Effects of MK-801 on Nicotine-Induced Locomotor Sensitization

Thomas Beer ’98

Advisor: John Kelsey

Previous research has shown that the coadministration of the glutamate NMDA receptor blocker MK-801 (dizocilpine) with drugs of addiction can prevent the expression of behavioral adaptations, such as tolerance and locomotor sensitization, produced by repeated administrations of these drugs alone. These findings have been interpreted to represent a blockade of the development of these adaptations via MK-80 l’s inhibitory actions on glutamate systems of the central nervous system. However, this interpretation is complicated by findings that MK-801 has locomotor activating and sensitizing effects of its own and may produce state-dependency. Four groups of male Long-Evans rats were given either saline, nicotine (0.4 mg/kg), MK-801 (0.25 mg/kg), or MK801/nicotine in combination and tested for open-field locomotion on alternate days for 5 sessions. As predicted, animals treated with nicotine showed enhanced activity with each subsequent injection, and the expression of sensitization to an injection of nicotine was prevented by the previous coadministration of MK-801 with nicotine. However, repeated injections of MK-801 alone or in combination with nicotine also increased locomotion across the 5 sessions, indicating that MK-80 I produced sensitization by itself. Moreover, the animals that received repeated injections of the drug combination were subsequently less activated by an injection of MK-80 1 than were animals that had previously received only MK-801, which is consistent with the possibility of state-dependency. Thus, while coadminstration of MK-80 1 can prevent the expression of locomotor sensitization to a subsequent injection of nicotine, this effect may represent an actual blockade of the development of sensitization to nicotine or a form of state-dependency.

Gonadal Steroids Have Differential Effects on the Hypothalamo-Pituitary-Adrenal (HPA) Response to Stress in Male and Female Rats

Lauren Cushing, ’98

Advisor: Cheryl McCormick

Sex hormones are known to regulate hypothalamic-pituitary-adrenal function: estrogens exert activational effects on the HPA axis, and cause an increase in release of CRH, ACTH, and Corticosterone (CORT) in response to a stressor (Burgess & Handa, 1992; Viau & Meaney, 1991). It is also well established that androgens inhibit hormonal release from the HPA axis in the presence of a stressor, and maintain stability of HPA function (McCormick et al., Viau & Meaney, 1996). In a study by Handa et al., 1994, the estrogen’s functional effects were similar in both male and female rats. Furthermore, the effects that androgens have on the HPA axis are not fully known, and its mechanism of action is also unclear. McCormick & Frye conducted a preliminary study (unpublished data) demonstrating that estradiol benzoate (EB) and the non-aromatizable androgen dihydrotestosterone (DHT) elicited an increase in HPA response in females when stress was induced. This present study involved gonadectomized male and female adult rats, who received either EB, DHT, testosterone propionate (TP), or cholesterol (CHOL) implants 16 days prior to stress testing. Catheter surgery was also performed 4 days before stress testing in order to obtain blood samples. Rats were stressed in plexiglass restrainers for 20 mm. and samples were taken at 5 different time points (pre & post stress). CORT levels were measured by radioimmunoassay. Both sex and hormone treatment had significant effects on weight gain over a 12 day period (p<.000l), where females showed a greater percent weight gain. TP and DHT treated rats showed the greatest marked percent weight gain in both groups. Fem~e rats secreted higher levels of plasma CORT at the pre-stress time point (p<.005). Hormone treatment was not significant at any of the 5 time points. The CORT values in the CHOL treated rats were unexpectedly higher than those of Eb treated rats. In general, the results of this study are inconclusive, and no assumptions can be made concerning the activational effects of androgens on HPA function in male and female rats.

Gonadal Steroids Have Differential Effects in the Hypothalamic Pituitary-Adrenal (HPA) axis and Immune Function in Male and Female Rats

