Jorge E. Quintero

GFP fluorescence reports Period 1 circadian gene regulation in the mammalian biological clock.

&NA; Endogenous cyclic activation of a specific set of genes, including Period 1 (Per1), drive circadian rhythms in the suprachiasmatic nucleus (SCN), a biological clock nucleus of the brain. We have produced transgenic mice in which a degradable form of recombinant jellyfish green fluorescent protein (GFP) is driven by the mouse Period 1 (mPer1) gene promoter. GFP protein is expressed in the circadian neural structures of the retina and SCN. Fluorescent signals are resolved at the level of individual neurons. mPer1‐driven GFP fluorescence intensity reports light‐induction and circadian rhythmicity in SCN neurons. This circadian reporter transgene captures the gene expression dynamics of living biological clock neurons and ensembles, providing a novel view of this brain function.

Diffuse Brain Injury Elevates Tonic Glutamate Levels and Potassium-Evoked Glutamate Release in Discrete Brain Regions at Two Days Post-Injury: An Enzyme-Based Microelectrode Array Study

Traumatic brain injury (TBI) survivors often suffer from a wide range of post-traumatic deficits, including impairments in behavioral, cognitive, and motor function. Regulation of glutamate signaling is vital for proper neuronal excitation in the central nervous system. Without proper regulation, increases in extracellular glutamate can contribute to the pathophysiology and neurological dysfunction seen in TBI. In the present studies, enzyme-based microelectrode arrays (MEAs) that selectively measure extracellular glutamate at 2 Hz enabled the examination of tonic glutamate levels and potassium chloride (KCl)-evoked glutamate release in the prefrontal cortex, dentate gyrus, and striatum of adult male rats 2 days after mild or moderate midline fluid percussion brain injury. Moderate brain injury significantly increased tonic extracellular glutamate levels by 256% in the dentate gyrus and 178% in the dorsal striatum. In the dorsal striatum, mild brain injury significantly increased tonic glutamate levels by 200%. Tonic glutamate levels were significantly correlated with injury severity in the dentate gyrus and striatum. The amplitudes of KCl-evoked glutamate release were increased significantly only in the striatum after moderate injury, with a 249% increase seen in the dorsal striatum. Thus, with the MEAs, we measured discrete regional changes in both tonic and KCl-evoked glutamate signaling, which were dependent on injury severity. Future studies may reveal the specific mechanisms responsible for glutamate dysregulation in the post-traumatic period, and may provide novel therapeutic means to improve outcomes after TBI.

A short half-life GFP mouse model for analysis of suprachiasmatic nucleus organization.

Period1 (Per1) is one of several clock genes driving the oscillatory mechanisms that mediate circadian rhythmicity. Per1 mRNA and protein are highly expressed in the suprachiasmatic nuclei, which contain oscillator cells that drive circadian rhythmicity in physiological and behavioral responses. We examined a transgenic mouse in which degradable green fluorescent protein (GFP) is driven by the mPer1 gene promoter. This mouse expresses precise free-running rhythms and characteristic light induced phase shifts. GFP protein (reporting Per1 mRNA) is expressed rhythmically as measured by either fluorescence or immunocytochemistry. In addition the animals show predicted rhythms of Per1 mRNA, PER1 and PER2 proteins. The localization of GFP overlaps with that of Per1 mRNA, PER1 and PER2 proteins. Together, these results suggest that GFP reports rhythmic Per1 expression. A surprising finding is that, at their peak expression time GFP, Per1 mRNA, PER1 and PER2 proteins are absent or not detectable in a subpopulation of SCN cells located in the core region of the nucleus.

Disruptions in the Regulation of Extracellular Glutamate by Neurons and Glia in the Rat Striatum Two Days after Diffuse Brain Injury

