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NIGROSTRIATAL DOPAMINE-NEURON FUNCTION FROM NEUROTROPHIC-LIKE PEPTIDE TREATMENT AND NEUROTROPHIC FACTOR DEPLETIONLittrell, Ofelia Meagan 01 January 2011 (has links)
Trophic factors have shown great promise in their potential to treat neurological disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent neurotrophic factor for midbrain dopamine (DA) neurons in the substantia nigra (SN), which lose function in Parkinson’s disease (PD). GDNF progressed to phase II clinical trials, which did not meet proposed endpoints. The large size and binding characteristics of GDNF have been suspected to contribute to some of the shortcomings of GDNF related to delivery to target brain regions. Smaller peptides derived from GDNF (Dopamine-Neuron Stimulating Peptides – DNSPs) have been recently investigated and appear to demonstrate trophic-like effects comparable to GDNF. In the described studies, a time course study was conducted to determine in vivo DA-release characteristics 1-, 2- and 4- weeks after peptide treatment. These studies determined the effects on DA terminals within striatal sub-regions using microelectrodes. A heterogeneous effect on striatal sub-regions was apparent with the maximum effect in the dorsal striatum – corresponding to terminals originating from the SN.
Dysregulation of GDNF or GDNF signaling is believed to contribute to motor dysfunction in aging and PD. Thus, it is hypothesized that GDNF is necessary for the maintenance and function of neurons. To extend this line of investigation, in vivo functional measures (DA-release and -uptake) and behavioral and cellular alterations were investigated in a transgenic mouse model (Gdnf+/-) with reduced GDNF protein levels. The described studies determined that both DA-uptake and -release properties were altered in middle-aged Gdnf+/- mice with only modest reductions in DA neurochemical levels. GDNF levels in Gdnf+/- mice were restored to levels comparable to wild-type (WT) counterparts by treatment with GDNF. GDNF protein supplementation led to enhanced motor behavior and increased markers for DA neurons in the SN of Gdnf+/- mice. Gdnf+/- mice appeared to show a heightened sensitivity to GDNF treatment compared to WT counterparts.
Overall, this body of work examines novel synthetic peptides with potential to enhance DA-neuron function and expands upon the current understanding of GDNF’s role in the nigrostriatal pathway.
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ROLE OF REACTIVE OXYGEN SPECIES PEROXYNITRITE IN TRAUMATIC SPINAL CORD INJURYXiong, Yiqin 01 January 2008 (has links)
Peroxynitrite (PN, ONOO-), formed by nitric oxide radical (•NO) and superoxide radical (O2•-), plays an important role in post-traumatic oxidative damage. In the early work, we determined the temporal characteristics of PN-derived oxidative damage in a rat spinal cord injury (SCI) model. Our results showed 3-nitrotyrosine (3-NT), a specific marker for PN, rapidly accumulated at early time points (1 hr, 3 hrs), after when it plateaued and the high level was sustained to 1 week post injury. The co-localization of 3-NT and lipid peroxidation derived-4-HNE observed in immunohistochemistry indicates PN is involved in lipid peroxidative as well as protein nitrative damage. PN-oxidative damage exacerbates intracellular Ca2+ overload, which activates Ca2+ dependent calpain-mediated cytoskeletal protein (α-spectrin) degradation. The 145 kD fragments of α-spectrin (SBDP 145), which are specifically generated by calpain, increased dramatically as early as 1 hr after injury although the peak increase did not occur until 72 hrs post injury. The high level waned back toward sham level at one week post injury.
We then carried out experiments to evaluate the beneficial effects of tempol, a scavenger of PN-derived radicals, following SCI. Three pathological events including PN-induced oxidative damage, mitochondrial dysfunction and cytoskeletal degradation were investigated. Immunoblotting and immunohistochemical studies indicated PN-mediated oxidative damage including protein nitration, protein oxidation and lipid peroxidation, were all reduced by a single dose of tempol (300mg/kg, i.p) after SCI. Spinal cord (SC) mitochondrial dysfunction in terms of the respiratory control ratio (RCR) significantly improved by both 150 mg/kg and 300 mg/kg tempol treatments. Moreover, calpain-mediated proteolysis was significantly decreased by tempol, with greater effects on calpain-specific SBDP 145 observed.
