus, exposure to PAF (200-600 nM) evoked depolarizing responses (8.2 +/- 3.8 mV) in 12.4% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.5% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology, whereas in the myenteric preparations, depolarizing responses were elicited by a similar concentration of PAF in 9.5% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.0% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology. The results suggest that subgroups of secreto- and musculomotor neurons in the submucosal and myenteric plexuses express PAFR. Coexpression of PAFR IR with ChAT IR in the myenteric plexus and ChAT IR and VIP IR in the submucosal plexus suggests that PAF, after release in the inflamed bowel, might act to elevate the excitability of submucosal secretomotor and myenteric musculomotor neurons. Enhanced excitability of motor neurons might lead to a state of neurogenic secretory diarrhea.     

Minimal Hodgkin–Huxley type models for different classes of cortical and thalamic neurons

We review here the development of Hodgkin–Huxley (HH) type models of cerebral cortex and thalamic neurons for network simulations. The intrinsic electrophysiological properties of cortical neurons were analyzed from several preparations, and we selected the four most prominent electrophysiological classes of neurons. These four classes are “fast spiking” “regular spiking” “intrinsically bursting” and “low-threshold spike” cells. For each class, we fit “minimal” HH type models to experimental data. The models contain the minimal set of voltage-dependent currents to account for the data. To obtain models as generic as possible, we used data from different preparations in vivo and in vitro, such as rat somatosensory cortex and thalamus, guinea-pig visual and frontal cortex, ferret visual cortex, cat visual cortex and cat association cortex. For two cell classes, we used automatic fitting procedures applied to several cells, which revealed substantial cell-to-cell variability within each class. The selection of such cellular models constitutes a necessary step towards building network simulations of the thalamocortical system with realistic cellular dynamical properties.     

'Non-synaptic' mechanisms in seizures and epileptogenesis

The role of 'non-synaptic' mechanisms (i.e. those mechanisms that are independent of active chemical synpases) in the synchronization of neuronal activity during seizures and their possible contribution to chronic epileptogenesis are summarized. These 'non-synaptic' mechanisms include electrotonic coupling through gap junctions, electrical field effects (i.e. ephaptic transmission), and ionic interactions (e.g. increases in the extracellular concentration of K(+)). Several lines of evidence indicate that granule cells and pyramidal cells of the hippocampus, and probably other cortical neurons, can generate synchronized electrical activity after active chemical synaptic transmission has been blocked. This synchronized activity is sensitive to alterations in the size of the extracellular space, thus suggesting that electrical field effects and ionic mechanisms contribute to this synchronized activity. Recent studies also indicate that 'non-synaptic' synchronization is quite prominent early in development. Electrophysiological data from hippocampal and neocortical slices have led to a re-interpretation of the fast prepotentials (i.e. partial spikes) recorded in cortical pyramidal cells, suggesting that they may not be due to dendritic spike generation. Improvement in freeze-fracture ultrastructural techniques have led to a re-assessment of previous data on gap junctions in the nervous system and opened new approaches to the quantitative analysis and characterization of gap junctions on glia and neurons. Finally, new methods of dye/tracer coupling have the potential to provide a more rigorous basis for evaluating gap junctions and electrotonic communication between neurons in the mammalian central nervous system. Therefore, recent data continue to suggest that gap junctions and electrotonic coupling play an important role in neural integration, although additional studies using new techniques will be needed to address some of the controversial issues that have arisen over the last several decades.     

Seasonal changes in intrinsic electrophysiological activity of song control neurons in wild song sparrows

Song behavior and its underlying neural substrate can change seasonally in adult songbirds. To test whether environmental cues induce seasonal changes in electrophysiological characteristics of song control neurons, we measured in vitro intrinsic neuronal activity in the song control nucleus RA of adult male song sparrows (Melospiza melodia) in both the fall non-breeding and spring breeding seasons. We found that RA neurons in spring-captured birds show a more than threefold increase in spontaneous firing rate compared to those from fall-captured birds. We conclude that environmental cues are sufficient to induce seasonal changes in electrophysiological properties of song control neurons, and that changes in these properties may underlie seasonal changes in song behavior.     

Anisotropic connectivity and cooperative phenomena as a basis for orientation sensitivity in the visual cortex

A computer simulation model of the neural circuitry underlying orientation sensitivity in cortical neurons is examined. The model consists of a network of 3000 neurons divided into two functionally distinct cell types: excitatory (E-cells) and inhibitory (I-cells). We demonstrate that both orientation sensitivity and shape selectivity can be accounted for by making the following assumptions: 1) thalamic afferents to a sheet of cortical neurons are retionotopically organized; 2) thalamic afferents come from a single neuron, or at most a few neurons, in the lateral geniculate nucleus; 3) cortical activity is cooperative, i.e. largely dependent on intracortical connections, some of which have anisotropies along directions parallel to the pial surface. Anisotropies are specified only by the distribution of cells which are postsynaptic to a particular neuron, without specifying the axonal or dendritic contributions. In this paper, orientation sensitivity arises through cooperative interactions among neurons having anisotropic excitatory, and isotropic inhibitory connections.     

Excitation of nigral dopamine neurons by the GABA(A) receptor agonist muscimol is mediated via release of glutamate

Previous electrophysiological studies have shown that the GABA(A)-receptor agonist muscimol is able to markedly increase the firing rate of rat nigral dopamine (DA) neurons. This action of the drug is paradoxical since local microiontophoretic application of the drug is associated with a clearcut inhibition of this neurons. In the present electrophysiological study, an attempt was made to analyze the mechanism of this action of the drug. Administration of muscimol (0.25-4.0 mg/kg, i.v.) was associated with a dose-dependent increase in firing rate as well as an increased bursting activity of the nigral DA neurons. Both these effects of muscimol were clearly antagonised by intravenous administration of the NMDA receptor antagonist MK 801(1 mg/kg) or by intracerebroventricular administration of the broad-spectrum excitatory amino acid receptor antagonist kynurenic acid. Furthermore, pretreatment with PNU 156561A (40 mg/kg, i.v., 5-8h), a compound that raised endogenous kynurenic acid levels about 9 times, also clearly antagonised the actions of muscimol. Indeed, this treatment reversed the excitatory action of muscimol into an inhibitory effect on the nigral DA neurons. Here, we report that the excitatory action of muscimol is mediated indirectly by release of glutamate.