Disrupted regulation of extracellular glutamate in the central nervous system contributes to and can exacerbate the acute pathophysiology of traumatic brain injury (TBI). Previously, we reported increased extracellular glutamate in the striatum of anesthetized rats 2 days after diffuse brain injury. To determine the mechanism(s) responsible for increased extracellular glutamate, we used enzyme-based microelectrode arrays (MEAs) coupled with specific pharmacological agents targeted at in vivo neuronal and glial regulation of extracellular glutamate. After TBI, extracellular glutamate was significantly increased in the striatum by (∼90%) averaging 4.1±0.6 μM compared with sham 2.2±0.4 μM. Calcium-dependent neuronal glutamate release, investigated by local application of an N-type calcium channel blocker, was no longer a significant source of extracellular glutamate after TBI, compared with sham. In brain-injured animals, inhibition of glutamate uptake with local application of an excitatory amino acid transporter inhibitor produced significantly greater increase in glutamate spillover (∼ 65%) from the synapses compared with sham. Furthermore, glutamate clearance measured by locally applying glutamate into the extracellular space revealed significant reductions in glutamate clearance parameters in brain-injured animals compared with sham. Taken together, these data indicate that disruptions in calcium-mediated glutamate release and glial regulation of extracellular glutamate contribute to increased extracellular glutamate in the striatum 2 days after diffuse brain injury. Overall, these data suggest that therapeutic strategies used to regulate glutamate release and uptake may improve excitatory circuit function and, possibly, outcomes following TBI.

Age-related changes in glutamate release in the CA3 and dentate gyrus of the rat hippocampus

The present studies employed a novel microelectrode array recording technology to study glutamate release and uptake in the dentate gyrus, CA3 and CA1 hippocampal subregions in anesthetized young, late-middle aged and aged male Fischer 344 rats. The mossy fiber terminals in CA3 showed a significantly decreased amount of KCl-evoked glutamate release in aged rats compared to both young and late-middle-aged rats. Significantly more KCl-evoked glutamate release was seen from perforant path terminals in the DG of late-middle-aged rats compared young and aged rats. The DG of aged rats developed an increased glutamate uptake rate compared to the DG of young animals, indicating a possible age-related change in glutamate regulation to deal with increased glutamate release that occurred in late-middle age. No age-related changes in resting levels of glutamate were observed in the DG, CA3 and CA1. Taken together, these data support dynamic changes to glutamate regulation during aging in subregions of the mammalian hippocampus that are critical for learning and memory.

Molecular Cloning of a Lobster Gαq Protein Expressed in Neurons of Olfactory Organ and Brain

Abstract: We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, hGαq, with >80% identity to mammalian and arthropod Gαq sequences. In brain and olfactory organ, hGαq mRNA was expressed predominantly in neurons, including virtually all the neuronal cell body clusters of the brain. Gαq protein was also expressed broadly, appearing on western blots as a single band of 46 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that hGαq plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, the expression of a single hGαq mRNA species (6 kb) in the olfactory organ, and the localization of hGαq mRNA predominantly in the olfactory receptor neurons of the olfactory organ strongly suggest that one function of hGαq is to mediate olfactory transduction.

Tonic and Phasic Release of Glutamate and Acetylcholine Neurotransmission in Sub-regions of the Rat Prefrontal Cortex Using Enzyme-based Microelectrode Arrays

The medial prefrontal cortex (mPFC) is an area of the brain critical for higher cognitive processes and implicated in disorders of the CNS such as drug addiction, depression and schizophrenia. Glutamate and acetylcholine are neurotransmitters that are essential for cortical functioning, yet little is known about the dynamic function of these neurotransmitters in subregions of the mPFC. In these studies we used a novel microelectrode array technology to measure resting levels (tonic release) of glutamate and acetylcholine as well as KCl-evoked release (stimulated phasic release) in the mPFC of the anesthetized rat to further our understanding of both tonic and phasic neurotransmission in the cingulate cortex, prelimbic cortex, and infralimbic cortex of the mPFC. Studies revealed homogeneity of tonic and phasic signaling among brain subregions for each neurotransmitter. However, resting levels of glutamate were significantly higher as compared to acetylcholine levels in all subregions. Additionally, KCl-evoked acetylcholine release in the cingulate cortex (7.1 μM) was significantly greater than KCl-evoked glutamate release in any of the three subregions (Cg1, 2.9 μM; PrL, 2.0 μM; IL, 1.8 μM). Interestingly, the time for signal decay following KCl-evoked acetylcholine release was significantly longer by an average of 240% as compared to KCL-evoked glutamate release for all three brain subregions. Finally, we observed a negative relationship between acetylcholine resting levels and KCl-evoked release in the Cg1. These data suggest a homogenous distribution of both glutamatergic and acetylcholinergic innervation in the mPFC, with alterations in tonic and phasic release regulation accounting for differences between these neurotransmitters.