Direct PN-scavenging effect of tempol was confirmed in vitro. Exposure of healthy SC mitochondria to SIN-1, a PN donor in vitro, impaired mitochondrial respiration in a dose-dependent manner. Tempol was able to protect mitochondria against SIN-1-induced damage by improving mitochondrial function and decreasing mitochondrial 3-NT formation. These findings strongly support the concept that PN is a crucial player in the secondary damage following SCI. And tempol, by scavenging PN-induced free radicals, provides a promising pharmocotherapeutic strategy for treating acute SCI.
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THE UNDERLYING MECHANISM(S) OF FASTING INDUCED NEUROPROTECTION AFTER MODERATE TRAUMATIC BRAIN INJURYDavis, Laurie Michelle Helene 01 January 2008 (has links)
Traumatic brain injury (TBI) is becoming a national epidemic, as it accounts for 1.5 million cases each year. This disorder affects primarily the young population and elderly. Currently, there is no treatment for TBI, which means that ~2% of the U.S. population is currently living with prolonged neurological damage and dysfunction. Recently, there have been many studies showing that TBI negatively impacts mitochondrial function. It has been proposed that in order to save the cell from destruction mitochondrial function must be preserved. The ketogenic diet, originally designed to mimic fasting physiology, is effective in treating epilepsy. Therefore, we have used fasting as a post injury treatment and attempted to elucidate its underlying mechanism. 24 hours of fasting after a moderate TBI increased tissue sparing, cognitive recovery, improved mitochondrial function, and decreased mitochondrial biomarkers of injury. Fasting results in hypoglycemia, the production of ketones, and the upregulation of free fatty acids (FFA). As such, we investigated the neuroprotective effect of hypoglycemia in the absence of fasting through insulin administration. Insulin administration was not neuroprotective and increased mortality in some treatment groups. However, ketone administration resulted in increased tissue sparing. Also, reduced reactive oxygen species (ROS) production, increased the efficiency of NADH utilization, and increased respiratory function. FFAs and uncoupling proteins (UCP) have been implicated in an endogenously regulated anti-ROS mechanism. FFAs of various chain lengths and saturation were screened for their ability to activate UCP mediated mitochondrial respiration and attenuate ROS production. We also measured FFA levels in serum, brain, and CSF after a 24 hour fast. We also used UCP2 transgenic overexpressing and knockout mice in our CCI injury model, which showed UCP2 overexpression increased tissue sparing, however UCP2 deficient mice did not show a decrease in tissue sparing, compared with their wild type littermates. Together our results indicate that post injury initiated fasting is neuroprotective and that this treatment is able to preserve mitochondrial function. Our work also indicates ketones and UCPs may be working together to preserve mitochondrial and cellular function in a concerted mechanism, and that this cooperative system is the underlying mechanism of fasting induced neuroprotection.
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DISRUPTIONS IN THE REGULATION OF EXTRACELLULAR GLUTAMATE IN THE RAT CENTRAL NERVOUS SYSTEM AFTER DIFFUSE BRAIN INJURYHinzman, Jason Michael 01 January 2012 (has links)
Glutamate, the predominant excitatory neurotransmitter in the central nervous system, is involved in almost all aspects of neurological function including cognition, motor function, memory, learning, decision making, and neuronal plasticity. For normal neurological function, glutamate signaling must be properly regulated. Disrupted glutamate regulation plays a pivotal role in the acute pathophysiology of traumatic brain injury (TBI), disrupting neuronal signaling, initiating secondary injury cascades, and producing excitotoxicity. Increases in extracellular glutamate have been correlated with unfavorable outcomes in TBI survivors, emphasizing the importance of glutamate regulation.
The aim of this thesis was to examine disruptions in the regulation of extracellular glutamate after experimental TBI. In these studies, we used glutamate-sensitive microelectrode arrays (MEAs) to examine the regulation of extracellular glutamate two days after diffuse brain injury. First, we examined which brain regions were vulnerable to post-traumatic increases in extracellular glutamate. We detected significant increases in extracellular glutamate in the dentate gyrus and striatum, which correlated to the severity of brain injury. Second, we examined the regulation of extracellular glutamate by neurons and glia to determine the mechanisms responsible for post-traumatic increases in extracellular glutamate. In the striatum of brain-injured rats, we detected significant disruptions in release of glutamate by neurons and significant decreases in the removal of glutamate from the extracellular space by glia. Third, we examined if a novel therapeutic strategy, a viral-vector mediated gene delivery approach, could improve the regulation of extracellular glutamate. Infusion of an adeno-associated virus expressing a glutamate transporter into the rat striatum produced significant improvements in glutamate clearance, identifying a novel strategy to reduce excitotoxicity. Lastly, we examined the translational potential of MEAs as novel neuromonitoring device for clinical TBI research. Overall, these studies have demonstrated the translational potential of MEAs to aid in the diagnosis and treatment of TBI survivors.
