Characterization oflnhibitory Glutamate Responses in Motor Neurons Involved in Feeding Behavior in the Pond Snail, Helisoma trivolvis

Carly A. Dell’Ova (Neuroscience)

Advisor: Nancy Kleckner

Neurons are organized in circuits, often referred to as central pattern generators (CPGs), that work together to produce behaviors. Studying the CPGs of an organism can help to elucidate how neuronal circuitry works to perform a specific behavior. The pond snailHelisoma trivo/vis uses a CPG composed of three parts, S1, S2 and S3, to control feeding. Two individual neurons, named B5 and B19, have been identified as motor neurons in phases S1 and S3 respectively. Glutamate, released from B2 interneurons, has been shown to inhibit spontaneous activity in both neurons. This inhibitory action of glutamate in Helisoma and other invertebrates contrasts its actions in vertebrate animals, which are almost always excitatory. As this inhibitory action of glutamate in pond snails is a fairly recent finding, not much is known about the pharmacology or mechanisms of the receptors mediating the effect. One way of characterizing newly discovered receptors is to determine which chemical analogs of the natural agonist interact with the receptor. The present study utilized intracellular recording techniques to measure inhibition of spontaneous action potentials in order to examine the selectivity of glutamate analogs for activation or inhibition of the glutamate receptors in cells B5 and B19. Quisqualate and (1S, 3R)-aminocyclopentane-l ,3-dicarboxylic acid (ACPD), vertebrate glutamate metabotropic receptor agonists, mimicked the actions of glutamate in B5 neurons, and ibotenate, a vertebrate ionotropic receptor agonist did not. Quisqualate also inhibited spontaneous action potential firing in B19 neurons, but ACPD and ibotenate did not significantly inhibit action potential firing. D-2-amino-5-phosphonovalerate (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), vertebrate glutamate ionotropic receptor antagonists, did not block the inhibition by glutamate in any of the B5 or B19 neurons tested. These data suggest that neurons B5 and B19 might contain different glutamate receptor subtypes, and that at least one of these receptors (B5) might resemble the invertebrate glutamate receptor in Aplysia which activates an outward potassium current. Further research will be done with non-selective metabotropic receptor agonists and antagonists as well as pertussis toxin and chloride channel blockers to help discern the mechanism by which glutamate, acting at its receptors, causes inhibition of neuronal firing.

The Spontaneously Hypertensive Rat: An Animal Model of Impulsivity in Attention Deficit Hyperactivity Disorder.

Geoffrey Stoner Ganem (Neuroscience)

Advisor: John Kelsey

It has been suggested the Spontaneously Hypertensive Rat (SHR) may provide a good animal model of attention-deficit hyperactivity disorder (ADHD; e.g., Sagvolden et al. 1992.). To further test the validity and usefulness of this model, in Experiment I we tested male SHR and Wistar Kyoto (WKY) rats, from which the SHR rats were derived, on a measure of impulsiveness, a key feature of ADHD. The rats were given a choice between entering immediately into one arm of a Y -maze where water reinforcement was available on only 30% of the trials or waiting 10 sec to enter the other arm where water reinforcement was always available (CRF arm). As expected, whereas the WKY control rats waited 10 sec to enter the CRF arm on 50-60% of the trials, the SHR waited on only 20-30% of the trials, reflecting impulsiveness. Moreover, i.p. injections of methylphenidate (Ritalin; 5 and 10 mg/kg) and d-amphetamine (1, but not 0.5 mg/kg) normalized the choice of the SHR rats. Nicotine (0.8, but not 0.4 mg/kg) also made the SHR rats less impulsive, although it did not normalize their choices. In experiment 2, the SHR rats locomoted more than twice as far in an open field as did the WKY rats, reflecting hyperactivity. Methylphenidate (10 mg/kg), amphetamine (0.5 and 1 mg/kg), and nicotine (0.8 mg/kg) increased locomotion in both groups and increased it more in the SHR rats. Thus the SHR rats were impulsive and hyperactive. Furthermore, drugs used to treat ADHD (methylphenidate and amphetamine) made the SHR rats less impulsive in the Y -maze, while making them more hyperactive in the open field. The SHR rat would appear to have the potential to serve as a good animal model of at least the impulsivity seen in ADHD.

