Mitochondria Ca2+ Accumulation: The roles of the Permeability Transition Pore, the Na+/Ca2+ Exchanger and Reactive Oxygen Species Generation in Glutamate -Mediated Excitotoxicity
Thomas H. Armet (Neuroscience)
Advisor: Nancy Kleckner
Glutamate (glut), one of the most important neurotransmitters present in the human brain is important in controlling all bodily functions. However, an over stimulation of glutamate can lead to a type of neuron death called excitotoxicity caused by increased intracellular levels of Ca2+ in neurons. Excitotoxicity has been linked to brain damage caused by stroke and neurodegenerative disease such as Alzheimer’s Disease and Parkinson’s Syndrome. It has been known for some time that both NMDA and non NMDA receptors play an extensive role in glut-mediated excitotoxicity. But current research points to the role of the mitochondria, more specifically the Na+/Ca2+ exchanger (NCE), the production of reactive oxygen species (ROS). The NCE and ROS have been shown to perpetuate the opening of the permeability transition pore (PTP), which has been linked to the secondary rise in [Ca2+]c deemed delayed calcium deregulation (DCD) which is believed to be a key player in excitotoxicity. Excitotoxicity was induced in the lab with O.5mM glut application to neuronal forebrain cultures of E 19 Sprague-Dawley rats. The resultant excitotoxicity was combated using NMDA receptor antagonists (100uM APV) and non-NMDA receptor antagonists (10uM CNQX) that both produced neuroprotective trends, although not significant (n=5). Due to time constraints, only one mitochondrial agent was tested, CGP-37157 a NCE inhibitor. CGP-37157 demonstrated a neuroprotective trend at 5uM and 25uM but not at 100uM. CGP-37157, APV and CNQX all provided similar levels of neuroprotection. This research suggests that blockage at the receptor level produces similar neuroprotective results as blockage at the mitochondrial level. Eventually this and similar research concerning excitotoxicity will lead to the production of more advanced neuroprotective agents.
Testing the Validity of a Possible Cycling Model of Bipolar Affective Disorder
Lisa Briand (Psychology- Honors)
Advisor: John Kelsey
Bipolar disorder, commonly known as manic depression, affects over 1.5 percent of the population and its current drug treatments include lithium, anticonvulsants, and calcium channel blockers. Because current drug treatments can be ineffective in up to 50% of patients, it is necessary that better drugs to be developed. The intent of my study was to further examine an animal model proposed by Antelman et al. (1998) to determine if this model might be useful in developing better therapies. In this model, repeated cocaine (15 mg/kg) administration lead to an oscillation in both physiological and behavioral measures, and was attenuated by lithium. In the first experiment, 8 animals each began testing after cocaine injections 2, 3, and 4 and being tested after all subsequent injections. Both the repeated and independent measures cold water swim analgesia data exhibited cycling in that animals tested after injections 2 and 4 were significantly more analgesic than animals tested after injection 3. The locomotor data showed no significant signs of cycling, although animals tested after injection 2 tended to be less active than animals tested after injection 3. This data indicate that Antelman’s cycling model can be replicated with a more typical analgesia procedure. In the second experiment, 12 animals were pre-treated with valproic acid, a commonly used treatment for bipolar disorder, administered through their food (0.7% VPA) and tested in the same manner as the first experiment. The valproic pre-treatment caused an attenuation of the cocaine-induced cycling. Further research should examine the effects of carbamazepine and calcium channel blockers as well as novel agents such as glutamatergic drugs.