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ROLE OF CYCLOPHILIN D IN SECONDARY SPINAL CORD AND BRAIN INJURYClark, Jordan Mills 01 January 2009 (has links)
In the hours and days following acute CNS injury, a secondary wave of events is initiated that exacerbate spinal tissue damage and neuronal cell death. A potential mechanism driving these secondary events is opening of the mitochondrial permeability transition pore (mPTP) and subsequent release of several cell death proteins. Previous studies have shown that inhibition of cyclophilin D(CypD), the key regulating component in mPTP opening, was protective against insults that induce necrotic cell death. We therefore hypothesized that CypD-null mice would show improved functional and pathological outcomes following spinal cord injury (SCI) and traumatic brain injury (TBI). Moderate and severe spinal contusion was produced in wild-type (WT) and CypD-null mice at the T-10 level using the Infinite Horizon impactor. Changes in locomotor function were evaluated using the Basso Mouse Scale (BMS) at 3 days post-injury followed by weekly testing for 4 weeks. Histological assessment of tissue sparing and lesion volume was performed 4 weeks post SCI. Calpain activity, measured by calpain-mediated spectrin degradation, was assessed in moderate injury only by western blot 24 hours post SCI. Results showed that following moderate SCI, CypD-null mice had no significant improvement in locomotor recovery or tissue sparing compared to wild-type mice. Following severe SCI, CypD-null mice showed significantly lower locomotor recovery and decreased tissue sparing compared to WT mice. Calpain-mediated spectrin degradation was not significantly reduced in CypD-null mice compared to WT mice 24h post moderate SCI. The lack of protective effects in CypD-null mice suggests that more dominant mechanisms are involved in the pathology of SCI. In addition, CypD may have a pro survival role that is dependent on the severity of the spinal cord injury.
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GLUTAMATE REGULATION IN THE HIPPOCAMPAL TRISYNAPTIC PATHWAY IN AGING AND STATUS EPILEPTICUSStephens, Michelle Lee 01 January 2009 (has links)
A positive correlation exists between increasing age and the incidence of hippocampal-associated dysfunction and disease. Normal L-glutamate neurotransmission is absolutely critical for hippocampal function, while abnormal glutamate neurotransmission has been implicated in many neurodegenerative diseases. Previous studies, overwhelmingly utilizing ex vivo methods, have filled the literature with contradicting reports about hippocampal glutamate regulation during aging. For our studies, enzyme-based ceramic microelectrode arrays (MEA) were used for rapid (2 Hz) measurements of extracellular glutamate in the hippocampal trisynaptic pathway of young (3-6 months), late-middle aged (18 mo.) and aged (24 mo.) urethane-anesthetized Fischer 344 rats. Compared to young animals, glutamate terminals in cornu ammonis 3 (CA3) showed diminished potassium-evoked glutamate release in aged rats. In late-middle aged animals, terminals in the dentate gyrus (DG) showed increased evoked release compared to young rats. The aged DG demonstrated an increased glutamate clearance capacity, indicating a possible age-related compensation to deal with the increased glutamate release that occurred in late-middle age.
To investigate the impact of changes in glutamate regulation on the expression of a disease process, we modified the MEA technology to allow recordings in unanesthetized rats. These studies permitted us to measure glutamate regulation in the hippocampal formation without anesthetic effects, which showed a significant increase in basal glutamatergic tone during aging. Status epilepticus was induced by local application of 4-aminopyridine. Realtime glutamate measurements allowed us to capture never-before-seen spontaneous and highly rhythmic glutamate release events during status epilepticus. A significant correlation between pre-status tonic glutamate and the quantity of status epilepticus-associated convulsions and glutamate release events was determined. Taken together, this body of work identifies the DG and CA3 as the loci of age-associated glutamate dysregulation in the hippocampus, and establishes elevated levels of glutamate as a key factor controlling severity of status epilepticus in aged animals.
