Search

The attribution of incentive salience to reward-predictive stimuli has been shown to be associated with substance abuse-like behavior such as increased drug taking. Evidence suggests that glutamate neurotransmission and sequential N-methyl-D-aspartate (NMDA) activation are involved in the attribution of incentive salience. Here, we further explore the role of second-by-second glutamate neurotransmission in the attribution of incentive salience to reward-predictive stimuli by measuring sign-tracking behavior during a Pavlovian conditioned approach procedure using ceramic-based microelectrode arrays configured for sensitive measures of extracellular glutamate in awake behaving Sprague-Dawley rats. Specifically, we show that there is an increase in extracellular glutamate levels in the prelimbic cortex (PrL) and the nucleus accumbens core (NAcC) during sign-tracking behavior to a food-predictive conditioned stimulus (CS+) compared to the presentation of a non-predictive conditioned stimulus (CS-). Furthermore, the results indicate greater increases in extracellular glutamate levels in the PrL compared to NAcC in response to the CS+, including differences in glutamate release and signal decay. Taken together, the present research suggests that there is differential glutamate signaling in the NAcC and PrL during sign-tracking behavior to a food-predictive CS+.

J. Neurochem. (2012) 122, 619–627. Abstract The potential anxiolytic effects of a novel positive allosteric modulator (PAM) of the metabotropic glutamate receptor subgroup 2 (mGluR2) were investigated using a self-referencing recording technique with enzyme-based microelectrode arrays (MEAs) that reliably measures tonic and phasic changes in extracellular glutamate levels in awake rats. Studies involved glutamate measures in the rat prefrontal cortex during subcutaneous injections of the following: vehicle, a mGluR2/3 agonist, LY354740 (10 mg/kg), or a mGluR2 PAM, 1-Methyl-2-((cis-(R,R)-3-methyl-4-(4-trifluoromethoxy-2-fluoro)phenyl)piperidin-1-yl)methyl)-1H-imidazo[4,5-b]pyridine ((+)-TFMPIP; 1.0 or 17.8 mg/kg). Studies assessed changes in tonic glutamate levels and the glutamatergic responses to a 5-min restraint stress. Subcutaneous injection of (+)-TFMPIP at a dose of 1.0 mg/kg (day 3: -7.1 ± 15.1 net AUC; day 5: -24.8 ± 24.9 net AUC) and 17.8 mg/kg (day 3: -46.5 ± 33.0 net AUC; day 5: 34.6 ± 36.8 net AUC) significantly attenuated the stress-evoked glutamate release compared to vehicle controls (day 3: 134.7 ± 50.6 net AUC; day 5: 286.6 ± 104.5 net AUC), whereas the mGluR2/3 agonist LY354740 had no effect. None of the compounds significantly affected resting glutamate levels, which we have recently shown to be extensively derived from neurons. Taken together, these data support that systemic administration of (+)-TFMPIP produces phasic rather than tonic release of glutamate that may play a major role in the effects of stress on glutamate neuronal systems in the prefrontal cortex.

l-glutamate (Glu) is the main excitatory neurotransmitter in the central nervous system (CNS) and is associated with motor behavior and sensory perception. While microdialysis methods have been used to record tonic levels of Glu, little is known about the more rapid changes in Glu signals that may be observed in awake rats. We have reported acute recording methods using enzyme-based microelectrode arrays (MEA) with fast response time and low detection levels of Glu in anesthetized animals with minimal interference. The current paper concerns modification of the MEA design to allow for reliable measures in the brain of conscious rats. In this study, we characterized the effects of chronic implantation of the MEA into the brains of rats. We were capable of measuring Glu levels for 7 days without loss of sensitivity. We performed studies of tail-pinch induced stress, which caused a robust biphasic increase in Glu. Histological data show chronic implantation of the MEAs caused minimal injury to the CNS. Taken together, our data show that chronic recordings of tonic and phasic Glu can be carried out in awake rats for up to 17 days in vivo allowing longer term studies of Glu regulation in behaving rats.

The potential anxiolytic effects of a novel positive allosteric modulator (PAM) of the metabotropic glutamate receptor subgroup 2 (mGluR2) were investigated using a self-referencing recording technique with enzyme-based microelectrode arrays (MEAs) that reliably measures tonic and phasic changes in extracellular glutamate levels in awake rats. Studies involved glutamate measures in the rat prefrontal cortex during subcutaneous injections of the following: vehicle, a mGluR2/3 agonist, LY354740 (10 mg/kg), or a mGluR2 PAM, 1-Methyl-2-((cis-(R,R)-3-methyl-4-(4-trifluoromethoxy-2-fluoro)phenyl)piperidin-1-yl)methyl)-1H-imidazo[4,5-b]pyridine ((+)-TFMPIP; 1.0 or 17.8 mg/kg). Studies assessed changes in tonic glutamate levels and the glutamatergic responses to a five minute restraint stress. Subcutaneous injection of (+)-TFMPIP at a dose of 1.0 mg/kg (day 3: −7.1 ± 15.1 net AUC; day 5: −24.8 ± 24.9 net AUC) and 17.8 mg/kg (day 3: −46.5 ± 33.0 net AUC; day 5: 34.6 ± 36.8 net AUC) significantly attenuated the stress-evoked glutamate release compared to vehicle controls (day 3: 134.7 ± 50.6 net AUC; day 5: 286.6 ± 104.5 net AUC), while the mGluR2/3 agonist LY354740 had no effect. None of the compounds significantly affected resting glutamate levels, which we have recently shown to be extensively derived from neurons. Taken together, these data support that systemic administration of (+)-TFMPIP produces phasic rather than tonic release of glutamate that may play a major role in the effects of stress on glutamate neuronal systems in the prefrontal cortex.

