Theresa Currier Thomas

Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra.

Glial cell line‐derived neurotrophic factor (GDNF) improves motor dysfunction associated with aging in rats and non‐human primates, in animal models of Parkinsons disease, and may improve motoric function in patients with advanced Parkinsons disease. These improvements are associated with increased dopamine function in the nigrostriatal system, but the molecular events associated with this increase are unknown. In these studies, 100 µg of GDNF was injected into the striatum of normal aged (24‐month‐old) male Fischer 344 rats. The protein levels and phosphorylation of TH, ERK1/2, and related proteins were determined by blot‐immunolabeling of striatum and substantia nigra harvested 30 days after injection. In GDNF‐treated rats, TH phosphorylation at Ser31 increased ∼40% in striatum and ∼250% in the substantia nigra. In the substantia nigra, there was a significant increase in ERK1 phosphorylation. In striatum, there was a significant increase in ERK2 phosphorylation. Microdialysis studies in striatum showed that both amphetamine‐ and potassium‐evoked dopamine release in GDNF recipients were significantly increased. These data show that GDNF‐induced increases in dopamine function are associated with a sustained increase in TH phosphorylation at Ser31, which is greatest in the substantia nigra and maintained for at least one month following a single striatal administration of GDNF. These findings, taken from the nigrostriatal system of normal aged rats, may help explain the long lasting effects of GDNF on dopamine function and prior studies supporting that a major effect of GDNF involves its effects on dopamine storage and somatodendritic release of dopamine in the substantia nigra.

Morphological and genetic activation of microglia after diffuse traumatic brain injury in the rat.

Traumatic brain injury (TBI) survivors experience long-term post-traumatic morbidities. In diffuse brain-injured rats, a chronic sensory sensitivity to whisker stimulation models the agitation of TBI survivors and provides anatomical landmarks across the whisker-barrel circuit to evaluate post-traumatic neuropathology. As a consequence of TBI, acute and chronic microglial activation can contribute to degenerative and reparative events underlying post-traumatic morbidity. Here we hypothesize that a temporal sequence of microglial activation states contributes to the circuit pathology responsible for post-traumatic morbidity, and test the hypothesis by examining microglial morphological activation and neuroinflammatory markers for activation states through gene expression and receptor-binding affinity. Adult male, Sprague-Dawley rats were subjected to a single moderate midline fluid percussion injury (FPI) or sham injury. Microglial activation was determined by immunohistochemistry, quantitative real-time PCR and receptor autoradiography in the primary somatosensory barrel field (S1BF) and ventral posterior medial nucleus (VPM) of the thalamus at 7 and 28 days following FPI. Morphological changes indicative of microglial activation, including swollen cell body with thicker, shrunken processes, were evident in S1BF and VPM at 7 and 28 days post-injury. Principally at 7 days post-injury in VPM, general inflammatory gene expression (major histocompatibility complex I, major histocompatibility complex II, translocator protein 18 kDa [TSPO]) is increased above sham level and TSPO gene expression confirmed by receptor autoradiography. Further, CD45, a marker of classical activation, and TGF-βI, an acquired deactivation marker, were elevated significantly above sham at 7 days post-injury. Daily administration of the anti-inflammatory ibuprofen (20mg/kg, i.p.) significantly reduced the expression of these genes. Evidence for alternative activation (arginase 1) was not observed. Thus, these data demonstrate concomitant classical activation and acquired deactivation phenotypes of microglia in diffuse TBI in the absence of overt contusion or cavitation. Anti-inflammatory treatment may further alleviate the neuropathological burden of post-traumatic inflammation.

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.

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.

