The present invention is directed to the treatment and prevention of epilepsy.
Epilepsy is a neurological disorder in which normal brain function is disrupted as a consequence of intensive burst activity from groups of neurons (Wyllie, E., “The Treatment of Epilepsy Principles and Practice,” (Lippincot, Williams, and Wilkins, New York (2001)). Epilepsies result from long-lasting plastic changes in the brain affecting the expression of receptors and channels, and involve sprouting and reorganization of synapses, as well as reactive gliosis (Heinemann et al., “Contribution of Astrocytes to Seizure Activity,” Adv. Neurol. 79:583-590 (1999); Rogawski et al., “The Neurobiology of Antiepileptic Drugs,” Nat. Rev. Neurosci. 5:553-564 (2004)). Epileptic seizures can result from a primary epileptic disorder, such as Rolandic epilepsy, Lennox Gastaut or West syndrome, and juvenile myoclonic epilepsies, petit mal, or idiopathic temporal lobe seizures, psychomotor epilepsy or mesial temporal sclerosis. Epileptic seizures can also result from pediatric or adult-onset hereditary metabolic disorders or as a manifestation or late sequela to stroke, traumatic brain injury, intracerebral hemorrhage, tumors, infection, vascular malformation, metabolic, endocrine or electrolyte disturbance, and coagulation dysfunction. Several lines of evidence suggest a key role of glutamate in the pathogenesis of epilepsy. Local or systemic administration of glutamate agonists triggers excessive neuronal firing, whereas glutamate receptor (GluR) antagonists have anticonvulsant properties (Meldrum, B. S., “Update on the Mechanism of Action of Antiepileptic Drugs,” Epilepsia 37 (Suppl.):6, S4-11 (1996)).
Paroxysmal depolarization shifts (PDSs) are abnormal prolonged depolarizations with repetitive spiking and are reflected as interictal discharges in the electroencephalogram (Heinemann et al., “Contribution of Astrocytes to Seizure Activity,” Adv. Neurol. 79:583-590 (1999); Rogawski et al., “The Neurobiology of Antiepileptic Drugs,” Nat. Rev. Neurosci. 5:553-564 (2004)).
Astrogliosis is a prominent feature of the epileptic brain, with autopsy and surgical resection specimens demonstrating that post-traumatic seizures and chronic temporal lobes epilepsy, may originate from gliotic scars (Tashiro et al., “Calcium Oscillations in Neocortical Astrocytes under Epileptiform Conditions,” J. Neurobiol. 50:45-55 (2002); Rothstein et al., “Knockout of Glutamate Transporters Reveals a Major Role for Astroglial Transport in Excitotoxicity and Clearance of Glutamate,” Neuron 16:675-686 (1996); Duffy et al., “Modulation of Neuronal Excitability by Astrocytes,” in Jasper's Basic Mechanisms of Epilepsies, Third Edition: Advances in Neurology, Vol 79, Delgado-Escueta et al., eds., Lippincott Williams & Wilkins, Philadelphia (1999)). In addition, astrocytes can modulate synaptic transmission through release of glutamate (Haydon, P. G., “GLIA: Listening and Talking to the Synapse,” Nat. Rev. Neurosci. 2:185-193 (2001)). For example, spontaneous astrocytic Ca2+ oscillations drive NMDA-receptor-mediated neuronal excitation in the rat ventrobasal thalamus and activate groups of neurons in hippocampus (Fellin et al., “Neuronal Synchrony Mediated by Astrocytic Glutamate Through Activation of Extrasynaptic NMDA Receptors,” Neuron 43:729-743 (2004); Angulo et al., “Glutamate Released from Glial Cells Synchronizes Neuronal Activity in the Hippocampus,” J. Neurosci. 24:6920-6927 (2004)). These and other studies have pointed to glutamate as a key transmitter of bi-directional communication between astrocytes and neurons (Nedergaard et al., “Beyond the Role of Glutamate as a Neurotransmitter,” Nat. Rev. Neurosci. 3:748-755 (2002); Haydon, P. G., “GLIA: Listening and Talking to the Synapse,” Nat. Rev. Neurosci. 2:185-193 (2001)). Nonetheless, experimental observations implicating astrocytes in initiation, maintenance, or spread of seizure activity, have not existed until now.
The present invention is directed to overcoming these and other deficiencies in the art.
A first aspect of the present invention relates to a method of treating or preventing epileptic seizures in a subject. The method involves administering an agent which interferes with glutamate, aspartate, and/or ATP release from astrocytes to the subject under conditions effective to treat or prevent epileptic seizures.
Another aspect of the present invention relates to a method of inhibiting hypersynchronous burst activity of a large group of neurons. The method involves administering an agent which interferes with glutamate, aspartate, and/or ATP release from astrocytes to the group of neurons under conditions effective to inhibit hypersynchronous burst activity.
A further aspect of the present invention relates to a method of identifying agents suitable for treating or preventing epileptic seizures. The method involves contacting astrocytes with one or more candidate compounds, evaluating the astrocytes for glutamate, aspartate, and/or ATP release, and then identifying the candidate compounds which interfere with glutamate, aspartate, and/or ATP release as agents potentially suitable for treating or preventing epileptic seizures.
According to the present invention, glutamate released by astrocytes can trigger PDSs in several models of experimental seizure. A unifying feature of seizure activity was its consistent association with antecedent astrocytic Ca2+ signaling. Oscillatory, tetrodotoxin (TTX)-insensitive increases in astrocytic Ca2+ preceded or occurred concomitantly with PDSs, and targeting astrocytes by photolysis of caged Ca2+ evoked PDSs. Furthermore, several anti-epileptic agents, including valproate, gabapentin, and phenyloin, potently reduced astrocytic Ca2+ signaling detected by 2-photon imaging in live animals. This suggests that pathologic activation of astrocytes likely play a central role in the genesis of epilepsy, as well as in the pathways targeted by current anti-epileptics. The observation that astrocytes release glutamate via a regulated Ca2+ dependent mechanism (Parpura et al., “Glutamate-Mediated Astrocyte-Neuron Signalling,” Nature 369:744-747 (1994); Bezzi et al., “Prostaglandins Stimulate Calcium-Dependent Glutamate Release in Astrocytes,” Nature 391:281-285 (1998); Fellin et al., “Neuronal Synchrony Mediated by Astrocytic Glutamate Through Activation of Extrasynaptic NMDA Receptors,” Neuron 43:729-743 (2004); Angulo et al., “Glutamate Released from Glial Cells Synchronizes Neuronal Activity in the Hippocampus,” J. Neurosci. 24:6920-6927 (2004), which are hereby incorporated by reference in their entirety) leads one to hypothesize that glutamate released by astrocytes plays a causal role in synchronous firing of large populations of neurons.
The present invention relates to a method of treating or preventing epileptic seizures in a subject. The method involves administering an agent which interferes with glutamate, aspartate, and/or ATP release from astrocytes to the subject under conditions effective to treat or prevent epileptic seizures.
