Journal of Pharmacology and Experimental Therapeutics, 2011
Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinicall... more Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K(+)-evoked glutamate release without affecting basal glutamate levels. PGB (0.1-100 μM) exhibited concentration-dependent inhibition of K(+)-evoked glutamate release with an IC(50) value of 5.3 μM. R-(-)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K(+)-evoked glutamate release. The decrease of K(+)-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α(2)δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α(2)δ-1 (subtype) subunit of VSCC, PGB did not affect K(+)-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α(2)δ-1 subunit of VSCC appears to mediate this effect.
Tyramine permeates chromaffin-granule membranes via a reserpine-insensitive mechanism. The rate i... more Tyramine permeates chromaffin-granule membranes via a reserpine-insensitive mechanism. The rate is unsaturable and increases with pH, indicating permeation of the unprotonated form of the amine. Reserpine-insensitive dopamine uptake is at least 10 times slower, consistent with dopamine's lesser lipophilicity. Dopamine is transported into chromaffin-granule membrane vesicles via a saturable, reserpine-sensitive, proton-linked mechanism. Tyramine inhibits dopamine transport with a Ki of 5-10 microM. Tyramine is not accumulated nearly as well as dopamine because inward transport is opposed by outward permeation. Nevertheless, the velocity of reserpine-sensitive tyramine transport can be deduced from the steady-state level of tyramine accumulation and the rate of permeation. Vmax for tyramine transport is about one-third of the value for dopamine transport. Therefore, two aromatic hydroxyls are not needed for monoamine transport but are required for efficient accumulation and storage.
Abstract L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous sy... more Abstract L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and is implicated in a number of brain disorders including Parkinsons disease (PD), cognitive disturbances, epilepsy, schizophrenia, attention deficit ...
To circumvent the challenges associated with delivering large compounds directly to the brain for... more To circumvent the challenges associated with delivering large compounds directly to the brain for the treatment of Parkinson's disease (PD), non-invasive procedures utilizing smaller molecules with protective and/or restorative actions on dopaminergic neurons are needed. We developed a methodology for evaluating the effects of a synthetic neuroactive peptide, DNSP-11, on the nigrostriatal system using repeated intranasal delivery in both normal and a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of PD. Normal rats repeatedly administered varying doses of DNSP-11 intranasally for 3 weeks exhibited a significant increase in dopamine (DA) turnover in both the striatum and substantia nigra (SN) at 300μg, suggestive of a stimulative effect of the dopaminergic system. Additionally, a protective effect was observed following repeated intranasal administration in 6-OHDA lesioned rats, as suggested by: a significant decrease in d-amphetamine-induced rotation at 2 weeks; a decrea...
We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neu... more We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neurons of C57BL/6 mice results in increased depolarization-induced glutamate release that eventually leads to selective neuronal injury and cell loss by 12 months of age. However, it is known that isogenic lines of Tg (transgenic) mice produced through back-crossing with one strain may differ in their phenotypic characteristics from those produced using another inbred mouse strain. Therefore, we decided to introduce the Glud1 transgene into the Balb/c strain that has endogenously lower levels of GLUD1 (glutamate dehydrogenase 1) enzyme activity in the brain as compared with C57BL/6. Using an enzyme-based MEA (microelectrode array) that is selective for measuring glutamate in vivo, we measured depolarization-induced glutamate release. Within a discrete layer of the striatum, glutamate release was significantly increased in Balb/c Tg mice compared with wt (wild-type) littermates. Furthermore...
Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectr... more Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both nonenzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantages over glutaraldehyde as we observe comparable selectivities and responses. These results support that glutaraldehyde-cross-linked glutamate oxidase retains sufficient enzyme specificity for accurate in vivo brain measures of tonic and phasic glutamate levels when immobilized using specific "wet" coating procedures.
During the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercel... more During the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercellular modulator of cell functions. In the brain, .NO is implicated in mechanisms of synaptic plasticity but it is also involved in cell death pathways underlying several neurological diseases. Because of its hydrophobicity, small size, and rapid diffusion properties, the rate and pattern of .NO concentration changes are critical determinants for the understanding of its diverse actions in the brain. .NO measurement in vivo has been a challenging task due to its low concentration, short half-life, and high reactivity with other biological molecules, such as superoxide radical, thiols, and heme proteins. Electrochemical methods are versatile approaches for detecting and monitoring various neurotransmitters. When associated with microelectrodes inserted into the brain they provide high temporal and spatial resolution, allowing measurements of neurochemicals in physiological environments in a real-time fashion. To date, electrochemical detection of .NO is the only available technique that provides a high sensitivity, low detection limit, selectivity, and fast response to measure the concentration dynamics of .NO in vivo. We have used carbon fiber microelectrodes coated with two layers of Nafion and o-phenylenediamine to monitor the rate and pattern of .NO change in the rat brain in vivo. The analytical performance of microelectrodes was assessed in terms of sensitivity, detection limit, and selectivity ratios against major interferents: ascorbate, dopamine, noradrenaline, serotonin, and nitrite. For the in vivo recording experiments, we used a microelectrode/micropipette array inserted into the brain using a stereotaxic frame. The characterization of in vivo signals was assessed by electrochemical and pharmacological verification. Results support our experimental conditions that the measured oxidation current reflects variations in the .NO concentration in brain extracellular space. We report results from recordings in hippocampus and striatum upon stimulation of N-methyl-d-aspartate-subtype glutamate receptors. Moreover, the kinetics of .NO disappearance in vivo following pressure ejection of a .NO solution is also addressed.
Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference, 2013
Conformal ceramic electrodes utilized in prior recordings of nonhuman primate prefrontal cortical... more Conformal ceramic electrodes utilized in prior recordings of nonhuman primate prefrontal cortical layer 2/3 and layer 5 neurons were used in this study to record tonic glutamate concentration and transient release in layer 2/3 PFC. Tonic glutamate concentration increased in the Match (decision) phase of a visual delayed-match-to-sample (DMS) task, while increased transient glutamate release occurred in the Sample (encoding) phase of the task. Further, spatial vs. object-oriented DMS trials evoked differential changes in glutamate concentration. Thus the same conformal recording electrodes were capable of electrophysiological and electrochemical recording, and revealed similar evidence of neural processing in layers 2/3 and layer 5 during cognitive processing in a behavioral task.
ABSTRACT L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous sy... more ABSTRACT L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and is implicated in a number of brain disorders including Parkinson’s disease (PD), cognitive disturbances, epilepsy, schizophrenia, attention deficit hyperactivity disorder (ADHD) and drug abuse. While microdialysis methods have been used extensively over the last decade to investigate minute-by-minute measures of L-glutamate, the rapid time dynamics of L-glutamate signaling in the CNS has warranted a technique to measure L-glutamate release on a second-by- second basis. A major goal of the research is to develop a recording technology for recording second-by-second measurements of L-glutamate and other neurotransmitters—specifically a mass-fabricated microelectrode technology that could be (1) mass produced such that other laboratories could utilize the same recording technology and (2) configured for “self-referencing” recordings, which allows for second-by-second cross-checking of the selectivity of the micro-electrode measures and improved signal-to-noise of the recording methods. The present chapter documents current capabilities of measuring L-glutamate and several other neurotransmitters on a second-by-second basis using mass-fabricated microelectrode arrays formed on ceramic. While not a comprehensive assessment of the technology, this chapter contains a large amount of information regarding the fabrication, use, and potential pitfalls of this technology. The reader should refer to numerous articles [1–6] for additional details regarding measuring neurotransmitters in the CNS.
Journal of Pharmacology and Experimental Therapeutics, 2011
Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinicall... more Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K(+)-evoked glutamate release without affecting basal glutamate levels. PGB (0.1-100 μM) exhibited concentration-dependent inhibition of K(+)-evoked glutamate release with an IC(50) value of 5.3 μM. R-(-)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K(+)-evoked glutamate release. The decrease of K(+)-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α(2)δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α(2)δ-1 (subtype) subunit of VSCC, PGB did not affect K(+)-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α(2)δ-1 subunit of VSCC appears to mediate this effect.
Tyramine permeates chromaffin-granule membranes via a reserpine-insensitive mechanism. The rate i... more Tyramine permeates chromaffin-granule membranes via a reserpine-insensitive mechanism. The rate is unsaturable and increases with pH, indicating permeation of the unprotonated form of the amine. Reserpine-insensitive dopamine uptake is at least 10 times slower, consistent with dopamine's lesser lipophilicity. Dopamine is transported into chromaffin-granule membrane vesicles via a saturable, reserpine-sensitive, proton-linked mechanism. Tyramine inhibits dopamine transport with a Ki of 5-10 microM. Tyramine is not accumulated nearly as well as dopamine because inward transport is opposed by outward permeation. Nevertheless, the velocity of reserpine-sensitive tyramine transport can be deduced from the steady-state level of tyramine accumulation and the rate of permeation. Vmax for tyramine transport is about one-third of the value for dopamine transport. Therefore, two aromatic hydroxyls are not needed for monoamine transport but are required for efficient accumulation and storage.
Abstract L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous sy... more Abstract L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and is implicated in a number of brain disorders including Parkinsons disease (PD), cognitive disturbances, epilepsy, schizophrenia, attention deficit ...
To circumvent the challenges associated with delivering large compounds directly to the brain for... more To circumvent the challenges associated with delivering large compounds directly to the brain for the treatment of Parkinson's disease (PD), non-invasive procedures utilizing smaller molecules with protective and/or restorative actions on dopaminergic neurons are needed. We developed a methodology for evaluating the effects of a synthetic neuroactive peptide, DNSP-11, on the nigrostriatal system using repeated intranasal delivery in both normal and a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of PD. Normal rats repeatedly administered varying doses of DNSP-11 intranasally for 3 weeks exhibited a significant increase in dopamine (DA) turnover in both the striatum and substantia nigra (SN) at 300μg, suggestive of a stimulative effect of the dopaminergic system. Additionally, a protective effect was observed following repeated intranasal administration in 6-OHDA lesioned rats, as suggested by: a significant decrease in d-amphetamine-induced rotation at 2 weeks; a decrea...