Randy DePaolo, ’98

Advisor: Cheryl McCormick

Sex hormones are known to regulate hypothalamic-pituitary-adrenal (HPA) function. Estrogen has been known to increase the release of CRH, ACTH and CORT, through its activational effects on HPA function, in response top a physiological stressor (Kitay, 1963; Burgess & Handa, 1992; Viau & Meaney, 1996). Males and females both respond in similar fashions to estrogen treatments (Handa et al., 1994). However, the effect androgens on HPA function in females have not been established. Preliminary observations (McCormick & Frye, unpublished data) indicated that in females, both estradiol benzoate (EB) and non-aromatizable androgen dihydrotestosterone (DHT) increased CORT release in response to stress in females. The present study consisted of adult males and females implanted with silastic capsules of EB, DHT, testosterone propionate (TP), or cholesterol (CHOL) for 16 days prior to undergoing 20 minute restraint stress in Plexiglass restrainers. Four days prior to the stress test animals were fitted with jugular catheters for blood sampling. On the day of restraint stress blood samples were obtained before restraint, immediately following stress, 30, 70 and 120 minutes after stress. Plasma CORT levels was determined by radioimmunoassay. Sex (p<.000l) and hormone treatment (p<.0001) had a marked effect on weight gain: Females showed a higher percent weight gain than males over 12 days. Rats treated with androgen (TP or DHT) had a higher weight gain than those treated with EB or CHOL. There was a sex effect at the pre-stress time point, with females having a higher pre-stress CORT level than males (p<.05), but no hormone effect at the pre-stress level. Due to the high post -stress CORT levels in females, and because of the interaction of sex and hormone treatment on CORT levels, the data was analyzed separately for males and females. For both males and females there was no significant effect of hormone on CORT secretion at any time point. From the data collected in this study, no tangible explanation can be made as to the activational effects of androgens in male and female rats.

Allosteric Modulation of the Glycine Binding Site on N-Methyl D-Aspartate Receptors by the NR2 Subunits

Daniel Graham, ’98

Advisor: Nancy Kleckner

The inotropic N-methyl D-aspartate (NMDA) receptor is ubiquitous within the vertebrate nervous system and plays a vital role in synaptic transmission along excitatory pathways. Furthermore, NMDA receptors have been shown to increase synaptic plasticity which mediates learning and nervous system development (Madison, 1991). Activation of NMDA receptors is initiated by binding of the amino acid neurotransmitter glutamate and the coagonist glycine to their respective recognition sites (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). Upon activation, the receptor ion channel opens causing an influx of Na+ and Ca2+. The overactivation of NMDA receptors has been shown to induce neuronal cell death by increasing intracellular calcium levels to toxic concentrations. Such excitotoxicity has been observed in myocardial infarctions, stroke, epilepsy, and several other neurological conditions (Choi, 1988).      Two types of subunits, NR1 and N~, interact to form the NMDA receptor complex. The NR1 class is composed of seven different isoforms labelled a-g. Similarly, the NR2 class consists of four different subunits labelled A-D. It is postulated that NMDA receptors are tetramers of these subunits which contain at least one NR1 subunit (Clements and Westbrook, 1991). The different subunit combinations account for the variability in characteristic properties of NMDA receptors. Specifically, NR1a-N~C has a 10-fold greater affinity for glycine than does NR1a-NR2A (Laurie, 1994). Evidence suggests that the N~ subunits allosterically interact with the glycine binding site on the NR1 subunit to regulate glycine affinity. It is likely that the extracellular N-terminus of the N~ subunits is responsible for this interaction. To test this, an attempt was made to exchange the extracellular N-terminus of the NR2A subunit with that of the NR2C subunit. However, more work is required to create these chimeric subunits. It was expected that the NR1a-N~A receptor with the N~C N- terminus (CTMIA) would behave like the NR1a-NR2C receptor in terms of glycine affinity and the NR1a-NR2C receptor with the NR2A N-terminus (ATMIC) would behave like the NR1a-NR2A receptor. In this scenario, it could be deduced that the extracellular N-terminus of the NR2 subunits was responsible for modulating glycine binding. If the ATMIC chimera exhibited glycine affinity similar to the N~C subunit and the CTMIA chimera exhibited glycine affinity similar to the N~C subunit, then some other region of the N~ subunits was responsible for modulating glycine binding at the NR1 subunit.The N-methyl-D-aspartate receptors (NMDA-R) are classified within the excitatory glutamate receptor family. These receptors are ionotropic receptors whose current is generated by the influx of Ca+2 and other cations. Research has shown that two different classes of subunits must come together in order for a functional NMDA receptor to form. One family, the NR1 subunit family is comprised of seven functional splice variants, NR1a-g. The second subunit that must be present for functional channels to form is the NR2 subunit. There are four known NR2 subunits (A-d); each is coded for by a separate gene (Moriyoshi et al. 1991).  Creation and pharmacological characterization of the chimeric subunits will provide a great deal of insight into the structure-function relationship of NMDA receptors. Furthermore, elucidating the three dimensional conformation of NMDA receptors may aid in the design of antagonistic drugs which show promise in preventing the toxic effects with excessive NMDA receptor activation.