Astrocytes are primarily viewed as passive support cells, which perform important but perfunctory housekeeping tasks to optimize the environment for neural transmission. New evidence has questioned this concept by demonstrating that astrocytes can actively modulate neuronal function. Indeed, astrocytes are required for synapse formation and, stability and can actively modulate synaptic transmission by release of glutamate by exocytosis (Volterra et al., “Astrocytes, From Brain Glue to Communication Elements: The Revolution Continues,” Nat. Rev. Neurosci. 6(8):626-640 (2005); Haydon, P. G., “GLIA: Listening and Talking to the Synapse,” Nat. Rev. Neurosci. 2(3):185-193 (2001), which are hereby incorporated by reference in their entirety). Astrocytes express several proteins that are required for exocytosis, and neurotoxins inhibit astrocytic glutamate release in cultures. Astrocytes also express functional vesicular glutamate transporters VGLUT1/2 and pharmacological inhibition of VGLUT1/2 reduced Ca2+-dependent glutamate release (Montana et al., “Vesicular Glutamate Transporter-Dependent Glutamate Release From Astrocytes,” J. Neurosci. 24(12):2633-2642 (2004); Bezzi et al., “Astrocytes Contain a Vesicular Compartment That is Competent for Regulated Exocytosis of Glutamate,” Nat. Neurosci. 7(6):613-620 (2004), which are hereby incorporated by reference in their entirety). However, other mechanisms by which astrocytes release glutamate likely exist. In addition, astrocytes possess multiple mechanisms for several key functions. For example, the important task of K+ buffering is undertaken by several K+ channels expressed by astrocytes, including KIR4.1 and rSlo K(Ca) (Price et al., “Distribution of rSlo Ca2+-Activated K+ Channels in Rat Astrocyte Perivascular Endfeet,” Brain Res. 956(2):183-193 (2002), which is hereby incorporated by reference in its entirety), but also by the K+—Na+—Cl− cotransporter (Su et al., “Contribution of Na(+)-K(+)-Cl(−) Cotransporter to High-[K(+)](o)-Induced Swelling and EAA Release in Astrocytes,” Am. J. Physiol. 282(5):C1136-C1146 (2002), which is hereby incorporated by reference in its entirety). The present invention utilizes these properties of astrocytes to treat and/or prevent epileptic seizures.
Glutamate is a small anion that permeates through several channels, including volume-sensitive channels (VSCs) (Mongin et al., “ATP Regulates Anion Channel-Mediated Organic Osmolyte Release From Cultured Rat Astrocytes via Multiple Ca2+-Sensitive Mechanisms,” Am. J. Physiol. 288(1):C204-C213 (2005), which is hereby incorporated by reference in its entirety). Furthermore, glutamate functions as an osmolyte and is released in large quantities by astrocytes in response to external hypotonicity (Kimelberg et al., “Swelling-Induced Release of Glutamate, Aspartate, and Taurine from Astrocyte Cultures,” J. Neurosci. 10(5):1583-1591 (1990), which is hereby incorporated by reference in its entirety). Cellular swelling leads to activation of VSCs and to the release of glutamate and other amino acids including aspartate, glutamine, and taurine, as a part of the regulatory volume decrease (Jentsch et al., “Molecular Structure and Physiological Function of Chloride Channels,” Physiol. Rev. 82(2):503-568 (2002), which is hereby incorporated by reference in its entirety). Ca2+-dependent astrocytic glutamate release has not been linked previously to the opening of VSCs, because these channels are activated by Ca2+-independent processes. The present invention shows that receptor-induced Ca2+ increase is associated with an increase in astrocytic cell volume, which leads to the activation of VSCs and, thereby, results in the Ca2+-dependent release of glutamate.
The methods of the present invention, when used to treat epilepsy, are particularly useful in reducing the incidence of and/or the spread of epileptic seizures. The agents which are administered can include those that do not suppress neural transmission.
In preferred embodiments, the agent interferes with glutamate release, aspartate release, and/or ATP release from astrocytes and includes compounds selected from those presented in Tables 1, 2, 3, 4, 5, or 6; all cited references are hereby incorporated by reference.
Agents of the present invention can be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. They may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
The active agents of the present invention may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, these active agents may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of active agent. The percentage of the agent in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of active agent in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions according to the present invention are prepared so that an oral dosage unit contains between about 1 and 250 mg of active agent.
The tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a fatty oil.
Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
These active agents may also be administered parenterally. Solutions or suspensions of these active agents can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
The agents of the present invention may also be administered directly to the airways in the form of an aerosol. For use as aerosols, the agents of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
The present invention also relates to a method of inhibiting hypersynchronous burst activity of a large group of neurons. The method involves administering an agent which interferes with glutamate, aspartate, and/or ATP release from astrocytes to the group of neurons under conditions effective to inhibit hypersynchronous burst activity. The method can be carried out either in vivo or in vitro. In carrying out the in vivo embodiment of the present invention, the above-described formulations and modes of administration can be utilized.
In preferred embodiments, the agent interferes with glutamate release, aspartate release, and/or ATP release from astrocytes and includes compounds selected from those presented in Tables 1, 2, 3, 4, 5, or 6, as presented above.
A further aspect of the present invention relates to a method of identifying agents suitable for treating or preventing epileptic seizures. The method involves contacting astrocytes with one or more candidate compounds, evaluating the astrocytes for glutamate, aspartate, and/or ATP release, and then identifying the candidate compounds which interfere with glutamate, aspartate, and/or ATP release as agents potentially suitable for treating or preventing epileptic seizures. Evaluation of astrocytes may also include detecting calcium release. Detection may be accomplished by monitoring changes in intracellular Ca2+ levels and Ca2+ release using the fluorescence of indicator dyes such as indo or fura, or using confocal Ca2+ imaging (Didier et al., “Ca2+ Blinks: Rapid Nanoscopic Store Calcium Signaling”, PNAS 102:3099-3104 (2005); Grimaldi et al., “Mobilization of Calcium from Intracellular Stores, Potentiation of Neurotransmitter-Induced Calcium Transients, and Capacitative Calcium Entry by 4-Aminopyridine,” J. Neurosci. 21:3135-3143 (2001), which are hereby incorporated by reference in their entirety).
Preferably, astrocytes are evaluated for glutamate release, aspartate release, and/or ATP release.
The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
Slice preparation, 2-photon laser scanning imaging, and photolysis: Hippocampal slices were prepared from Sprague-Dawley (SD) rats (P14-18) as previously described (Kang et al., “Astrocyte-Mediated Potentiation of Inhibitory Synaptic Transmission,” Nat. Neurosci. 1:683-692 (1998); Zonta et al., “Neuron-to-Astrocyte Signaling is Central to the Dynamic Control of Brain Microcirculation,” Nat. Neurosci. 6:43-50 (2003); Liu et al., “Astrocyte-Mediated Activation of Neuronal Kainate Receptors. Proc. Natl. Acad. Sci. USA 101:3172-3177 (2004), which are hereby incorporated by reference in their entirety). The slices were mounted in a perfusion chamber and viewed by a custom built laser scanning microscope (BX61WI, FV300, Olympus) attached to Mai Tai laser (SpectraPhysics, Inc.). For Ca2+ measurements, slices were loaded with the Ca2+ indicator, fluo-4/AM (10 M, 1.5 h; Molecular Probes). For uncaging experiments, NP-EGTA/AM (200 μM; Molecular Probes) was co-incubated with fluo-4 AM. Photolysis was carried out by a 3 μm diameter UV pulse delivered as 10 trains (2 pulses with a duration of 10 ms and an interval of 50 ms; 100-500 μW) (DPSS lasers, Inc; 355 nm, 1.0 W).