d-Tetrandrine’s Selective Inhibition on NMDA NRl/2A Subtypes Expressed in Xenopus laevis Frog Oocytes
Carolyn Su Chew (Neuroscience)
Advisor: Nancy Kleckner
Ever since the Yung-Chia period (307-313 AD.), Fang ji has served in anti-rheumatic, analgesic, diuretic, and anti-inflammatory roles. The bisbenzylisoqinoline alkaloid which is isolated from the Fang ji, d-tetrandrine (TET), is responsible for its main therapeutic actions. TET’s anti-inflammatory was studied against NMDA NR1/2A and NRl/2B receptors, receptors that may induce cell death in strokes, ischemia, hypoxia, and anoxia. In the experimental study done with Xenopus laevis oocytes expressing recombinant NMDA NRl/2A and NRl/2B subtypes, TET inhibited with a greater efficacy the NMDA NRl/2A subtype ( 90.2 +/- 1.6% (n=4)) over NR1/2B subtype receptors (39.6 +/- 9.4% (n=4)). TET also gave an IC50 of 0.03 +/- 18.80 mM against the NR1/2A subtype. The dose-response relationship between 100 uM NMDA/1 uM glycine-induced current at increasing concentrations of TET demonstrated a weak dose dependent response dependency (r2=0.39) due to TET’s multi-phasic behavior. Results of the voltage sensitivity and trapping experiments suggested that TET may not act within the channel. However, the trapping experiment showed that 0.3 mM of TET, TET could be trapped within the NMDA NRl/2A subtype channel. A significant inhibition was seen in the NMDA/glycine-induced current following the application of NMDA/Glycine/TET and just TET (p=0.0361, n= 4). As a result, TET does have the potential to exhibit neuroprotective effects. Further studies should be conducted to examine the mechanistic properties of TET with the NMDA NRI/NR2A subtype channel and also look at if TET crosses the blood-brain-barrier. If can cross the blood-brain-barrier, TET could be used therapeutically for brain NMDA-induced neurotoxicity.
The Effects of Sex Hormones and Lesions of the Bed Nucleus of the Stria Terminalis on Anxiety in Female Rats
Alex Cutler (Neuroscience)
Advisor: Cheryl McCormick
Past research has shown that the amygdala is involved in mediating anxiety. Given the close anatomical and neurochemical relationships between the bed nucleus of the stria terminalis (BNST) and the amygdala, combined with a large body of research implicating the amygdala in anxiety, it is expected that the BNST will also play a role in anxiety. The BNST has been connected with autonomic and hormonal reactions to fearful stimuli, however there have been few published studies that directly examine the effects of BNST lesions on untrained behavioral reactions to anxiety provoking stimuli: Furthermore, past research shows gonadal hormones and their metabolites (such as testosterone) may playa role in responses to anxiety producing stimuli (Bitran et al., 1993). There are many testosterone targets in the BNST (Lisciotto & Morrel, 1994). The present study investigates whether the behavioral effects of lesions to the BNST are altered by hormonal status. Female Long Evans rats were given either electrolytic or sham lesions to the BNST using stereotaxic placements derived from Paxinos and Watson (1986). At that time, rats were also ovariectomized, and approximately half of each surgery group received subcutaneous silastic implants of dihydrotestosterone (DHT), while the others received testosterone propionate (TP). Following eight days of recovery, rats underwent 5 minutes of testing on the elevated plus maze (a validated test of anxiety). The results revealed that lesioned rats spent a greater percentage of time in the open arms than did sham lesioned rats. In addition, TP treated rats had a greater percentage of entries into the open arms than did DHT treated rats suggesting that TP is being converted to estrogen by aromatase which has an anxiolytic effect. These data suggest that the BNST does playa role in mediating anxiety in an unconditioned paradigm.