Neuroprotective Effects of Androgens in the Granule Cell Layer of the Dentate Gyrus of Long- Term Adrenalectomized Rats

Kate M. Lang  (Neuroscience)

Advisor: Cheryl McCormick

Pyknotic cells are visible in the granule layer of the dentate gyrus in as little as 2-4 days post adrenalectomy (ADX), which can be attenuated by low-dose replacement of corticosterone or aldosterone (Conde et al., 1998). This model of specific hippocampal degeneration has also been used in demonstrating the neuroprotective effects of testosterone (T), dihydrotestosterone (DHT), and 3-α diol (5α-androstane-3α, 17β-diol) in the suprapyramidal blade of the dentate gyrus in comparison with vehicle-administered ADX rats (Frye et al., 1999; and Frye et al., 2000). The mechanism by which androgens exert neuroprotective effects is unknown, as is the duration and mechanism of their protection. These questions were addressed by examining the long-term effects of testosterone in n = 40 bilaterally ADX, gonadectomized (GDX) rats. Cannulae were implanted into the shoulders of male rats and filled either with the testosterone propoinate (TP), oil vehicle (VEH), finasteride (FIN), or FIN + TP. Two months following surgery rats were sacrificed and trunk blood was collected for plasma corticosterone levels as a measurement of efficacy of ADX. Histological analysis was used in determining the total area of the dentate gyrus as well as quantifying the number of pyknotic cells in the arch of the suprapyramidal blade. A significant main effect of T as a long-term neuroprotectant in the dentate was shown in the minimal number of pyknotic cells counted in comparison with VEH. Post hoc tests supported that the rats receiving VEH had significantly more pyknotic cells than those subjects who received TP. FIN treated animals maintained less pyknotic cells than FIN + TP treated animals, which does not clarify whether T exerts its long-term neuroprotective effects prior to or following its conversion to DHT and 3α-diol.

MK-801 Appears to Block Learning Involved in the Development of Nicotine Locomotor Sensitization, But Not Its Consolidation

Eleanor Lee (Neuroscience)

Advisor: John Kelsey

Co-administration of NMDA receptor antagonists, such as MK-801, has been found to block the development of locomotor sensitization to repeated administration of psychomotor stimulants, such as amphetamine. Researchers suggest that this may be due to a blockade of the learning involved in the development of locomotor sensitization. However, other researchers argue that effects ofMK-801 reflect state dependency. In Experiment 1, male Long Evans rats were injected with either saline/saline, saline/nicotine (0.4 mg/kg), MK-801 (0.075 mg/kg)/saline, or MK-801lnicotine before placement in an activity box for 5 1-hr sessions on alternate days. Co-administration of MK-801 blocked the development of nicotine locomotor sensitization, but not the expression of already acquired sensitization. In order to assess whether MK-801 also affects the consolidation of sensitization, a second experiment was conducted. In Experiment 2, rats were divided into four groups as in Experiment 1 with the exception that 0.2 mg/kg MK-801 was given 20 min after each 1-hr session in the activity box. In contrast to Experiment 1, post-session injections of MK-801 did not block the development of nicotine locomotor sensitization, suggesting that MK-801 did not block consolidation. However, several factors, including a small sample size and a potentially low dose of MK-801, imply that further research is needed before firm conclusions about the role of NMDA receptors in consolidation of sensitization can be made. At any rate, these results imply that activation of NMDA receptors is essential for the development, if not the consolidation or expression, of the neural changes that underlie locomotor sensitization to nicotine.

Chronic caffeine increases sensitivity to the locomotor depressant effects of adenosine A1agonists

Alissa Menovich (Neuroscience)

Advisor: John Kelsey

Resnick (1999) suggests that long term prenatal caffeine exposure could contribute to postnatal suppression of activity and increase of timid, fearful and emotional behaviors, assumed to be representative of a behavioral pattern called behavioral inhibition. Specifically he hypothesizes that chronic caffeine exposure leads to an adaptive long term, upregulation of adenosine receptors, which enhances the inhibitory effect of the natural ligand adenosine. The present study was designed to investigate the effects of adult chronic caffeine exposure, on locomotion and anxiety, and the mediating effects of adenosine receptors. Rats were exposed to caffeine (205 mg/kg/day in drinking water) or regular drinking water for 20 days. Animals exposed to chronic caffeine subsequently demonstrated a long lasting increase in sensitivity to the locomotor depressant effects of the adenosine Al agonist CPA (1, .5, .25, .1, and .025 mg/kg), but not to the suppressant effects of the A2a agonist CGS- 21680 (1 mg/kg), or to the stimulant effects of caffeine (5,2.5 mg/kg). Anxiety testing did not reveal differential effects in the caffeine-treated animals, in response to any drug. Therefore the present study suggests that chronic caffeine exposure leads to a long lasting increase in sensitivity to the locomotor depressant effects of the Al agonist possibly reflecting a compensatory upregulation of the A1 receptor.