Decreased Dopamine D4 Receptor Expression Increases Extracellular Glutamate and Alters Its Regulation in Mouse Striatum

To better understand the effect of the dopamine D4 receptor (DRD4) on glutamate (Glu) neurotransmission in the brain, we utilized transgenic mice with partial or complete removal of functional DRD4 plasma membrane expression (DRD4+/− and DRD4−/−, respectively). We measured resting extracellular Glu levels, Glu clearance kinetics, and KCl-evoked release of Glu in the striatum and nucleus accumbens core of these mice using in vivo amperometry coupled to a novel microelectrode array configured for sub-second detection of Glu. Recordings from DRD4−/− and DRD4+/− mice were compared with their wild-type littermates (DRD4+/+). Resting extracellular levels of Glu were increased in the striatum of DRD4−/− mice (p<0.01). Glu clearance kinetics were significantly decreased in the dorsal striatum of DRD4−/− mice (p<0.05). KCl-evoked overflow of Glu was reliably measured but unchanged in the striatum of the three groups. By contrast, no changes in resting Glu, Glu uptake kinetics, or KCl-evoked release of Glu were observed in the nucleus accumbens core among the three genotypes. These data indicate that the DRD4 receptor is involved in modulation of Glu neurotransmission, primarily in the striatum. A better understanding of Glu control by the DRD4 may improve our understanding of the physiological role of the DRD4 in disorders such as attention-deficit/hyperactivity disorder and schizophrenia.

Dopamine D4 receptor knockout mice exhibit neurochemical changes consistent with decreased dopamine release

Dopamine D4 receptor (D4R) knockout mice (D4R-/-) provided for unique neurochemical studies designed to understand D4R contributions to dopamine (DA) regulation. In this study, post-mortem brain tissue content of DA did not differ between D4R+/+ and D4R-/- mice in the striatum (Str) or nucleus accumbens core (NAc). However, there was a significant decrease (82%) in the content of 3,4-dihydoxyphenylacetic acid (DOPAC), a major metabolite of DA, in the NAc of D4R-/- mice. Microdialysis studies performed in a region of brain spanning of the dorsal Str and NAc showed lower baseline levels of DA and a significant reduction in KCl-evoked overflow of DA in the D4R-/- mice. Baseline extracellular levels of DOPAC and homovanillic acid were also significantly lower in the D4R-/- mice. In vivo chronoamperometric recordings of KCl-evoked release of DA also showed decreased release of DA in the Str and NAc of the D4R-/- mice. These studies demonstrate a role of D4Rs in presynaptic DA regulation and support the hypothesis that alterations in D4Rs may lead to diminished DA function.

Hypersensitive Glutamate Signaling Correlates with the Development of Late-Onset Behavioral Morbidity in Diffuse Brain-Injured Circuitry

In diffuse brain-injured rats, robust sensory sensitivity to manual whisker stimulation develops over 1 month post-injury, comparable to agitation expressed by brain-injured individuals with overstimulation. In the rat, whisker somatosensation relies on thalamocortical glutamatergic relays between the ventral posterior medial (VPM) thalamus and barrel fields of somatosensory cortex (S1BF). Using novel glutamate-selective microelectrode arrays coupled to amperometry, we test the hypothesis that disrupted glutamatergic neurotransmission underlies the whisker sensory sensitivity associated with diffuse brain injury. We report hypersensitive glutamate neurotransmission that parallels and correlates with the development of post-traumatic sensory sensitivity. Hypersensitivity is demonstrated by significant 110% increases in VPM extracellular glutamate levels, and 100% increase in potassium-evoked glutamate release in the VPM and S1BF, with no change in glutamate clearance. Further, evoked glutamate release showed 50% greater sensitivity to a calcium channel antagonist in brain-injured over uninjured VPM. In conjunction with no changes in glutamate transporter gene expression and exogenous glutamate clearance efficiency, these data support a presynaptic origin for enduring post-traumatic circuit alterations. In the anatomically-distinct whisker circuit, the injury-induced functional alterations correlate with the development of late-onset behavioral morbidity. Effective therapies to modulate presynaptic glutamate function in diffuse-injured circuits may translate into improvements in essential brain function and behavioral performance in other brain-injured circuits in rodents and in humans.