Based upon the potential ability to discriminate brain areas experiencing seizure (i.e. synchronized spontaneous glutamate release) from areas not, we have initiated the development of a MEA for human use during temporal lobe resection surgery. The final studies presented here document the development and testing of a human microelectrode array prototype in non-human primates.
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CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUSTalauliker, Pooja Mahendra 01 January 2010 (has links)
An overarching goal of the Gerhardt laboratory is the development of an implantable neural device that allows for long-term glutamate recordings in the hippocampus. Proper L-glutamate regulation is essential for hippocampal function, while glutamate dysregulation is implicated in many neurodegenerative diseases. Direct evidence for subregional glutamate regulation is lacking in previous in vivo studies because of limitations in the spatio-temporal resolution of conventional experimental techniques. We used novel enzyme-coated microelectrode arrays (MEAs) for rapid measurements (2Hz) of extracellular glutamate in urethane-anesthetized rats. Potassium-evoked glutamate release was highest in the cornu ammonis 1 (CA1) subregion and lowest in the cornu ammonis 3 (CA3). In the dentate gyrus (DG), evoked-glutamate release was diminished at a higher potassium concentration but demonstrated faster release kinetics. These studies are the first to show subregion specific regulation of glutamate release in the hippocampus.
To allow for in vivo glutamate measurements in awake rats, we have adapted our MEAs for chronic use. Resting glutamate measurements were obtained up to six days post-implantation but recordings were unreliable at later time points. To determine the cause(s) for recording failure, a detailed investigation of MEA surface characteristics was conducted. Scanning electron microscopy and atomic force microscopy showed that PT sites have unique surface chemistry, a microwell geometry and nanometer-sized features, all of which appear to be favorable for high sensitivity recordings. Accordingly, studies were initiated to improve enzyme coatings using a computer-controlled microprinting system (Microfab Technologies, Plano, TX). Preliminary testing showed that microprinting allowed greater control over the coating process and produced MEAs that met our performance criteria.
Our final studies investigated the effects of chronic MEA implantation. Immunohistochemical analysis showed that the MEA produced minimal damage in the hippocampus at all time points from 1 day to 6 months. Additionally, tissue attachment to the MEA surface was minimal. Taken together with previous electrophysiology data supporting that MEAs are functional up to six months, these studies established that our chronic MEAs technology is capable of maintaining a brain-device interface that is both functional and biocompatible for extended periods of time.
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AGE MAY BE HAZARDOUS TO OUTCOME FOLLOWING TRAUMATIC BRAIN INJURY: THE MITOCHONDRIAL CONNECTIONGilmer, Lesley Knight 01 January 2009 (has links)
Older individuals sustaining traumatic brain injury (TBI) experience a much higher incidence of morbidity and mortality. This age-related exacerbated response to neurological insult has been demonstrated experimentally in aged animals, which can serve as a model to combat this devastating clinical problem. The reasons for this worse initial response are unknown but may be related to age-related changes in mitochondrial respiration.
Evidence is shown that mitochondrial dysfunction occurs early following traumatic brain injury (TBI), persists long after the initial insult, and is severitydependent. Synaptic and extrasynaptic mitochondrial fractions display distinct respiration capacities, stressing the importance to analyze these fractions separately. Sprague- Dawley and Fischer 344 rats, two commonly used strains used in TBI and aging research, were found to show very similar respiration profiles, indicating respiration data are not strain dependent. Neither synaptic nor extrasynaptic mitochondrial respiration significantly declined with age in naïve animals. Only the synaptic fraction displayed significant age-related increases in oxidative damage, measured by 3-nitrotyrosine (3- NT), 4-hydroxynonenal (4-HNE), and protein carbonyls (PC). Alterations in respiration with age appear to be more subtle than previously thought. Subtle declines in respiration and elevated levels of oxidative damage may not to be sufficient to produce detectable deficits until the system is challenged.
Following TBI, synaptic mitochondria exhibit dysfunction that increased significantly with age at injury, evident in lower respiratory control ratio (RCR) values and declines in ATP production rates. Furthermore, synaptic mitochondria displayed increased levels of oxidative damage with age and injury, while extrasynaptic mitochondria only displayed significant elevations following the insult. Age-related synaptic mitochondrial dysfunction following TBI may contribute to an exacerbated response in the elderly population.