Characterization of the site of action of ifenprodil on N-methyl-D-aspartate receptors composed of NR1/NR2A and NR1/N2B subunits in Xenopus  oocytes

Josh Hill, ’98

Advisor: Nancy Kleckner

The NMDA receptors are clinically important because they are thought to be involved in processes such as neuro-degenerative disease and acute brain damage caused by stroke (Rothman et al. 1987). An over-stimulation of the NMDA receptor by excess glutamate release has been shown to result in a neuronal cell death. Therefore, drugs that exhibit antagonism at the NMDA receptor are of clinical importance.    Ifenprodil is a drug that displays potent antagonism at the NMDA receptor. Furthermore, previous studies have shown that the 1C50 of ifenprodil at receptors comprised of NR1INR2A is different from the 1C50 at NR1INR2B receptors. Gallagher al. (1996) found the affinity of ifenprodil for NR1INR2B receptors to be 300 times higher than its affinity for NR1/NR2A receptors. Williams (1993) found that ifenprodil exhibits different modes of action at NR1INR2A (voltage dependent) and NR1/NR2B (glycine dependent). It is thought that the binding site for ifenprodil is near one of the three polyamine sites on the NR2 subunit (Williams et al. 1995).  The present study sought to determine whether the binding site of ifenprodil was different receptors comprised of NR2A and NR2B subunits. It

The Effect of Voltage on the Inhibitory Action of the Antipsychotics Clozapine and Chiorpromazine at the NMDA Receptor

Kristen Kosswig, 98

Advisor: Nancy Kleckner

Antipsychotic drugs are known antagonists of the NMDA receptor, but their mechanism of action at this receptor has not yet been characterized. The present study examined the voltage dependence of the inhibition of the typical antipsychotic clozapine and the atypical antipsychotic chiorpromazine at the NMDA receptor. Based on previous studies, it was assumed that both drugs would act within the ion channel of the receptor and would thus demonstrate voltage dependence. NR1 /NR2A and NR1 /NR2B NMDA receptor subtypes were expressed in Xeno pus laevis oocytes by injecting the cells with mRNA encoding the subunits forming these receptors. Electrophysiological recording was used to determine the effect of clozapine and chlorpromazine on the NMDA/glycine response at holding voltages of -30 mV, -60 mV, and -90 mV, measured as percent block by the antagonist. Clozapine demonstrated a trend toward an increase of block with hyperpolarization at both the NR1 /NR2A and NR1 /NR2B receptor subtypes. At the NR1 /N~A subtype, the mean percent block of NMDA and glycinemediated currents by 150 ~M clozapine varied from 68.5 f 8.8% at -90 mV to 49.4 f 25.9% at -30 mV. The mean percent block at the NR1 /NR2B receptor subtype varied from 64.6 1 5.2% at -90 mV to 47.9 1 20.6% at -30 mV. Chlorpromazine exhibited significant effects due to voltage at both subtypes. At the NR1 /NR2A subtype (n=6), the mean percent block of NMDA and

Prior Inescapable and Escapable Shock and Restraint All Depress the Development of Locomotor Sensitization to Repeated Injections of Amphetamine

Justin Lander, ’98

Advisor: John Kelsey

A large body of literature indicates that prior exposure to mild stress can potentiate addictive behaviors such as self administration, conditioned place preference, and locomotion in response to addictive drugs. The learned helplessness literature indicates that a history of exposure to uncontrollable, but not controllable shock, can have serious detrimental psychological effects on an organism, and that controllable stress can occasionally have protective effects. To test the prediction that drug addiction can be potentiated by uncontrollable, and perhaps inhibited by controllable shock, three experiments using locomotor sensitization to repeated injections of amphetamine as the measure of reward were designed. Trios of rats received a series of 80 or 100 escapable tail shocks, inescapable tail shocks, or no shock in wheel-turn boxes. After 2 days or 1 week, rats received the first of seven injections of saline or amphetamine (0.3 mg/kg) and were tested for activity. The remaining injections were given every other day, and were also followed by locomotor testing. A test trial in which all animals received amphetamine was conducted 1 week after the final sensitization trial. The results indicate that, in contrast to the previous literature, the initial locomotor response to amphetamine was not affected by prior stress, and that subsequent locomotor sensitization was, in fact, suppressed in comparison to a home cage control. The degree of control over the prior shock did not matter. Subsequent research is required to examine the mechanisms for the suppression and the likely consequence of this prior stress on other measures of addictive behavior.