Culture preparation and Ca2+ imaging: Cultured astrocytes were prepared from P1 rat pups as previously described (Arcuino et al., “Intercellular Calcium Signaling Mediated by Point-Source Burst Release of ATP,” Proc. Natl. Acad. Sci. USA 99:9840-9845 (2002), which is hereby incorporated by reference in its entirety). Confluent monolayer cultures were loaded with the Ca2+ indicator fluo-4 (5 μM for 1 h) and Ca2+ signaling monitored by confocal microscopy (BioRad, MRC1034) (Takano et al., “Glutamate Release Promotes Growth of Malignant Gliomas,” Nat. Med. 7:1010-1015 (2001), which is hereby incorporated by reference). Maximum increase in fluo-4 intensity following stimulation occurred within 20-30 s and was normalized relative to baseline fluorescence.
Electrophysiology: Whole-cell recordings from CA1 pyramidal neurons and stratum radiatum astrocytes in hippocampal slices were performed as previously described (Liu et al., “Astrocyte-Mediated Activation of Neuronal Kainate Receptors. Proc. Natl. Acad. Sci. USA 101:3172-3177 (2004), which is hereby incorporated by reference in its entirety). The perfusion artificial cerebrospinal fluid (ACSF) contained (in mM): 125 NaCl, 5 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 10 glucose and 25 NaHCO3, pH 7.4 when aerated with 95% O2, 5% CO2 (Valiante et al., “Coupling Potentials in CA1 Neurons During Calcium-Free-Induced Field Burst Activity,” J. Neurosci. 15:6946-6956 (1995), which is hereby incorporated by reference in its entirety). Membrane potentials were filtered at 1 kHz, digitized at 5 kHz by using an Axopatch 200B amplifier, a pCLAMP 8.2 program and DigiData 1332A interface (Axon Instruments, Foster City, Calif.). Field potential recordings were made in stratum radiatum and stratum pyramidale of CA1 in hippocampal slices as previously described (Valiante et al., “Coupling Potentials in CA1 Neurons During Calcium-Free-Induced Field Burst Activity,” J. Neurosci. 15:6946-6956 (1995), which is hereby incorporated by reference in its entirety). Recording signals were filtered at 1 kHz, digitized at 5 kHz. All experiments were performed at 32-34° C.
Microdialysis, EEG recordings, and HPLC Analysis of Amino Acid Release: Adult SD rats (220-250 g) were anesthetized by ketamine (60 mg/kg) and xylazine (10 mg/kg). Microdialysis probes with a built-in electrode for EEG recordings (Applied Neuroscience, London, UK) were stereotaxically implanted into the right dorsal hippocampus (from bregma: 3.0 mm rostral; 2.0 mm lateral; from dura: 3.5 mm vertical) and fixed to the skull using dental cement and perfused using a microinjection pump (Harvard Apparatus Inc. USA), at a rate of 2 μl/min (Mena et al., “In vivo Glutamine Hydrolysis in the Formation of Extracellular Glutamate in the Injured Rat Brain,” J. Neurosci. Res. 60:632-641 (2000), which is hereby incorporated by reference in its entirety). Seizure activity was induced by delivering 4-AP (5 mM) through the microdialysis probe. The amino acid content was analyzed after reaction with ophthaldialdehyde utilizing fluorometric detection (Mena et al., “In vivo Glutamine Hydrolysis in the Formation of Extracellular Glutamate in the Injured Rat Brain,” J. Neurosci. Res. 60:632-641 (2000), which is hereby incorporated by reference in its entirety). EEG (1-100 Hz) was recorded continuously by an amplifier (DP-311, Warner Instruments, Inc) (Ayala et al., “Expression of Heat Shock Protein 70 Induced by 4-Aminopyridine Through Glutamate-Mediated Excitotoxic Stress in Rat Hippocampus In vivo,” Neuropharmacology 45:649-660 (2003); Urenjak et al., “Kynurenine 3-Hydroxylase Inhibition in Rats: Effects on Extracellular Kynurenic Acid Concentration and N-Methyl-D-Aspartate-Induced Depolarisation in the Striatum,” J. Neurochem. 75:2427-2433 (2000), which are hereby incorporated by reference in their entirety), a pCLAMP 9.2 program and DigiData 1332A interface with an interval of 200 μs.
In vivo two-photon Imaging: Adult mice (25-30 g) were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg) injection and a femoral artery catheterized. A custom made metal frame was glued to the skull with dental acrylic cement. A craniotomy (3 mm in diameter), centered 1-2 mm posterior to bregma and 2-3 mm from midline was performed. Dura was removed and the exposed cortex loaded with fluo-4/am (2 mM, 1 hr) and in selected experiments, sulforhodamine 101 (100 μM, 10 min) (Nimmerjahn et al., “Sulforhodamine 101 as a Specific Marker of Astroglia in the Neocortex In vivo,” Nature Methods 1:1-7 (2004), which is hereby incorporated by reference in its entirety). Agarose (0.75%) in saline was poured into the craniotomy and a coverslip mounted. Valproate was administred i.p. 450 mg/kg, 30 min before imaging; gabapentin 200 mg/kg, 60 min before imaging; and Na+ phenyloin, 100 mg/kg, 90 min before imaging (Boothe, D. M., “Anticonvulsant Therapy in Small Animals,” Vet. Clin. North. Am. Small Anim. Pract. 28:411-448 (1998), which is hereby incorporated by reference in its entirety). A custom built microscope attached to Tsunami/Millinium laser (SpectraPhysics, Inc.) and a scanning box (FV300, Olympus) was utilized for two-photon imaging experiments. Electrodes filled with saline containing 100 mM 4-AP were inserted 100-150 μm from the pial surface for cortical EEG (CoEEG) recordings. CoEEG (1-100 Hz) was recorded continuously by an amplifier (700A, Axon Instruments Inc.) (Ayala et al., “Expression of Heat Shock Protein 70 Induced by 4-Aminopyridine Through Glutamate-Mediated Excitotoxic Stress in Rat Hippocampus In vivo,” Neuropharmacology 45:649-660 (2003); Urenjak et al., “Kynurenine 3-Hydroxylase Inhibition in Rats: Effects on Extracellular Kynurenic Acid Concentration and N-Methyl-D-Aspartate-Induced Depolarisation in the Striatum,” J. Neurochem. 75:2427-2433 (2000), which are hereby incorporated by reference in their entirety), and a pCLAMP 9.2 program and DigiData 1332A interface with an interval of 200 μs. The seizure was induced by puffing 4-AP (5-10 pulses of 5-10 ms at 10 psi, Picospitzer). ATP (50 mM) was delivered iontophoretically (100 nA, 15 sec) with an electrode (100-150 μm from surface).