The effect of bilateral lateral amygdaloid lesions on the development of associative and non-associative tolerance to nicotine analgesia in rats
Peter A.Groblewski (Neuroscience)
Advisor: John Kelsey
Associative tolerance is said to have occurred when tolerance to a drug is greater in an environment previously associated with the drug than in an environment that has not been paired with that drug. Recent studies have indicated that the basolateral amygdala is involved in the development and expression of associative morphine tolerance. Other studies have shown that associative tolerance to nicotine analgesia can be developed. This study attempts to combine these findings by examining the role of the amygdala in the development and expression of associative tolerance to nicotine. Experiment 1 produced too much biological (non-associative) tolerance and no indication of the development of associative tolerance. The data from Experiment 2 indicated the development of associative tolerance, characterized by a shorter tail-flick latencies in response to nicotine (1.5 mg/kg) in paired rats than in unpaired rats. Amygdala lesions reduced the development of associative tolerance by reducing the difference between the paired and unpaired groups. Moreover, the lesions enhanced the deviation of analgesia produced by nicotine. These data indicate that the amygdala is not only involved in the mediation of analgesia, but also in the development and/or expression of associative nicotine tolerance.
Lesions of The Basolateral/Central Amygdala Enhance Context-Specific Locomotor Sensitization to Nicotine
Rejean M. Guerriero (Neuroscience)
Advisor: John Kelsey
Present research suggests that the mesocorticolimbic dopamine system is a major mediator of the rewarding effects of many drugs, including locomotor sensitization. In this experiment I investigated the role of a related structure, the amygdala, in mediating the context-specificity of locomotor sensitization produced by repeated injections of nicotine. Context-specificity refers to findings that drugs often have different effects, e.g., larger sensitization, in a environment where they have been previously administered, as compared with an environment where they are being administered drug for the first time. Contrary to my predictions, I found that amygdala lesions enhanced context-specific locomotor sensitization to repeated injections of nicotine (0.4 mg/kg). Specifically, when all groups were given a challenge dose of nicotine (0.4 mg/kg) following six sessions of repeated injections of nicotine (or saline), either paired or unpaired with the testing environment, the lesion-paired rats locomoted more than the sham-paired rats, while the lesion-unpaired rats locomoted less than the sham-unpaired rats. Both paired and unpaired groups locomoted more in response to the challenge of nicotine than did the two groups (sham and lesion) that had previously received only saline. Although amygdala lesions appear to reduce or not affect locomotor sensitization to psychostimulants such as amphetamine, my data suggest that these lesions enhance context-specific sensitization to nicotine, implying that nicotine produces sensitization by a different mechanism than do other psychostimulants.
Ability of Glutamate Antagonists to Reduce Akinesia and L-dopa- Induced Dyskinesia in 6-0HDA Lesioned Rats
Reyna Pijanowski (Neuroscience)
Steve Mague (Biology)
Advisors: Nancy Kleckner & John Kelsey
Recent research suggests that Parkinson’s disease (PD) is associated with enhanced glutamate transmission as well as a decrease in dopamine function. This glutamate overactivity is assumed to worsen akinesia and may also be implicated in L-dopa-induced dyskinesia; therefore glutamate antagonists should help alleviate these problems. In Experiment 1, chronic administration of 25 mg/kg L-dopa for 23 days failed to produce dyskinesias, as measured by changes in L-dopa-induced contralateral rotational behavior in unilaterally 6-0HDA-lesioned rats. In Experiment 2, akinesia was measured in unilaterally 6-0HDA rats by a decrease in stepping with the contralateral paw. As expected, acute injections of L-dopa (8 and 16 mg/kg L-dopa with 6.25 mg/kg benserazide) significantly increased, but did not normalize contralateral stepping. Indicating the involvement of glutamate, acute injections of (+)-5-methyl-10,11-dihydro -5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (0.15, 0.2 mg/kg), and L -dopa/MK-801 combination MK-801, a glutamate N-methyl-D-aspartate (NMDA) receptor blocker, alone or in combination with 8-16 mg/kg L-dopa also increased but did not normalize contralateral stepping. However, a 2.5 mg/kg dose of 1-[4-aminophenyl] -4-methyl-7,8-methylene-dioxy-5H-2,3-benzodiazepine, GYKI 52466, a glutamate Alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor blocker, was ineffective at increasing stepping on the contralateral side. Our results suggest there are beneficial consequences to using NMDA antagonists in the acute treatment of akinesia in PD, supporting the hypothesis that glutamate overactivity plays a significant role in PD.