We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neu... more We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neurons of C57BL/6 mice results in increased depolarization-induced glutamate release that eventually leads to selective neuronal injury and cell loss by 12 months of age. However, it is known that isogenic lines of Tg (transgenic) mice produced through back-crossing with one strain may differ in their phenotypic characteristics from those produced using another inbred mouse strain. Therefore, we decided to introduce the Glud1 transgene into the Balb/c strain that has endogenously lower levels of GLUD1 (glutamate dehydrogenase 1) enzyme activity in the brain as compared with C57BL/6. Using an enzyme-based MEA (microelectrode array) that is selective for measuring glutamate in vivo, we measured depolarization-induced glutamate release. Within a discrete layer of the striatum, glutamate release was significantly increased in Balb/c Tg mice compared with wt (wild-type) littermates. Furthermore...
Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectr... more Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both nonenzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantages over glutaraldehyde as we observe comparable selectivities and responses. These results support that glutaraldehyde-cross-linked glutamate oxidase retains sufficient enzyme specificity for accurate in vivo brain measures of tonic and phasic glutamate levels when immobilized using specific "wet" coating procedures.
During the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercel... more During the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercellular modulator of cell functions. In the brain, .NO is implicated in mechanisms of synaptic plasticity but it is also involved in cell death pathways underlying several neurological diseases. Because of its hydrophobicity, small size, and rapid diffusion properties, the rate and pattern of .NO concentration changes are critical determinants for the understanding of its diverse actions in the brain. .NO measurement in vivo has been a challenging task due to its low concentration, short half-life, and high reactivity with other biological molecules, such as superoxide radical, thiols, and heme proteins. Electrochemical methods are versatile approaches for detecting and monitoring various neurotransmitters. When associated with microelectrodes inserted into the brain they provide high temporal and spatial resolution, allowing measurements of neurochemicals in physiological environments in a real-time fashion. To date, electrochemical detection of .NO is the only available technique that provides a high sensitivity, low detection limit, selectivity, and fast response to measure the concentration dynamics of .NO in vivo. We have used carbon fiber microelectrodes coated with two layers of Nafion and o-phenylenediamine to monitor the rate and pattern of .NO change in the rat brain in vivo. The analytical performance of microelectrodes was assessed in terms of sensitivity, detection limit, and selectivity ratios against major interferents: ascorbate, dopamine, noradrenaline, serotonin, and nitrite. For the in vivo recording experiments, we used a microelectrode/micropipette array inserted into the brain using a stereotaxic frame. The characterization of in vivo signals was assessed by electrochemical and pharmacological verification. Results support our experimental conditions that the measured oxidation current reflects variations in the .NO concentration in brain extracellular space. We report results from recordings in hippocampus and striatum upon stimulation of N-methyl-d-aspartate-subtype glutamate receptors. Moreover, the kinetics of .NO disappearance in vivo following pressure ejection of a .NO solution is also addressed.
Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference, 2013
Conformal ceramic electrodes utilized in prior recordings of nonhuman primate prefrontal cortical... more Conformal ceramic electrodes utilized in prior recordings of nonhuman primate prefrontal cortical layer 2/3 and layer 5 neurons were used in this study to record tonic glutamate concentration and transient release in layer 2/3 PFC. Tonic glutamate concentration increased in the Match (decision) phase of a visual delayed-match-to-sample (DMS) task, while increased transient glutamate release occurred in the Sample (encoding) phase of the task. Further, spatial vs. object-oriented DMS trials evoked differential changes in glutamate concentration. Thus the same conformal recording electrodes were capable of electrophysiological and electrochemical recording, and revealed similar evidence of neural processing in layers 2/3 and layer 5 during cognitive processing in a behavioral task.
ABSTRACT L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous sy... more ABSTRACT L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and is implicated in a number of brain disorders including Parkinson’s disease (PD), cognitive disturbances, epilepsy, schizophrenia, attention deficit hyperactivity disorder (ADHD) and drug abuse. While microdialysis methods have been used extensively over the last decade to investigate minute-by-minute measures of L-glutamate, the rapid time dynamics of L-glutamate signaling in the CNS has warranted a technique to measure L-glutamate release on a second-by- second basis. A major goal of the research is to develop a recording technology for recording second-by-second measurements of L-glutamate and other neurotransmitters—specifically a mass-fabricated microelectrode technology that could be (1) mass produced such that other laboratories could utilize the same recording technology and (2) configured for “self-referencing” recordings, which allows for second-by-second cross-checking of the selectivity of the micro-electrode measures and improved signal-to-noise of the recording methods. The present chapter documents current capabilities of measuring L-glutamate and several other neurotransmitters on a second-by-second basis using mass-fabricated microelectrode arrays formed on ceramic. While not a comprehensive assessment of the technology, this chapter contains a large amount of information regarding the fabrication, use, and potential pitfalls of this technology. The reader should refer to numerous articles [1–6] for additional details regarding measuring neurotransmitters in the CNS.
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