Substantia nigra vulnerability after a single moderate diffuse brain injury in the rat

Dementia and parkinsonism are late-onset symptoms associated with repetitive head injury, as documented in multiple contact-sport athletes. Clinical symptomatology is the likely phenotype of chronic degeneration and circuit disruption in the substantia nigra (SN). To investigate the initiating neuropathology, we hypothesize that a single diffuse brain injury is sufficient to initiate SN neuropathology including neuronal loss, vascular disruption and microglial activation, contributing to neurodegeneration and altered dopamine regulation. Adult, male Sprague-Dawley rats were subjected to sham or moderate midline fluid percussion brain injury. Stereological estimates indicated a significant 44% loss of the estimated total neuron number in the SN at 28-days post-injury, without atrophy of neuronal nuclear volumes, including 25% loss of tyrosine hydroxylase positive neurons by 28-days post-injury. Multi-focal vascular compromise occurred 1-2 days post-injury, with ensuing microglial activation (significant 40% increase at 4-days). Neurodegeneration (silver-stain technique) encompassed on average 21% of the SN by 7-days post-injury and increased to 29% by 28-days compared to sham (1%). Whole tissue SN, but not striatum, dopamine metabolism was altered at 28-days post-injury, without appreciable gene or protein changes in dopamine synthesis or regulation elements. Together, single moderate diffuse brain injury resulted in SN neurovascular pathology potentially associated with neuroinflammation or dopamine dysregulation. Compensatory mechanisms may preserve dopamine signaling acutely, but subsequent SN damage with aging or additional injury may expose clinical symptomatology of motor ataxias and dementia.

Differential effects of amphetamine isomers on dopamine release in the rat striatum and nucleus accumbens core

RationaleCurrent medications for attention-deficit/hyperactivity disorder (ADHD) include some single isomer compounds [dextroamphetamine (d-amphetamine, dexedrine) and dexmethylphenidate (Focalin)] and some racemic compounds [methylphenidate and mixed-salts amphetamine (Adderall)]. Adderall, which contains approximately 25% l-amphetamine, has been successfully marketed as a first-line medication for ADHD. Although different clinical effects have been observed for d-amphetamine, Adderall, and benzedrine; potential psychopharmacological differences on the level of neurotransmission between d-amphetamine and l-amphetamine have not been well characterized.ObjectivesTo evaluate potential differences in the isomers, we used the technique of high-speed chronoamperometry with Nafion-coated single carbon-fiber microelectrodes to measure amphetamine-induced release of dopamine (DA) in the striatum and nucleus accumbens core of anesthetized male Fischer 344 rats. Amphetamine solutions were locally applied by pressure ejection using micropipettes.ResultsThe presence of l-amphetamine in the d,l-amphetamine solutions did not cause increased release of DA but did change DA release kinetics. The d,l-amphetamine-evoked signals exhibited significantly faster rise times and shorter signal decay times. This difference was also observed in the nucleus accumbens core. When l-amphetamine was locally applied, DA release was not significantly different in amplitude, and it exhibited the same rapid kinetics of d,l-amphetamine.ConclusionsThese data support the hypothesis that amphetamine isomers have different effects on release of DA from nerve endings. It is possible that l-amphetamine may have unique actions on the DA transporter, which is required for the effects of amphetamine on DA release from nerve terminals.

Using anesthetics and analgesics in experimental traumatic brain injury.

Valid modeling of traumatic brain injury (TBI) requires accurate replication of both the mechanical forces that cause the primary injury and the conditions that lead to secondary injuries observed in human patients. The use of animals in TBI research is justified by the lack of in vitro or computer models that can sufficiently replicate the complex pathological processes involved. Measures to reduce nociception and distress must be implemented, but the administration of anesthetics and analgesics can influence TBI outcomes, threatening the validity of the research. In this review, the authors present evidence for the interference of anesthetics and analgesics in the natural course of brain injury in animal models of TBI. They suggest that drugs should be selected for or excluded from experimental TBI protocols on the basis of IACUC-approved experimental objectives in order to protect animal welfare and preserve the validity of TBI models.