Animals were artificially ventilated with a ventilator (SAR-830, CWE) and blood gasses, pCO2 (30-50 mm Hg), O2 (100-150 mm Hg), and pH (7.25-7.45), monitored with a pH/blood gas analyzer (Rapidlab 248, Bayer, samples 40 μl). Body temperature was maintained at 37° C. by a homeothermic blanket system (Harvard Apparatus). All experiments were approved by the Institution Animal Care and Use Committee of University of Rochester.
To examine the cellular mechanism underlying PDSs, CA1 pyramidal neurons in rat hippocampal slices exposed to 4-aminopyridine (4-AP) were patch clamped. 4-AP is a K+ channel blocker that induces intense electrical discharges in slices (Luhmann et al., “Generation and Propagation of 4-AP-Induced epileptiform activity in neonatal intact limbic structures in vitro. Eur. J. Neurosci. 12, 2757-2768 (2000), which is hereby incorporated by reference in its entirety) and seizure activity in experimental animals (Yamaguchi et al., “Effects of Anticonvulsant Drugs on 4-Aminopyridine-Induced Seizures in Mice,” Epilepsy Res. 11:9-16 (1992), which is hereby incorporated by reference in its entirety). All slices exposed to 4-AP (61 slices from 23 rats) exhibited epileptiform bursting activity expressed as transient episodes of neuronal depolarizations eliciting trains of action potentials (
Combined, these experiments demonstrated that PDSs can be triggered by an action potential-independent mechanism. Neurons exhibited a 16±5 mV (n=24) depolarization shift, whereas astrocytes only display a modest change in membrane potential (0.5±0.2 mV, n=22) during PDSs in the presence of TTX.
To examine the role of glutamate released from action potential-independent sources in PDSs, the occurrence of PDSs in the presence of TTX and GluR antagonists was quantified. The PDSs evoked by 4-AP resulted primarily from activation of ionotropic glutamate receptors, because APV and CNQX potently reduced both the frequency and the amplitude of the PDSs, in accordance with earlier studies (
Addition of TTX and CNQX (no APV) resulted in a significant decrease in the occurrence of PDSs compared with TTX alone (
In all experiments thus far, TTX was first added after the hippocampal slices had been exposed for 20 min to 4-AP (FIGS. 1 and 2A-H). To test the possibility that astrocytic activation was secondary to neuronal bursting activity triggered by 4-AP, TTX (10-15 min) was added before exposing the slices to 4-AP (
Combined, these observations show that TTX decreased the relative frequency of PDSs by 32±8% (P=0.001) compared with 4-AP alone (
Seizures can be induced by a variety of inciting agents with apparently unrelated mechanisms of action. The traditionally defined mechanisms of epileptogenesis involve either the facilitation of excitatory synaptic activity, or the suppression of inhibitory transmission. To assess whether glutamate release from action potential-independent sources plays a role in experimental epilepsy, the dependence of PDSs upon TTX and glutamate receptor antagonists in several seizure models was analyzed. A common approach to induce hypersynchronous burst activity of large groups of neurons is to enhance excitatory synaptic activity by removing extracellular Mg2+. The epileptogenic action of Mg2+ depletion has been attributed to the activation of NMDA receptors at the resting membrane potential (Schuchmann et al., “Nitric Oxide Modulates Low-Mg2+-Induced Epileptiform Activity in Rat Hippocampal-Entorhinal Cortex Slices,” Neurobiol. Dis. 11:96-105 (2002), which is hereby incorporated by reference in its entirety). In accordance with earlier reports, Mg2+-free solution triggered repeated PDSs (
Thus, in all experimental models of seizure analyzed, including exposure to 4-AP, Mg2+-free solution, bicuculline, penicillin, and removal of extracellular Ca2+, PDSs were largely insensitive to TTX. Depending upon the model, TTX (and VGCC blockers) reduced the frequency of PDSs to 70-90% of total, demonstrating that the majority of PDSs was evoked by action potential-independent pathways. Another key observation was that glutamate is the principal mediator of TTX-insensitive PDS, because combined exposure to APV/CNQX/MCPG decreased the frequency of PDSs to 5-20%. The TTX- and APV/CNQX/MCPG-insensitive PDS might be elicited by other action potential-independent mechanisms, including gap junctions (Perez-Velazquez et al., “Modulation of Gap Junctional Mechanisms During Calcium-Free Induced Field Burst Activity: A Possible Role for Electrotonic Coupling in Epileptogenesis,” J. Neurosci. 14:4308-4317 (1994), which is hereby incorporated by reference in its entirety) and purinergic receptor activation possibly mediated by release of ATP by astrocytes (Cotrina et al., “Connexins Regulate Calcium Signaling by Controlling ATP Release,” Proc. Natl. Acad. Sci. USA 95:15735-15740 (1998); Cotrina et al., “ATP-Mediated Glia Signaling,” J. Neurosci. 20:2835-2844 (2000), which are hereby incorporated by reference in their entirety).
Recordings in hippocampal slices indicated that the cellular hallmark of epileptic discharge, PDSs, is caused by prolonged episodes (˜500 ms) of neuronal depolarization triggered by glutamate release from a non-synaptic source. Since a number of studies have documented that astrocytes can release glutamate in a Ca2+-dependent manner (Bezzi et al., “Prostaglandins Stimulate Calcium-Dependent Glutamate Release in Astrocytes,” Nature 391:281-285 (1998); Fellin et al., “Neuronal Synchrony Mediated by Astrocytic Glutamate Through Activation of Extrasynaptic NMDA Receptors,” Neuron 43:729-743 (2004); Angulo et al., “Glutamate Released from Glial Cells Synchronizes Neuronal Activity in the Hippocampus,” J. Neurosci. 24:6920-6927 (2004), which are hereby incorporated by reference in their entirety), whether activation of astrocytic Ca2+ signaling was a unifying feature of epileptogenesis was examined. Hippocampal slices were loaded with the Ca2+ indicator, fluo-4/AM and viewed by two-photon laser scanning microscopy. The preferential loading of fluorescent acetoxymethyl esters indicators by astrocytes has been extensively reported (Kang et al., “Astrocyte-Mediated Potentiation of Inhibitory Synaptic Transmission,” Nat. Neurosci. 1:683-692 (1998); Zonta et al., “Neuron-to-Astrocyte Signaling is Central to the Dynamic Control of Brain Microcirculation,” Nat. Neurosci. 6:43-50 (2003); Liu et al., “Astrocyte-Mediated Activation of Neuronal Kainate Receptors. Proc. Natl. Acad. Sci. USA 101:3172-3177 (2004), which are hereby incorporated by reference in their entirety). Bath application of 4-AP potently initiated astrocytic Ca2+ signaling expressed as infrequent Ca2+ oscillations (
Astrocytes within the cortex and hippocampus are organized in essentially non-overlapping microdomains with an average diameter of 40-70 μm, reviewed in Nedergaard et al., “New Roles for Astrocytes: Redefining the Functional Architecture of the Brain,” Trends Neurosci. 26:523-530 (2003), which is hereby incorporated by reference in its entirety). Since Ca2+ oscillations are restricted to 1-3 neighboring astrocytes, it is expected that the PDSs are limited to small (<50-200 μm) regions. To establish the spatial territories of PDSs, recording with two field electrodes in the stratum radiatum of CA1 was performed (
Taken together, these observations demonstrate that astrocytic Ca2+ signaling is evoked in 5 different models of acute seizure. In all paradigms studied, astrocytic Ca2+ signaling was insensitive to TTX indicating direct stimulation of astrocytes, rather than a secondary response to neuronal bursting activity. Furthermore, PDSs were spatially restricted to a few hundred micrometers and increments in cytosolic Ca2+ of astrocytes always preceded PDSs by in the stratum radiatum.
