The Role of Calcium in Kainate-Induced Toxicity of Cultured Rat Fetal Forebrain Neurons

Cristina A. Cellurale (Neuroscience- Honors)

Advisor: Nancy Kleckner

Glutamate, the major excitatory neurotransmitter in the brain, is thought to cause excitotoxicity through calcium influx at NMDA receptors. Other receptors, such as kainate-sensitive AMPA/KA receptors, are also implicated in excitotoxic cell death. A particular subset of these receptors, calcium-permeable AMPA/KA receptors, are believed to playa large role in kainate-induced toxicity of both cortical and cerebellar granule neurons due to their permeability to calcium. In cerebellar granule neurons exposed to kainate, calcium entry via voltage-gated calcium channels and release from intracellular stores appears to be protective. The present study investigates the role of calcium in kainate-induced cell death of rat cortical neurons in culture. The pharmacological agents nifedipine, a blocker of L-type voltage-gated calcium channels, and ryanodine and thapsigargin, two inhibitors of intracellular calcium release, were used to assess the role of calcium via these pathways. In addition, CNQX, an inhibitor of non­NMDA receptors, as well as Joro Spider Toxin, a selective inhibitor of calcium permeable AMPA/KA receptors, were used to elucidate the receptors through which kainate induces cell death. Cells were exposed to 100 μM kainate alone and in the presence of drug for one hour, with appropriate controls. MK-801 (10 μM) was added to all conditions to prevent activation of NMDA receptors. Following twenty-four hours, cells were assayed for viability using the trypan blue exclusion method and the colorimetric assay for conversion of MTT to formazan by live cells. Kainate caused death in approximately 10-20% of cortical neurons. Nifedipine (7 μM) and ryanodine (0.5 μM) applied in conjunction with kainate exerted no effect on cell viability, whereas thapsigargin (0.5 μM) alone decreased cell viability. In the presence ofkainate, CNQX (10 μM) effectively blocked kainate-induced death, whereas Joro Spider Toxin (500 nM) exerted no effect on viability. The results of the present study suggest that kainate induces death of a specific subset of cortical neurons via a pathway other than calcium influx through calcium-permeable AMP AIKA receptors. In addition, cortical neurons appear vulnerable to depletion of intracellular calcium stores as a result of thapsigargin treatment. Unlike cerebellar granule neurons, in the specific subpopulation of cortical neurons that is susceptible to kainate toxicity, calcium entry through voltage-gated calcium channels and release from intracellular stores are neither deleterious nor protective in this paradigm.

Effects of Dihydrotestosterone and Estradiol on Acquisition and Retention on Hippocampus based Spatial Memory Tasks

Carissa Gottlieb  (Psychology)

Advisor: Cheryl McCormick

There are a variety of methods of spatial navigation. The most cognitively challenging, and perhaps most effective, is termed place or true spatial learning. The ability to create spatial maps required for successful navigation using place learning seems to be influenced by sex hormones. However, the effect of sex hormones on cognitive performance may also depend on specific parameters of the spatial task. For example, estrogen tends to improve general memory aspects but impair spatial mapping aspects of performance. Additionally, factors such as motivation, sensory ability, and/or stress may be influenced by sex hormones, in turn altering performance on spatial navigation tasks. This study examined the strategies underlying performance of male rats in several versions of the Morris water maze, a standard test for spatial ability in rats. Gonadectomized rats were given either estradiol benzoate (E2), dihydrotestosterone (DHT), or vehicle (VEH) and tested under the following conditions: (1) a cued navigation version of the water maze involving neither memory nor spatial mapping; (2) a true spatial mapping version of the water maze involving no memory component; and 3) a probe test of memory retention. There was no effect of sex hormones on memory or on secondary performance-related factors such as motor or sensory effects. However, DHT­ treated animals performed significantly better than both E2 and VEH-treated groups on the spatial mapping task. Subsequent experiments examined the aspects of the spatial mapping task that were sensitive to DHT. It appears that DHT-treated rats may be more sensitive to learning of intramaze and extramaze cues. A preliminary experiment to test whether DHT’s effects are mediated by actions at androgen receptors or due to its conversion to androstanediol was inconclusive.