Glutamate in the prefrontal cortex (PFC) plays a significant role in several mental illnesses, including schizophrenia, addiction and anxiety. Previous studies on PFC glutamate-mediated function have used techniques that raise questions on the neuronal versus astrocytic origin of glutamate. The present studies used enzyme-based microelectrode arrays to monitor second-by-second resting glutamate levels in the PFC of awake rats. Locally applied drugs were employed in an attempt to discriminate between the neuronal or glial components of the resting glutamate signal. Local application of tetrodotoxin (sodium channel blocker), produced a significant (∼ 40%) decline in resting glutamate levels. In addition significant reductions in extracellular glutamate were seen with locally applied ω-conotoxin (MVIIC; ∼ 50%; calcium channel blocker), and the mGluR(2/3) agonist, LY379268 (∼ 20%), and a significant increase with the mGluR(2/3) antagonist LY341495 (∼ 40%), effects all consistent with a large neuronal contribution to the resting glutamate levels. Local administration of D,L-threo-β-benzyloxyaspartate (glutamate transporter inhibitor) produced an ∼ 120% increase in extracellular glutamate levels, supporting that excitatory amino acid transporters, which are largely located on glia, modulate clearance of extracellular glutamate. Interestingly, local application of (S)-4-carboxyphenylglycine (cystine/glutamate antiporter inhibitor), produced small, non-significant bi-phasic changes in extracellular glutamate versus vehicle control. Finally, pre-administration of tetrodotoxin completely blocked the glutamate response to tail pinch stress. Taken together, these results support that PFC resting glutamate levels in rats as measured by the microelectrode array technology are at least 40-50% derived from neurons. Furthermore, these data support that the impulse flow-dependent glutamate release from a physiologically -evoked event is entirely neuronally derived.

3,4-Dihydroxyphenyl-l-alanine (l-DOPA)-induced dyskinesia often develops as a side effect of chronic l-DOPA therapy. This study was undertaken to investigate dopamine (DA) release upon l-DOPA treatment. Chronoamperometric measurements were performed in unilaterally DA-depleted rats, chronically treated with l-DOPA, resulting in dyskinetic and non-dyskinetic animals. Normal and lesioned l-DOPA naive animals were used as controls. Potassium-evoked DA releases were significantly reduced in intact sides of animals undertaken chronic l-DOPA treatment, independent on dyskinetic behavior. Acute l-DOPA further attenuated the amplitude of the DA release in the control sides. In DA-depleted striata, no difference was found in potassium-evoked DA releases, and acute l-DOPA did not affect the amplitude. While immunoreactivity to serotonin uptake transporter was higher in lesioned striata of animals displaying dyskinetic behavior, no correlation could be documented between serotonin transporter-positive nerve fiber density and the amplitude of released DA. In conclusions, the amplitude of potassium-evoked DA release is attenuated in intact striatum after chronic intermittent l-DOPA treatment. No change in amplitude was found in DA-denervated sides of either dyskinetic or non-dyskinetic animals, while release kinetics were changed. This indicates the importance of studying DA release dynamics for the understanding of both beneficial and adverse effects of l-DOPA replacement therapy.

l-glutamate (Glu) is the main excitatory neurotransmitter in the central nervous system (CNS) and is associated with motor behavior and sensory perception. While microdialysis methods have been used to record tonic levels of Glu, little is known about the more rapid changes in Glu signals that may be observed in awake rats. We have reported acute recording methods using enzyme-based microelectrode arrays (MEA) with fast response time and low detection levels of Glu in anesthetized animals with minimal interference. The current paper concerns modification of the MEA design to allow for reliable measures in the brain of conscious rats. In this study, we characterized the effects of chronic implantation of the MEA into the brains of rats. We were capable of measuring Glu levels for 7 days without loss of sensitivity. We performed studies of tail-pinch induced stress, which caused a robust biphasic increase in Glu. Histological data show chronic implantation of the MEAs caused minimal injury to the CNS. Taken together, our data show that chronic recordings of tonic and phasic Glu can be carried out in awake rats for up to 17 days in vivo allowing longer term studies of Glu regulation in behaving rats.

L-glutamate (Glu) is the predominant excitatory neurotransmitter in the mammalian central nervous system. It plays major roles in normal neurophysiology and many brain disorders by binding to membrane-bound Glu receptors. To overcome the spatial and temporal limitations encountered in previous in vivo extracellular Glu studies, we employed enzyme-coated microelectrode arrays to measure both basal and potassium-evoked release of Glu in the anesthetized rat brain. We also addressed the question of signal identity, which is the predominant criticism of these recording technologies. In vivo self-referencing recordings demonstrated that our Glu signals were both enzyme- and voltage-dependent, supporting the identity of L-glutamate. In addition, basal Glu was actively regulated, tetrodotoxin (TTX)-dependent, and measured in the low micromolar range (approximately 2 microm) using multiple self-referencing subtraction approaches for identification of Glu. Moreover, potassium-evoked Glu release exhibited fast kinetics that were concentration-dependent and reproducible. These data support the hypothesis that Glu release is highly regulated, requiring detection technologies that must be very close to the synapse and measure on a second-by-second basis to best characterize the dynamics of the Glu system.