To demonstrate that astrocytic activation is not only correlated with, but sufficient for generation of negative depolarization shifts, photo release of caged Ca2+ (NP-EGTA) in astrocytes was performed (
One of the characteristics of Ca2+-dependent astrocytic glutamate release is that other amino acids, including aspartate, glutamine, and taurine also are released (Jeremic et al., “ATP Stimulates Calcium-Dependent Glutamate Release from Cultured Astrocytes,” J. Neurochem. 77:664-675 (2001); Nedergaard et al., “Beyond the Role of Glutamate as a Neurotransmitter,” Nat. Rev. Neurosci. 3:748-755 (2002), which are hereby incorporated by reference in their entirety). These amino acids exit through volume sensitive channels (VSC) expressed by astrocytes, whereas other amino acids, including asparagine, isoleucine, leucine, phenylalanine and tyrosine, are released to a lesser extent. To test the idea that astrocytes release glutamate during epileptic seizures, a microdialysis probe with a built-in electrode for EEG recording (Obrenovitch et al., “Evidence Disputing the Link Between Seizure Activity and High Extracellular Glutamate,” J. Neurochem. 66:2446-2454 (1996), which is hereby incorporated by reference in its entirety), was implanted in the hippocampus and perfused with artificial cerebrospinal fluid (ACSF) containing 4-AP. The basal extracellular concentration of glutamate was low in accordance with earlier reports (0.5-1.5 μM) (Mena et al., “In vivo Glutamine Hydrolysis in the Formation of Extracellular Glutamate in the Injured Rat Brain,” J. Neurosci. Res. 60:632-641 (2000), which is hereby incorporated by reference in its entirety), but increased to 6-10 μM approximately 10 min after addition of 4-AP. Consistent with the idea that glutamate is released by astrocytes, a 3-8 fold increase in release of amino acid osmolytes, including glutamate, aspartate, glutamine, and taurine, was observed (
Given that photolysis experiments and HPLC analysis indicated that astrocytes contribute to elevations in extrasynaptic glutamate in epileptic tissue, it was predicted that compounds that reduce astrocytic glutamate release would suppress epileptiform activity. Based on culture experiments, it has been documented that anion channel inhibitors, including 5-nitro-2-(3-phenylpropylamino) benzoic acid (“NPPB”) and flufenamic acid (“FFA”), reduce glutamate release from astrocytes (Nedergaard et al., “Beyond the Role of Glutamate as a Neurotransmitter,” Nat. Rev. Neurosci. 3:748-755 (2002), which is hereby incorporated by reference in its entirety). To evaluate the effect of anion channel inhibition upon epileptic discharges, FFA or NPPB were bath applied to hippocampal slices exhibiting 4-AP induced seizures. Both NPPB and FFA markedly reduced the frequency and amplitude of PDSs (
Combined, these observations indicate: 1) that targeting astrocytes by photolysis of caged Ca2+ triggered PDSs, whereas similar stimulation of neurons had no effect upon the field potential; 2) that the footprint of amino acids released during 4-AP induced seizures was similar to Ca2+-dependent amino acids released from cultured astrocytes, and; 3) that anion channel inhibitors reduce the frequency and amplitude of PDSs. Together, these findings support the idea that astrocytes contribute to action potential-independent glutamate release in 4-AP evoked seizures.
To test the importance of astrocytic activation in generation of seizures in live animals, two-photon imaging of Ca2+ signaling in the exposed cortex of adult mice was used. The primary somatosensory cortex was loaded with fluo-4/AM prior to imaging. In initial experiments, Fluo-4/AM was loaded concomitant with the astrocyte specific marker Sulforhodamine 101 (Nimmerjahn et al., “Sulforhodamine 101 as a Specific Marker of Astroglia in the Neocortex In vivo,” Nature Methods 1:1-7 (2004), which is hereby incorporated by reference in its entirety). Fluo-4 and Sulforhodamine 101 were co-localized, indicating that fluo-4 is preferentially taken up by astrocytes in live exposed cortex as previously reported (
Post-traumatice epilepsy induced by intracortical Iron Injection: Adult mice (2 months) can be anesthetized using a ketamine (100 mg/kg) and xylazine (25 mg/kg) mixture and positioned in a stereotaxic frame. Following a small craniotomy and opening of the dura, 1.0 μl of 100 mM ferrous chloride solution can be injected into sensorimotor cortex (1.5-2.0 mm posterior to bregma, 1.0-1.5 mm lateral to midline, and 0.5-1.0 mm below the cortical surface) at a rate of 1.0 μl/min using a microprocessor controlled syringe pump (Model 210, Stoelting Co. IL, USA) (Willmore et al., “Chronic Focal Epileptiform Discharges Induced by Injection of Iron into Rat and Cat Cortex”, Science 200:1501-1503, (1978)). Electroencephalography can be obtained at 2 months after intracortical injections of ferric chloride (Shah et al., “Seizure-induced Plasticiy of H Channels in Entorhinal Corical Layer III Pyramidal Neurons”, Neuron 44:495-508 (2004)). More than 90% animal will develop spontaneous epileptiform EEG-activity after intracortical injection of ferrous chloride.
Genetic epilepsy: Genetic epilepsy mice-tottering mice (B6.D2-cacna1atg/J), which are genetically predisposed to epilepsy due to a mutation in the voltage gated calcium channel subunit α1A (Tg−) (Fletcher et al., “Absence Epilepsy in Tottering Mutant mice is Associated with Calcium Channel Defects”, Cell 87:607-617 (1996)), were obtained from the Jackson Laboratory (JAX #000544). Onset of seizures occurs usually 3-4 weeks of age and symptoms persist throughout life. The Tottering mouse has a characteristic wobbly gait and display bilaterally synchronous spike-wakes in EEG recordings of 1-10 seconds in duration many times during a day. Stereotypic partial motor seizures with abnormal ECG activity also occur once or twice a day and are usually 20-30 minutes in duration.