Ability of competitive and non-competitive NMDA antagonists to ameliorate akinesia in 6-OHDA lesioned rats

Ryan Harris (Neuroscience)

Advisor: John Kelsey

Glutamate-induced overactivity in the basal ganglia is one of the mechanisms believed to underlie akinesia associated with human Parkinson’s disease (PD).  This study explored the prospect of treating PD by administering N-methyl-d-aspartate (NMDA) glutamate antagonists as a monotherapy or as an adjuvant to L-Dopa.  Forelimb akinesia in unilaterally 6-OHDA lesioned rats, as measured by stepping tests, bears a direct similarity to the limb akinesia experienced by Parkinson’s patients, and thus, serves as a useful model.  Consistent with previous results from our lab,  Experiment 1 demonstrated that the lesions decreased stepping with the contralateral paw, and that the non-competitive NMDA antagonist dizocilpine (MK-801) at a dose of .3 mg/kg increased stepping in the affected paw.  In Experiments 2 and 3, L-Dopa (1-16 mg/kg) and competitive NMDA antagonist CPP (1.5-6.25 mg/kg) dose dependently improved stepping with the affected paw.  The L-Dopa/CPP combinations, at all doses, failed to improve stepping more than either drug alone, and no drug completely normalized stepping.  This study demonstrates the potential usefulness of NMDA antagonists as monotherapy in treating akinesia associated with Parkinson’s, but also acknowledges that that there appears is an upper limit to the degree of improvement that L-Dopa and the NMDA antagonists produce in the stepping paradigm, either alone or in combination. Future studies should focus on more selective NMDA antagonists that might be capable of producing even greater improvement.

Routes to Recognition:  Hemisphere Bias and Holistic/Featural Processing of Faces, Words, and Objects

Lisa C. Lucia (Psychology)

Advisor: Cheryl McCormick

Right-handed females were tested on recognition accuracy of face, house, and pseudo-word stimuli in normal (whole) or scrambled (part) configurations. Stimuli were presented to either the right or left visual field. For all stimuli, significantly greater accuracy occurred in whole-to-whole trials than in whole-to-part trials. The magnitude of the whole versus part advantage was greatest for face stimuli and smallest for word stimuli. Asymmetry scores indicated a greater left visual field (right hemisphere) advantage for faces and houses in whole conditions and a right visual field (left hemisphere) advantage for pseudo-words in part conditions. Processing of faces and houses in the part condition and words in the whole condition did not show hemispheric biases. The results suggest a relationship between hemisphere and process used in recognition.

A Role of Serotonin in a PCP Model of Schizophrenia

Tyler Moran (Neuroscience)

Advisor: John Kelsey

Schizophrenic symptoms are generally categorized according to three main groups: positive, negative, and cognitive. Administration of phencyclidine (PCP) has been shown to induce both positive and negative symptoms in healthy humans, and has subsequently been used in animal models to reproduce many of these symptoms. The present study has shown that acute injections of PCP (4 mg/kg) in rats can induce both increased activity and reduced exploratory behavior in the hole board apparatus, presumably reflecting the positive and negative symptoms of schizophrenia, respectively. In addition, my data suggest that the expression of positive and negative schizophrenic symptoms in this PCP model is due, in part, to elevated serotonin transmission. In particular, the 5-HT2Aantagonist ketanserin improved both positive and negative symptoms induced by PCP in rats, and high doses of the 5-HT1A antagonist pindolol blocked the negative symptoms. Conversely, drugs that enhance 5-HT function, the SSRI fluoxetine and the 5-HT1Aagonist 8-OH-DPAT, enhanced both the positive and negative symptoms produced by PCP. Future research should investigate the effects of more selective serotonergic drugs and the role of other serotonergic receptors in the PCP animal model of schizophrenia. In addition, the role of other non-serotonergic systems, including glutamatergic, dopamine D4, and varying kinetics at the D2 receptor, should be examined.