Amperometric measures of age-related changes in glutamate regulation in the cortex of rhesus monkeys

l-glutamate (glutamate) is the principal excitatory neurotransmitter of the central nervous system and is involved in altered neural function during aging and in neurodegenerative diseases. Relatively little is known about the mechanisms of glutamate signaling in the primate brain, in part, because there is an absence of a method capable of rapidly measuring glutamate in either a non-clinical or a clinical setting. We have addressed this paucity of information by measuring extracellular glutamate at 1 Hz in the pre-motor and motor cortices of young, middle-aged, and aged monkeys using a minimally invasive amperometric recording method. In the motor cortex, mean resting glutamate levels were five times higher in the aged group compared to the young group while the pre-motor cortex showed an increasing trend in resting glutamate levels that was not statistically significant. In addition, we measured rapid, phasic glutamate release after local pressure-ejection of nanoliter volumes of either isotonic 70 mM potassium (to stimulate glutamate release) or 1 mM glutamate (to study glutamate uptake) into the pre-motor and motor cortex. In the pre-motor cortex, we measured reproducible glutamate uptake signals that had a significantly decreased (47%) rate of glutamate uptake in aged animals compared to young animals. However, following a 70 mM potassium delivery, we did not observe any consistent changes in evoked release between young versus aged animals. Using these non-clinical microelectrodes to measure glutamate signaling in the brain, our results support the hypothesis that the glutamatergic system undergoes reorganization with aging of the central nervous system.

Methodology for rapid measures of glutamate release in rat brain slices using ceramic-based microelectrode arrays: Basic characterization and drug pharmacology

Excessive excitability or hyperexcitability of glutamate-containing neurons in the brain has been proposed as a possible explanation for anxiety, stress-induced disorders, epilepsy, and some neurodegenerative diseases. However, direct measurement of glutamate on a rapid time scale has proven to be difficult. Here we adapted enzyme-based microelectrode arrays (MEA) capable of detecting glutamate in vivo, to assess the effectiveness of hyperexcitability modulators on glutamate release in brain slices of the rat neocortex. Using glutamate oxidase coated ceramic MEAs coupled with constant voltage amperometry, we measured resting glutamate levels and synaptic overflow of glutamate after K(+) stimulation in brain slices. MEAs reproducibly detected glutamate on a second-by-second time scale in the brain slice preparation after depolarization with high K(+) to evoke glutamate release. This stimulus-evoked glutamate release was robust, reproducible, and calcium dependent. The K(+)-evoked glutamate release was modulated by ligands to the α(2)δ subunit of voltage sensitive calcium channels (PD-0332334 and PD-0200390). Meanwhile, agonists to Group II metabotropic glutamate (mGlu) receptors (LY379268 and LY354740), which are known to alter hyperexcitability of glutamate neurons, attenuated K(+)-evoked glutamate release but did not alter resting glutamate levels. This new MEA technology provides a means of directly measuring the chemical messengers involved in glutamate neurotransmission and thereby helping to reveal the role multiple glutamatergic system components have on glutamate signaling.

Real-time monitoring of extracellular adenosine using enzyme-linked microelectrode arrays

Throughout the central nervous system extracellular adenosine serves important neuroprotective and neuromodulatory functions. However, current understanding of the in vivo regulation and effects of adenosine is limited by the spatial and temporal resolution of available measurement techniques. Here, we describe an enzyme-linked microelectrode array (MEA) with high spatial (7500 µm(2)) and temporal (4 Hz) resolution that can selectively measure extracellular adenosine through the use of self-referenced coating scheme that accounts for interfering substances and the enzymatic breakdown products of adenosine. In vitro, the MEAs selectively measured adenosine in a linear fashion (r(2)=0.98±0.01, concentration range=0-15 µM, limit of detection =0.96±0.5 µM). In vivo the limit of detection was 0.04±0.02 µM, which permitted real-time monitoring of the basal extracellular concentration in rat cerebral cortex (4.3±1.5 µM). Local cortical injection of adenosine through a micropipette produced dose-dependent transient increases in the measured extracellular concentration (200 nL: 6.8±1.8 µM; 400 nL: 19.4±5.3 µM) [P<0.001]. Lastly, local injection of dipyridamole, which inhibits transport of adenosine through equilibrative nucleoside transporter, raised the measured extracellular concentration of adenosine by 120% (5.6→12.3 µM) [P<0.001]. These studies demonstrate that MEAs can selectively measure adenosine on temporal and spatial scales relevant to adenosine signaling and regulation in normal and pathologic states.