Paroxysmal depolarization shifts are abnormal prolonged depolarizations with repetitive spiking and are reflected as interictal discharges in the electroencephalogram (Heinemann et al., “Contribution of Astrocytes to Seizure Activity”, Adv. Neurol. 79:583-590 (1999)). Here, it has been demonstrated that glutamate released from astrocytes can trigger paroxysmal depolarization shifts in several models of acute experimental seizure (Tian et al., “An Astrocytic Basis of Epilepsy” Nature Med. 11:973-981 (2005)). A unifying feature of seizure activity was its consistent association with antecedent astrocytic Ca2+ signaling. Oscillatory, TTX-insensitive increases in astrocytic Ca2+ preceded or occurred concomitantly with paroxysmal depolarization shifts, and targeting astrocytes by photolysis of caged Ca2+ evoked paroxysmal depolarization shifts. Furthermore, several anti-epileptic agents, including valproate, gabapentin, and phenyloin, potently reduced astrocytic Ca2+ signaling detected by 2-photon imaging in live animals. This suggests that pathologic activation of astrocytes may play a central role in the genesis of epilepsy, as well in the pathways targeted by current anti-epileptics.
It has been observed that paroxysmal depolarization shifts preceded epileptiform EEG in mice with intracortical injection of ferric chloride 2 months prior (
It has also been observed that Cx43 formed large plaques in the cortex of mice with intracortical injection of ferric chloride (
According to the present invention, prolonged episodes of neuronal depolarization evoked by astrocytic glutamate release contribute to epileptiform discharges. Synchronized population spikes are key concomitants to seizure. Prior studies have demonstrated that multisynaptic excitatory pathways can trigger synchronized burst activity in picrotoxin-induced seizure activity (Miles et al., “Single Neurones Can Initiate Synchronized Population Discharge in the Hippocampus,” Nature 306:371-373 (1983), which is hereby incorporated by reference in its entirety), whereas other evidence has been presented for roles of both recurrent inhibition and gap junction coupling (Perez-Velazquez et al., “Modulation of Gap Junctional Mechanisms During Calcium-Free Induced Field Burst Activity: A Possible Role for Electrotonic Coupling in Epileptogenesis,” J. Neurosci. 14:4308-4317 (1994), which is hereby incorporated by reference in its entirety).
According to the present invention, additional mechanism exists indicating that an action potential-independent source of glutamate can trigger local depolarization events and synchronized bursting activity. That other cells, including neurons, contribute to extrasynaptic glutamate release cannot be excluded, but several observations point to astrocytes as the primary source. First, the existence of a Ca2+-dependent mechanism of astrocytic glutamate release has been documented by several groups (Haydon, P. G., “GLIA: Listening and Talking to the Synapse,” Nat. Rev. Neurosci. 2:185-193 (2001), which is hereby incorporated by reference in its entirety). Second, photolysis of caged Ca2+ in astrocytes was sufficient to trigger PDSs, as shown in
According to the present invention, 70-90% of PDSs were TTX-insensitive, indicating that a non-synaptic mechanism played a predominant role in generating seizure activity in the 5 models of experimental epilepsy studied. This observation does not exclude that astrocytes may play a role in seizure activity that originate in neurons. Astrocytes may amplify, maintain, and expand neurogenic seizure activity. Excessive neuronal firing is associated with marked alterations in the composition of the extracellular ions, most notably an increase in K+ and a reduction of Ca2+ (Heinemann et al., “Extracellular Calcium and Potassium Concentration Changes in Chronic Epileptic Brain Tissue,” Adv. Neurol. 44:641-461 (1986), which is hereby incorporated by reference in its entirety). Lowering of extracellular Ca2+ potently elicits astrocytic Ca2+ signaling (Stout et al., “Modulation of Intercellular Calcium Signaling in Astrocytes by Extracellular Calcium and Magnesium,: Glia 43:265-273 (2003), which is hereby incorporated by reference in its entirety) and glutamate release (Ye et al., “Functional Hemichannels in Astrocytes: A Novel Mechanism of Glutamate Release,” J. Neurosci. 23:3588-3596 (2003), which is hereby incorporated by reference in its entirety), and secondary engagement of astrocytes may convert an otherwise self-limited episode of intense neuronal firing into a seizure focus. It is also possible that spillover of glutamate from excitatory synapses contributes to activation of astrocytic Ca2+ signaling by binding to mGluR (Zonta et al., “Neuron-to-Astrocyte Signaling is Central to the Dynamic Control of Brain Microcirculation,” Nat. Neurosci. 6:43-50 (2003), which is hereby incorporated by reference in its entirety). Thus, astrocytes may initially be activated by excessive neuronal activity, but once activated, neuronal firing may no longer be required for continued activity of astrocytes, and thereby for maintenance and propagation of abnormal electrical activity.
Similar action potential-independent mechanisms may underlie local expansion of a seizure focus. Lowering of extracellular Ca2+ triggers propagation of astrocytic Ca2+ waves that spread into adjacent tissue (Arcuino et al., “Intercellular Calcium Signaling Mediated by Point-Source Burst Release of ATP,” Proc. Natl. Acad. Sci. USA 99:9840-9845 (2002), which is hereby incorporated by reference in its entirety). Long-distance astrocytic Ca2+ waves excite neurons along their path by release of glutamate (Nedergaard et al., “Beyond the Role of Glutamate as a Neurotransmitter,” Nat. Rev. Neurosci. 3:748-755 (2002), which is hereby incorporated by reference in its entirety). In turn, neuronal activity lowers extracellular Ca2+ resulting in activation of astrocytes in increasing distances from the seizure focus (Bikson et al., “Modulation of Burst Frequency, Duration, and Amplitude in the Zero-Ca(2+) Model of Epileptiform Activity,” J. Neurophysiol. 82:2262-2270 (1999), which is hereby incorporated by reference in its entirety). Thus, a cascade of events in which astrocytic Ca2+ signaling plays a key role may cause conversion of normal brain tissue remote from the center of seizure initiation into an epileptic focus.
The new observation reported here is that astrocytic activation can directly trigger seizure activity and that epilepsy thereby, at least in part, may originate in astrocytes.
It is proposed that seizure activity may have an astrocytic basis, in addition to the well-established neurogenic mechanisms. The primary argument for existence of an astrocytic basis for seizure is that the larger fraction (70-90%) of PDSs was TTX-insensitive in five experimental models of seizure studied (
Existing drugs available for treatment of epilepsy fall into three categories. Na+ channel blockers attenuate high-frequency firing by reducing the amplitude and rate of rise of action potentials. GABA receptor agonists mimic the action of GABA, thereby increasing inhibitory synaptic transmission. Lastly, glutamate receptor antagonists block ionotopic glutamate receptors thereby reducing excitatory synaptic transmission (Rogawski et al., “The Neurobiology of Antiepileptic Drugs for the Treatment of Nonepileptic Conditions,” Nat. Med. 10:685-692 (2004), which is hereby incorporated by reference in its entirety). The downside of these drugs is that the therapeutic mechanisms of action also suppress normal neural activity. Valproate, gabapentin, and phenyloin all reduced astrocytic Ca2+ signaling in animals exposed to 4-AP. Even more intriguing, valproate, gabapentin, and phenyloin directly depressed astrocytic Ca2+ signaling evoked by purinergic receptor activation, demonstrating a direct effect on the ability of astrocytes to mobilize Ca2+ and/or transmit intercellular Ca2+ signaling. Thus, the anticonvulsive activity of valproate, gabapentin, and phenyloin, may be mediated by directly depressing astrocytic activity. Since the results of the above experiments suggest that epileptic discharges are secondary to glial pathology, astrocytes may represent a promising new target for epileptogenic interventions. Pharmacotherapy directed specifically at suppressing glial Ca2+ signaling or decreasing TTX-insensitive glutamate release may achieve seizure control, without the suppression of neural transmission associated with current treatment options.