The Effects of Estrogen on Gonadectomized Stressed and Unstressed Rats in the Y-Maze

Leslie Moser  (Neuroscience)

Advisor: Cheryl McCormick

The present study examines the impact of estrogen on chronically stressed and non-stressed gonadectomized male and female rats. Eighty adult Long-Evans rats were used in the present experiment in which twenty males and twenty females received estrogen replacement (the remaining forty received blanks). From the entire eighty rats, forty underwent a 3-week restraint stress for six hours a day after which all rats were tested on the Y-maze task. The main findings are as follows. There was a significant difference observed in the percentage body weight change between males and females (F(1, 56) = 10.95, p = .002). In addition, a significant interaction between stress and hormone (F(1, 56) = 5.51, p = .02) was observed. When analyzing the time spent in the novel and familiar arms, there was a significant interaction between both stress and sex (F(1, 56) = 4.61, p = .04) and stress and hormone (F(1, 56) =3.90, p = .05) detected. Finally, a significant interaction was reported between sex and hormone (F(1, 56) = 3.45, p = .06) as well as sex and stress (F(1, 56) = 5.86, p =.02) when analyzing the locomotion data. These results are discussed and possible interpretations of the data are offered.

The Effects of Fluvoxamine and Chlordiazepoxide on Rats with Chronic Quinpirole Treatment on Compulsive Checking: A Possible Animal Model of OCD

Justin Pagnotta (Psychology)

Advisor: John Kelsey

Obsessive Compulsive Disorder (OCD) is a psychiatric anxiety disorder that affects up to 3% of the world’s population and is a major worldwide health problem. Typical symptoms include intrusive thoughts (obsessions) and repetitive behaviors (compulsions) that hinder everyday life. Szechtman, Sulis, and Eilam (1998) proposed a face valid animal model of compulsive checking in rats: the most common symptom of OCD. I replicated their model and found that rats (n = 11) treated chronically with quinpirole (0.5 mg/kg) revisited 2 of 4 objects in an open field more often and for a longer duration than other objects/locales compared to saline controls (n = 5).  Acute injections of fluvoxamine (2.5-10.0 mg/kg), an SSRI typically used to treat OCD, decreased the number and percent of returns to these 2 objects, and tended to increase the average duration of time spent at 2 other objects.  The anxiolytic benzodiazepine chlordiazepoxide (CDZ) (2.0- 8.0 mg/kg), which is usually ineffective in treating OCD, dose-dependently decreased returns to all locations, without consistently altering the percentage of returns to any location.  Thus, fluvoxamine’s ability to selectively reduce checking or preferred objects was not mimicked by CDZ, which appeared to only sedate them without causing a selective reduction of the checking of preferred objects.  Thus, this model appears to have promise as an animal model of some key features of OCD, perhaps aiding in the development of even better therapies.

The effects of estradiol and dihydrotestosterone on neurogenesis in the dentate gyrus of intact and adrenalectomized adult rats

Jennifer M. Strahle  (Neuroscience- Honors)

Advisor: Cheryl McCormick

The production of new neurons (neurogenesis) in the central nervous system is normally confined to a distinct developmental period. However, it is now accepted that neurogenesis occurs in the dentate gyrus of the hippocampus and in the olfactory bulbs of adult animals. Within the dentate gyrus, neuronal birth is regulated by glucocorticoids. The neurons in the dentate gyrus also require these adrenal hormones for survival, as selective cell loss in the dentate gyrus is seen by removal of glucocorticoids by adrenalectomy. The sex hormone estrogen also effects neuronal birth and death in the hippocampus, by stimulating a transitory increase in the number of new neurons in the dentate gyrus. It is still unknown how androgens affect hippocampal neurogenesis. This thesis set out to investigate the comparative roles of estrogen and dihydrotestosterone (an androgen) in mediating neuronal birth in intact animals and in response to adrenalectomy-induced cell death in the dentate gyrus. The commonly accepted way to mark neurogenesis is by immunohistochemical labeling with the thymidine analog bromodeoxyuridine (BrdU). Its incorporation into the DNA of proliferating cells indicates that the cell is mitotically active and thus dividing. This thesis study used BrdU labeling to quantify proliferating cells in the dentate gyrus. However, labeling was sparse and sporadic in the tissues analyzed. Since the time when BrdU immunohistochemistry was first used to mark cells in the central nervous system, the applications of its use have grown to include various species of animals and different areas in the central nervous system. Previously designed BrdU immunohistochemical protocols may no longer be applicable to all experimental cases, as has been demonstrated by the debate between the laboratories of Pasko Rakic and Elizabeth Gould. The latter part of this thesis report examines the methodological problems encountered with BrdU labeling and the problems surrounding neurogenesis quantification today.