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THE REGULATION AND PACKAGING OF SYNAPTIC VESICLES RELATED TO RECRUITMENT WITHIN CRAYFISH AND FRUIT FLY NEUROMUSCULAR JUNCTIONS: VARIATIONS IN LOW- AND HIGH-OUTPUT TERMINALSWu, Wenhui 01 January 2013 (has links)
Glutamate is the main excitatory neurotransmitter in the CNS and at the neuromuscular junctions (NMJs) of invertebrate. The characteristic similarities to CNS glutamatergic synapses in vertebrate and the anatomical simplicity of invertebrate NMJs favor the investigation of glutamatergic synaptic functions in this system. This dissertation mainly aimed to physiologically separate two functional vesicle groups, the reserve pool (RP) and readily releasable pool (RRP) within presynaptic nerve terminals of Procambarus Clarkii and Drosophila melanogaster. This was addressed in part by blocking the vesicular glutamate transporter (VGlut) with bafilomycin A1. Various frequencies of motor nerve stimulation, exposure time, and concentration of bafilomycin A1 were examined. The low-output tonic opener NMJs in crayfish exposed to 4μM bafilomycin A1 and 20Hz continuous stimulation decreased the EPSP amplitude to 50% in ∼30min with controls lasting 3h. After activity and bafilomycin A1-induced synaptic depression, the EPSPs were rapidly revitalized by serotonin (5-HT, 1μM) in the crayfish preparations. The 5-HT action can be blocked almost completely with a PLC inhibitor, but partially with a cAMP activator. The higher output synapses of the larval Drosophila NMJ when stimulated at 1Hz or 5Hz and exposed to 4μM of bafilomycin A1 showed a depression rate of 50% within ∼10min with controls lasting ∼40min. After low frequency depression and/or exposure to bafilomycin A1 a burst of higher frequency (10Hz) can recruit vesicles from the RP to the RRP. Physiological differences in low- (tonic like) and high-output (phasic like) synapses match many of the expected anatomical features of these terminals, part of this dissertation highlights physiological differences and differential modulation and/or extent of the vesicles in a RP for maintaining synaptic output during evoked depression of the RRP in crayfish abdomen extensor preparation. With the use of bafilomycin A1, the tonic terminal is fatigue resistant due to a large RRP, whereas the phasic depresses rapidly upon continuous stimulation. Upon depression of the tonic terminal, 5-HT has a large RP to act on to recruit vesicles to the RRP; whereas, the phasic terminal, 5-HT can recruit RP vesicles to the RRP prior to synaptic depression but not after depression.
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INJURY ESTABLISHES CONSTITUTIVE µ-OPIOID RECEPTOR ACTIVITY LEADING TO LASTING ENDOGENOUS ANALGESIA AND DEPENDENCECorder, Gregory F 01 January 2013 (has links)
Injury causes increased pain sensation in humans and animals but the mechanisms underlying the emergence of persistent pathological pain states, which arise in the absence of on-going physical damage, are unclear. Therefore, elucidating the physiological regulation of such intractable pain is of exceptional biomedical importance. It is well known that endogenous activation of µ-opioid receptors (MORs) provides relief from acute pain but the consequences of prolonged endogenous opioidergic signaling have not been considered. Here we test the hypothesis that the intrinsic mechanisms of MOR signaling promote pathological sensitization of pain circuits in the spinal cord. We found that tissue inflammation produces agonist-independent MOR signaling in the dorsal horn of the spinal cord, which tonically represses hyperalgesia for months, even after complete recovery from injury and re-established normal pain thresholds. Disruption of this constitutive activity with MOR inverse agonists reinstated pain and precipitated cellular, somatic and aversive signs of physical withdrawal. This phenomenon required N-methyl-D-aspartate receptor activation of calcium-sensitive adenylyl cyclase type 1. Thus, we present a novel mechanism of long-lasting opioid analgesia that regulates the transition from acute to chronic pain while, in parallel, generates physical dependence. In conclusion we propose that the prevalence of chronic pain syndromes may result from a failure in constitutive signaling of spinal MORs and a loss of endogenous analgesic control.
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