Cortical astrocyte cultures were made from P1 Sprague-Dawley rat pups. Heterozygotes of the Cx43 knockout line were obtained from The Jackson Laboratory (Lin et al., “Connexin Mediates Gap Junction-Independent Resistance to Cellular Injury,” J. Neurosci. 23(2):430-441 (2003), which is hereby incorporated by reference in its entirety). Astrocytes were loaded with calcein/acetoxymethyl ester (AM) (5 μM for 30 min) and visualized by confocal microscopy (Schreiber et al., “The Cystic Fibrosis transmembrane Conductance Regulator Activates Aquaporin 3 in Airway Epithelial Cells,” J. Biol. Chem. 274(17):11811-11816 (1999), which is hereby incorporated by reference in its entirety). The fluorescence dilution technique was performed on astrocytes loaded with fura-2/AM (5 μM for 30 min). The volume of astrocytes in suspension was analyzed with a Coulter counter.
An enzymatic fluorescence detection assay for monitoring glutamate was used (Bezzi et al., “Prostaglandins Stimulate Calcium-Dependent Glutamate Release in Astrocytes,” Nature 391(6664):281-285 (1998), which is hereby incorporated by reference in its entirety). For analysis by high-performance liquid chromatography (HPLC), confluent cultures were mounted in a perfusion chamber. The amino acid content was analyzed after reaction with o-phthaldialdehyde by using fluorometric detection (Shank et al., “Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism,” J. Neurochem. 61(1):315-323 (1993), which is hereby incorporated by reference in its entirety). Acutely isolated cortical or hippocampus slices prepared from 14- to 18-day-old Sprague-Dawley rats were used for electrophysiological recordings (Kang et al., “Astrocyte-Mediated Potentiation of Inhibitory Synaptic Transmission,” Nat. Neurosci. 1(8):683-692 (1998), which is hereby incorporated by reference in its entirety).
To test the hypothesis that a Ca2+ increase is associated with a transient increase in astrocytic cell volume, relative changes in cell volume were measured using three different approaches.
First, a confocal x-z layer scanning microscope was used Schreiber et al., “The Cystic Fibrosis Transmembrane Conductance Regulator Activates Aquaporin 3 in Airway Epithelial Cells,” J. Biol. Chem. 274(17):11811-11816 (1999), which is hereby incorporated by reference in its entirety). Vertical sections of cultured astrocytes loaded with calcein/AM (5 μM for 30 min) were constructed from repetitive x-z line scans (
Second, the use of the fluorescence-dilution method (Hanson, E., “Metabotropic Glutamate Receptor Activation Induces Astroglial Swelling,” J. Biol. Chem. 269:21955-21961 (1994), which is hereby incorporated by reference in its entirety) detected a 9.6±1.3% decrease in fluorescence-dilution emission in fura-2-loaded cultured astrocytes 1 min after ATP (100 μM) exposure compared with a 1.1±1.3% increase in emission in vehicle-controls (n=5; P<0.001, t test).
Third, exposure of astrocytes in suspension culture to ATP and subsequent assay of cell volume with a Coulter counter (Raat et al., “Measuring Volume Perturbation of Proximal Tubular Cells in Primary Culture with Three Different Techniques,” Am. J. Physiol. 271(1 Pt 1):C235-C241 (1996), which is hereby incorporated by reference in its entirety) showed a significant, reversible increase in astrocytic cell volume, averaging 3.5±0.6% at 30 sec (P<0.0003) and 3.0±0.5% at 60 sec (P<0.001); cell volume returned to prestimulation values 2 min after stimulation (
Each of these independent approaches to measure cell volume demonstrated a transient increase in astrocytic cell volume in response to purinergic activation. ATP-induced swelling was modest, in the range of 3-10%, compared with the 25-60% increase in cell volume in response to hypotonicity.
To determine whether ATP and hypotonicity induce glutamate release by the same mechanism, glutamate release from cultured rat cortical astrocytes was analyzed by using a highly sensitive enzymatic assay (Bezzi et al., “Prostaglandins Stimulate Calcium-Dependent Glutamate Release in Astrocytes,” Nature 391(6664):281-285 (1998), which is hereby incorporated by reference in its entirety). Application of 100 μM ATP resulted in the release of 2.49±0.21 fmol glutamate per cell. ATP-induced glutamate release depended on increases of cytosolic Ca2+, because BAPTA/AM (20 μM) for 30 min) and thapsigargin (1 μM for 10 min) attenuated the release, whereas removal of extracellular Ca2+ had no effect. In comparison, BAPTA and thapsigargin failed to affect glutamate release evoked by a hypoosmotic challenge (
Connexin (Cx) hemichannels have been implicated in astrocytic glutamate release after removal of extracellular divalent cations (such as Ca2+ and Mg2+) (Ye et al., “Functional Hemichannels in Astrocytes: A Novel Mechanism of Glutamate Release,” J. Neurosci. 23(9):3588-3596 (2003), which is hereby incorporated by reference in its entirety). To evaluate the role of Cx43 (the predominant member of the Cx family expressed by astrocytes), ATP-induced glutamate release from cultured astrocytes prepared from Cx43 KO mice and matched wild-type littermates was compared. ATP (100 μM) induced glutamate release of 3.02 fmol per cell from Cx43 KO astrocytes, which was 90.7% of astrocytes prepared from wild-type littermates (
To determine whether cell swelling was required for Ca2+-dependent astrocytic glutamate release, ATP was applied simultaneously with either increasing extracellular osmolarity (inhibition of cell swelling) or decreasing osmolarity (potentiation of cell swelling). ATP-induced glutamate release from cultured astrocytes was an inverse function of extracellular osmolarity shift (regression curve: y=0.181x+2.656, R2=0.994) and completely attenuated when osmolarity was raised by 15% (
One of the characteristics of swelling-induced glutamate release is that other osmolytes, including taurine, aspartate, and glutamine, are also released in parallel (Kimelberg et al., “Swelling-Induced Release of Glutamate, Aspartate, and Taurine from Astrocyte Cultures,” J. Neurosci. 10(5):1583-1591 (1990), which is hereby incorporated by reference in its entirety). To compare the mechanism of ATP-induced, Ca2+-dependent astrocytic glutamate release with swelling-induced release, the extracellular concentrations of amino acids released from cultured astrocytes by using HPLC was analyzed (
To provide direct evidence for purinergic-mediated opening of a glutamate-permeable channel, whole-cell currents in cultured astrocytes were recorded. To eliminate inward cation conductances, extracellular ions were replaced by sucrose (250 mM; osmolarity, 290 mEq) in the external solution, whereas the pipette solution contained 123 mM Cs+ Glutamate (Cl−-free). Under these ion conditions with a holding potential of −60 mV, glutamate is the only ion that can cause inward current due to its efflux. ATP (100 μM) triggered an inward current in 5 of 12 astrocytes, with average amplitude of 177±37 pA (range of 90-260 pA) (