Lesions of the Bed Nucleus of the Stria Terminalis and in Female Rats
Eliza Scott (Neuroscience- Honors)
Advisor: Cheryl McCormick
Past research has shown that the amygdala is involved in mediating anxiety. In addition to the amygdala, the lateral anterior BNST is beginning to be discussed as having a role in mediating anxiety as well. The BNST is considered to be part of the so called “extended amygdala” because it is highly similar to the central nucleus of the amygdala in terms of transmitter content, cell morphology, and efferent connections (Alheid, de Olmos & Beltramino, 1995). The bed nucleus of the stria terminalis has been implicated in autonomic and hormonal reactions to fearful stimuli, however there have been few published studies that directly examine the effects of L-BNST lesions on untrained behavioral reactions to anxiety provoking stimuli. Moreover, past research shows gonadal hormones and their metabolites (such as estradiol) may play a role in responses to anxiety producing stimuli (Wilson et al., 1996). Several areas of the LBNST have been shown to contain estrogen receptors, therefore making the LBNST a good target for examining the gonadal control of the anxiety response. The present study investigates whether the behavioral effects of lesions of the anterior/lateral BNST are altered by hormonal status. Female Long Evans rats were given either electrolytic or sham lesions to the anterior/lateral BNST using stereotaxic placements derived from Paxinos and Watson (1986). At that time, rats were also gonadectomized and half of each surgery group received subcutaneous silastic implants of estradiol or blank implants. Following one week of recovery, rats underwent 5 minutes of testing on the elevated plus maze (a validated test of anxiety). A two-factor ANOVA revealed that lesioned rats spent a greater percent of time in the open arms than did sham-lesioned rats, F (1,28) = 33.39, p < .0001. There was no effect of estrogen treatment, and no interaction of lesion and estrogen treatment. The results of this study indicate that the bed nucleus of the stria terminalis modifies performance on the elevated plus maze in female rats. Also, EB implants did not significantly alter any index of anxiety on the elevated plus maze in either the sham or the lesion groups. These data suggest that the anterior/lateral-BNST does play a role in mediating anxiety in an unconditioned paradigm.
An Investigation of the Subtype-Selective Antagonism of N-Methyl-D-Aspartate Receptors by new Antiepileptic Agent, R-2-Acetamido-N-Benzyl-3-Methoxypropionamide
Gregory M. Troughton (Biology)
Advisor: Nancy Kleckner
Harkoseride is a new anticonvulsant that shows much promise as an anti epileptic agent. Preliminary tests with Harkoseride report that it produced a time and dose dependent reduction of behavioral seizure score of hippocampal kindled rats. Previous experiments with Harkoseride have provided evidence that argues both for and against interactions at the NMDA receptor and the strychnine-insensitive glycine site. Recombinant NMDA receptors expressed in Xenopus laevis oocytes were used in effort to determine if Harkoseride’s clinical profile was linked to the strychnine-insensitive glycine site and the NR1-2A or NR1-2B receptors. Harkoseride reduced NMDA and glycine induced currents in NR1-2B receptors (IC50=1.89 mM) and showed no block of NMDA and glycine currents at NR1-2A receptors. Inhibition of NRl-2B receptors was independent of glycine concentration. H-209, a possible metabolite of Harkoseride, exhibited similar properties to Harkoseride, also inhibtiting NMDA and glycine induced currents at NRl -2B receptors. Harkoseride’s moderate potency for NRl-2B receptors and its independence of the strychnine-insensitive glycine site suggest that its mechanism of action may be similar to other noncompetitive NMDA antagonists. Further research with Harkoseride should focus on NRl- 2A, NRl-2C and NRl-2D NMDA receptors. Research should also be conducted on the inhibitory profile of H-209.
Exploring the PCP Model of Schizophrenia
Jodi Winterton (Neuroscience, 2001)
Advisor: John Kelsey