The Neuropsychology of Turner Syndrome

Jessica S. Lord, ’98

Advisor: Cheryl McCormick

Turner Syndrome (TS), a genetic disorder that affects approximately 1 in 2000 live-born females, is characterized by aberrations of the XX chromosomal pair. Along with certain physical stigmata, research has identified specific cognitive deficits in individuals with TS that have lead many to hypothesize that there are differences in brain lateralization and a right-hemisphere dysfunction in this population. To confirm and expand upon existing research, the present study investigated the performance of 10 TS patients 6-16 years of age and 12 age-matched control subjects on a comprehensive battery of neuropsychological tests. Results showed that girls with TS are significantly impaired compared to control subjects on tests of parietal lobe function such as arithmetic, visual-spatial and visual-motor tasks, visual memory, and facial recognition. The relevance of these findings to recent brain imaging studies and theories of cognitive development are discussed.

Inhibitory Glutamate Receptors in Helisoma trivolvis: Pharmacological Characterization and Mechanistic Implications

Anne K. Perry, 98

Advisor: Nancy Kleckner

Inhibitory glutamate receptors in invertebrates constitute an unusual and incompletely characterized class of glutamate receptors.  Inhibitory glutamate receptors are present extrajunctionally in insect muscle, and in the central nervous system of insects, molluscs, and crustaceans, where they often play a key role in neural networks.  Pharmacology of invertebrate glutamate receptors is dissimilar to vertebrate pharmacology, and often conflicting among invertebrates.  However, some general trends do occur.  Ibotenate seems to activate ionotropic inhibitory glutamate channels, and quisqualate often indicates a putative metabotropic mechanism.  This study further explores the pharmacology as it relates to mechanism in Helisoma trivolvis, a freshwater mollusc.  1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), a vertebrate metabotropic glutamate agonist, will also be used to explore the possibility of metabotropic mechanisms in this species.  In addition, a putative inhibitory glutamate receptor antagonist, proposed to be selective for both ionotropic and metabotropic receptors, was tested in order to further expand the pharmacology of invertebrate inhibitory glutamate receptors, and possibly confirm them as a separate subclass of glutamate receptors. I hypothesize that Helisoma inhibitory glutamate receptors are quisqualate-sensitive, ibotenate-insensitive, ACPD-sensitive, and alpha-amino-pimelate sensitive. No drug trials were performed due to time limitations and equipment problems, but the inhibitory glutamate response was confirmed compared with known responses.

The Effects of Repeated One Hour Neonatal Isolation on the Hypothalamic-Pituitary- Adrenal Axis of Juvenile and Adult Rats

Ben Rood, 98

Advisor: Cheryl McCormick

The first few weeks of life are a sensitive time in the development of the hypothalamic-pituitary-adrenal (HPA) axis in the rat. In general, these first few weeks of life are characterized by a stress hyporesponsive period which allows the brain to develop without the detrimental effects of high levels of glucocorticoids. It have been found however that several neonatal manipulations such ether stress, cold stress, and maternal separation can evoke a stress response in these neonates. The focus of this study is neonatal isolation which is a one hour separation for the first week of life.  It has been found previously that this repeated one hour neonatal isolation causes a sensitization of corticosterone release to isolation stress on postnatal day (PND) nine (McCormick, Kehoe & Kovacs, in press). The present study looks at this neonatal isolation paradigm and whether its effect on the HPA axis is evident in juvenile and adult animals. This was done by isolating pups for one hour per day for the first week of life in a temperature controlled (at 3O~C) room in which there were no familiar cues, and then obtaining blood samples both before and after a 20 minute restraint stress in juvenile rats (PND 30) and in adult rats (PND 70). It was found that isolated animals had a smaller increase in corticosterone in response to stress than nonhandled animals (p = .01) as juveniles, but there were no differences between the treatment groups as adults. Females had greater corticosterone release than males in response to stress at both juvenile and adult ages. In an open-field test in the juvenile rats, it was found that there are sex differences in locomotion, but there were no effects of neonatal isolation.