Because ATP-activated glutamate-permeable channel also exhibited permeability to Na+ and K+, characterization of the ion permeability of the channel was performed. Reversal potentials under different ionic conditions were measured. Ramp commands before and after the application of ATP was first applied. The net I-V current was obtained by subtracting the I-V current before ATP application from the I-V current after ATP application (
With 123 mM Cs-glutamate in the pipette and sucrose outside, the reversal potential of the ATP-induced current was +17.0±2.5 mV (
Together, these observations on ion permeability are consistent with the recordings in
To determine whether stimulation of ATP is associated with a transient increase in astrocytic Ca2+ concentration and activation of glutamate-permeable channels in intact tissue, ATP-induced activation of astrocytes in situ was next characterized by using two-photon microscopy and whole-cell current-clamp approach. Astrocytes in hippocampal slices were loaded with Ca2+ indicator dye, Fluo-4 am (Kang et al., “Astrocyte-Mediated Potentiation of Inhibitory Synaptic Transmission,” Nat. Neurosci. 1(8):683-692 (1998); Kang et al., “Imaging Astrocytes in Acute Brain Slices,” Plainview, N.Y.: Cold Spring Harbor Lab. Press (1999), which are hereby incorporated by reference in their entirety). Application of ATP (1001) evoked a 131±13% increase in the fluo-4 signal over baseline that lasted an average of 8.7±1.4 sec in the vast majority of cells (>95%, n=250) (
The main observation is that receptor-mediated astrocytic Ca2+ increases are associated with transient cell swelling, resulting in the activation of volume-sensitive channels and the release of cytosolic glutamate. This demonstrates that glutamate release is intimately linked to dynamic changes in astrocytic cell volume and activation of VSC. Another important observation is that cytosolic glutamate can be released in a regulated, Ca2+-dependent manner and, therefore, constitute a potential transmitter pool.
Direct evidence for channel-mediated efflux of glutamate was obtained by whole-cell recordings of cultured astrocytes. ATP activated a glutamate-permeable channel. The property of channel opening closely mimicked the characteristics of astrocytic glutamate release. BAPTA, NPPB, FFA, and glossypol potently inhibited both channel activation and glutamate release. Importantly, increasing both osmolarity by 15% strongly inhibited channel activation and eliminated glutamate release (
Four other possible mechanisms of glutamate release to explain the data were considered.
First, opening of Ca2+-activated Cl− channels may provide a pathway for glutamate efflux. However, the inner pore diameter of Ca2+-activated Cl− channels may not be large enough to allow permeation of glutamate (6.5×10.8 Å), because diphenylamine-2-carboxylic acid (DPC, 6.0×9.4 Å) failed to permeate (Qu et al., “Functional Geometry of the Permeation Pathway of Ca2+-Activated Cl-Channels Inferred From Analysis of Voltage-Dependent Block,” J. Biol. Chem. 276(21):18423-18429 (2001), which is hereby incorporated by reference in its entirety). Also, the dependence of astrocytic glutamate release upon medium osmolarity (
Second, P2×7 receptor-gated channels have been implicated in Ca2+-independent efflux of glutamate from astrocytes (Duan et al., “P2X7 Receptor-Mediated Release of Excitatory Amino Acids From Astrocytes,” J. Neurosci. 23(4):1320-1328 (2003), which is hereby incorporated by reference in its entirety). The lack of action of BzATP and OxATP lend no support to a significant contribution of P2×7 receptors in Ca2+-dependent glutamate release (
Third, Ransom and coworkers (Ye et al., “Functional Hemichannels in Astrocytes: A Novel Mechanism of Glutamate Release,” J. Neurosci. 23(9):3588-3596 (2003), which is hereby incorporated by reference in its entirety) have recently reported the removal of divalent cations that open Cs-hemichannels, resulting in efflux of cytosolic glutamate. It was confirmed that removal of both extracellular divalent cations Mg2+ and Ca2+ resulted in sustained basal release but failed to potentiate ATP-induced glutamate release. Furthermore, astrocytes prepared from Cx43 KO and wild-type mice released comparable amount of glutamate, lending no support for the idea that Cs-hemichannels play a role in Ca2+-dependent glutamate release from astrocytes. This observation does not exclude that Cx-hemichannel may play important roles in glutamate release in pathological conditions, including ischemia and epilepsy (Ye et al., “Functional Hemichannels in Astrocytes: A Novel Mechanism of Glutamate Release,” J. Neurosci. 23(9):3588-3596 (2003); Tian et al., “An Astrocytic Basis of Epilepsy,” Nat. Med. 11(9):973-981 (2005), which are hereby incorporated by reference in their entirety).
Fourth, Ca2+-dependent exocytosis of glutamate from cultured astrocytes has been demonstrated by several groups (Montana et al., “Vesicular Glutamate Transporter-Dependent Glutamate Release From Astrocytes,” J. Neurosci. 24(12):2633-2642 (2004); Bezzi et al., “Astrocytes Contain a Vesicular Compartment That is Competent for Regulated Exocytosis of Glutamate,” Nat. Neurosci. 7(6):613-620 (2004); Kreft et al., “Properties of Ca(2+)-Dependent Exocytosis in Cultured Astrocytes,” Glia 46(4):437-445 (2004); Zhang et al., “Fusion-Related Release of Glutamate from Astrocytes,” J. Biol. Chem. 279(13):12724-12733 (2004), which are hereby incorporated by reference in their entirety). Although the present observations do not directly address the role of exocytosis in glutamate release, a number of the observations are not consistent with exocytosis constituting the primary pathway of astrocytic glutamate release. First, several anion channel blockers attenuated Ca2+-dependent glutamate release (
The finding that astrocytes release glutamate by a regulated pathway that is sensitive to several anion channel inhibits offers an opportunity to manipulate synaptic transmission both in normal physiology and in conditions that involve the pathological activation of astrocytes, including neurodegenerative diseases. Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/627,847, filed Nov. 15, 2004, which is hereby incorporated by reference in its entirety.
The subject matter of this application was made with support from the National Institute of Health under Grant No. 5-28926. The U.S. Government may have certain rights.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US05/41058 | 11/14/2005 | WO | 00 | 8/22/2008 |
Number | Date | Country | |
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60627847 | Nov 2004 | US |