The Modulatory Effect of Androgens on the Lateral Bed Nucleus of the Stria Tenninalis in the HPA Axis in Male Rats

Nick Miller  (Neuroscience-Honors)

Advisor: Cheryl McCormick

The lateral bed nucleus of the stria terminalis (BNST) is involved in the control of hypothalamic-pituitary-adrenal (HP A) function. However, the effects of stimulation and lesions of the lateral BNST on HPA function appear to depend on whether the animal is examined under basal or stress conditions, the type of stressor used, and the sex of the animal (e.g., Herman et a1., 1997; Gray et a1., 1993; Feldman et a1., 1990; Dunn, 1987). The present study investigates whether androgens influence the role of the lateral BNST in the control of the HPA axis in response to restraint stress. Male Long Evans rats were given either electrolytic or sham lesions to the 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 dihydrotestosterone (DHT) or blank implants. Following one week of recovery, rats underwent a 20 min period of restraint stress and tail blood was collected at various timepoints. Plasma samples were assayed for levels of corticosterone using radioimmunoassay. Post hoc analysis indicated that among DHT-treated groups, those with lesions to the lateral BNST had significantly lower corticosterone levels than shams. Among blank-implant groups, those with lesions of the lateral BNST had significantly higher corticosterone levels than shams. These data suggest that the effects of lesions to the lateral BNST are contingent upon circulating androgen levels.

Signaling pathways involved in “run-up” of NMDA receptor-mediated currents inXenopus oocytes expressing recombinant NMDA receptors

Kate-Elizabeth Reed (Biochemistry)

Advisor: Nancy Kleckner

Glutamate is an excitatory amino acid involved in synaptic transmission in neuronal pathways in the mammalian nervous system. The N-methyl-D-aspartate (NMDA) receptor, a subtype of glutamate receptor, plays a major role in a variety of physiological processes including neuronal development and long term potentiation of synaptic transmission in the hippocampus. The NMDA receptor contains multiple binding sites at which antagonists, agonists and modulators can interact to affect the activity of the receptor. Due to inconsistent sensitivities in pharmacological studies, it was determined that the receptor can be assembled from different subunits: NR1 (a-h) and NR2 (A-D). NR1-NR2A type of NMDA receptors express well in Xenopus oocytes but exhibit a phenomenon called run-up in electrophysiological studies which confounds research using oocytes. To determine the cause of run-up, various intracellular processes were considered including the activation of protein kinase C, the activation of calcium dependent chloride channels and the role of post synaptic density (PSD) proteins on the NMDA receptor complex. Subtypes of the NMDA receptor (NR1-NR2A) were expressed in Xenopus oocytes and were tested for their sensitivities to NMDA and glycine using pharmacological and electrophysiological techniques. Run-up was seen in the application of NMDA/glycine in barium (Fig.1) and in the application of NMDA/glycine in calcium (Fig.2). However, run-up was never seen when the oocytes had been exposed to either one of the divalents previously. Run-up also occurred in the oocytes after injection with mRNA encoding PSD-93 and PSD-95 when exposed to NMDA/glycine in barium (Fig.3). The exact mechanism behind the run-up phenomenon remains unknown. Further research should involve the elimination of barium and calcium, the inhibition of PKC and the inhibition of the calcium dependent chloride channels.

Menstrual cycle and gender differences in spatial ability: Relation to salivary cortisol levels

Sarah Teillon  (Psychology)

Advisor: Cheryl McCormick

There are sex differences in cognitive ability in which women perform better on tasks of perceptual speed and fine-motor skills and men perform better on spatial tasks. The sex difference tends to be greatest on tests of spatial ability, particularly tasks involving mental rotation. Fluctuations in estrogen and progesterone levels across the menstrual cycle have been suggested as a source of intra-sex variations, and several studies, although not all studies, have found menstrual cycle differences in performance on cognitive tests which typically elicit sex differences. These mixed findings suggest that other factors may be mediating the relationship between cognitive ability and the menstrual cycle. Stress is one possibility. Cortisol levels fluctuate over the menstrual cycle due to changes in estrogen levels, and cortisol levels have also been linked to cognitive performance. In the present study, undergraduate students (n = 82) were given the Primary Mental Abilities (PMA) Test of Spatial Relations, a mental rotation test. Salivary samples were collected for analysis of cortisol levels upon arrival and immediately following the test. Participants also completed the Profile of Mood States (POMS) and questionnaires about their perceived stress and perceived success on the PMA test. Participants were divided into five groups: Men, women oral contraceptive users, women in the menstrual phase, women in the follicular phase, and women in the luteal phase of the cycle. There were significant group differences in performance on the PMA spatial test. Men performed better than all groups of women, although the difference between men and the menstrual group was not significant. Further, women in the luteal phase of the cycle had lower scores than the menstrual, follicular, and oral contraceptive groups. The latter three groups of women did not differ significantly. There were group differences in arrival cortisol levels, but these were not related to performance on the PMA spatial test. Group differences in spatial performance were not associated with perceived stress, perceived success, or mood. Thus, perceived stress, perceived success, mood, and cortisol levels do not seem to predict the low spatial scores of women in the luteal phase of the menstrual cycle. Some factor of the menstrual cycle is important in mediating group differences on the PMA Spatial Relations Test. A likely factor is estrogen and/or progesterone levels. Nevertheless, there are likely several other variables, both sociocultural and physiological, involved in mediating individual differences in spatial performance.