The Effects of Electrolytic Lesions of the Nucleus Accumbens Core on a Nicotine Conditioned Taste Aversion

Tom Berry (Neuroscience)

Advisor: John Kelsey

Drugs of abuse are rewarding in large part because they increase dopamine (DA) levels in the nucleus accumbens (NAc). Surprisingly, drugs of abuse can also have aversive effects, and that some of these aversive effects may also be mediated by DA in the NAc. Recent evidence suggests, however, that the aversive and rewarding effects may be anatomically segregated within the NAc. In particular, it has been suggested that while the rewarding effects are mediated by dopamine in the NAc shell, the aversive effects may be mediated by dopamine in the NAc core. To further examine this hypothesis, I examined the effects of electrolytic lesions of the NAc core in rats on the development of the conditioned taste aversion (CTA) that occurs when consumption a novel Kool-Aid flavor is followed by an i.p. injection of nicotine (0.08 mg/kg). Robust aversion to the nicotine-paired flavor in both the sham and lesion rats was found, but no difference was found between the two groups. Possible ceiling effects and insensitivity of the CTA paradigm might account for this.

Locating and profiling cells within the CNS of H. trivolvis that send processes through the esophageal trunks

Jay Bladon (Neuroscience)

Advisor: Nancy Kleckner

A three phase central pattern generator (CPG) located within the buccalganglia drives the motor program of feeding in H. trivolvis. The buccal CPG is adaptable, and both sends signals to and receives signals from the mouth, or buccal mass, via the buccal nerves. The buccal A cluster (BAC) of neurons resides in the buccal ganglia, and modulates feeding in H. trivolvis. Activity in BAC cells has been found to cause a change in the pattern of the buccal CPG from one of normal feeding to one resembling ejestion, or a variety of other behaviors. One determining characteristic of BAC cells is that they have processes that traverse the esophageal trunks (ETs). The ETs are not required for the feeding sequence, but are required for proper termination of feeding and recognition of inedible food. Intracellular tracers were backfilled through the ETs to further characterize BAC cells, and to determine whether there were other clusters of cells within the H. trivolvis CNS with processes through this nerve. Results from this experiment reinforced the existing body of knowledge about the morphology of cells B5 and B4. B5 was found to have processes through the ipsilateral ET whereas B4 was found to have processes through both ETs. A cell with processes to both the ipsilateral ET and cerebral ganglion provided evidence for a putative feedback loop traversing the ET for signals originating in the cerebral ganglia. Many BAC like cells were found to have processes through the ETs, further implicating these areas of the buccal ganglia in sending and receiving input from the ETs. Two novel clusters of cells were discovered, and one of these clusters putatively named the medial cluster, may have bilateral electrical coupling across the central commissure. Backfilling the ETs in conjunction with immunohistochemistry showed that some cells within the novel ventromedial cluster were immunoreactive to anti‐GluR5/6/7 antibodies. A taxonomically similar species of snail, Biomphalaria glabrata was found to have similar neuronal organization of buccal neurons traversing the ETs to H. trivolvis. Isolation of a portion of the ET in this organism found that the BAC like neurons as well as cells within the ventromedial and medial clusters had processes innervating the esophagus. Due to other similarities found between these two species, it was postulated that these cell groups innervate the esophagus in H. trivolvis as well. Furthermore, the neurons found within the BAC cluster and those found within the ventromedial cluster may have similar or identical functions, as both contain cells that are immunoreactive to anti Glu R5/6/7 antibodies. These two cell clusters as well as the medial cluster are also proposed to innervate the esophagus, and to participate in monitoring food intake in both B. glabrata and H. trivolvis. Nerve backfilling was found to be an effective method of locating cells whose processes traverse a given nerve, and further research should involve this technique in conjunction with electrophysiology, and immunohistochemistry to further understand the modulation of CPGs in invertebrates.

The Neurogenetic Basis of Alexithymia and Executive Dysfunction: The DAT1 Gene

Lear Carr  (Neuroscience-Honors)

Advisor: Nancy Koven

Alexithymia, a multifaceted trait, is marked by the reduced capacity to identify, distinguish, and describe emotions. Although not a diagnosable clinical syndrome per se, alexithymia has been implicated as a risk factor for numerous medical and psychiatric conditions. Previous studies indicate frontal lobe impairment in alexithymia, suggesting concomitant weaknesses in executive functions such as abstract thinking, creativity, and cognitive flexibility. Little is known about the genetic contributions to the cognitive components and emotion dysregulation in the alexithymic population. With dopaminergic innervation in the frontal lobe, the present study examines the relationships between the genetic basis of dopamine reuptake, alexithymia, and cognition. This study utilizes an unselected sample of community participants who completed self-report measures of alexithymia, a broadband self-report measure of executive functioning, a variety of neuropsychological measures of executive functioning, and who were assessed for the 40 base-pair variable number of tandem repeat (VNTR) polymorphism of the dopamine transporter gene (DAT1). In examining participants with the 9/10 genotype (n=53) and 10/10 genotype (n=61), results suggest a significant role of dopamine in aspects of cognitive and emotional functioning, with implications for unique neural pathways across facets of emotion processing.

Electrical and dye coupling in dorso-medial buccal A cluster neurons of the pond snail Helisoma trivolvis

Mayur M. Contractor (Neuroscience- Honors)

Advisor: Nancy Kleckner

The feeding behavior of Helisoma trivolvis is controlled via a tri-phasic central pattern generator (CPG), made up of S1, S2, and S3 neural subunits that drive the protraction, retraction, and hyperretraction movements of the radula respectively. Each of the neuronal subunits is considered to be an independent conditional oscillator, which can be independently activated by a different subunit, or become active again once released from inhibition of another. A group of neurons in the buccal ganglia of the H. trivolvis central nervous system called the buccal A cluster (BAC) is known to play a role in modulating the feeding CPG. They are implicated in having the ability to change or modulate an established motor system to smoothly transition from one behavior to another, a phenomenon called “motor program switching”. BAC neurons have been observed to facilitate motor program switching by inducing fictive regurgitation or satiation behavior, as well as by a result of Lyst-NPF perfusion on the H. trivolvis CNS. Furthermore, one of the defining characteristics of BAC neurons is their ability to electrically couple. The goal of this study is to target and characterize one specific group of BAC neurons in the dorso-medial area of the buccal ganglia. Characterization included determination of electrical coupling magnitude via intracellular recordings between BAC neurons, calculations of coupling coefficients to compare among the pairs of BAC neurons, Iontophoretic dyes injections into BAC neurons to help determine thier morphology and to observe any pattern of dye coupling in the dorsomedial group. Lastly, dopamine, Lyst– NPF and kainate were perfused onto CNS preparations to determine pharmacological characteristics of BACs in the dorso-medial group. Coupling coefficients between BAC neurons that were found to be electrically coupled had a range of 0.0-0.5, and an average of 0.069 for responses to negative current injection and 0.037 for response to positive current injections. Five potentially different types of electrical coupling were identified in the dorso-medial group. Furthermore, most BAC neurons that were found to show electrical coupling also showed dye coupling to the neuron it was paired with. All BAC neurons that were perfused with 30 ìM kainate displayed increased frequency of slow EPSP activity. One BAC neuron displayed firing activity in response to 100nM Lyst-NPF. Type 1 and type 2 BAC neuron morphologies were seen most often within the area most likely to be the dorso-medial group. Results that show type 1 and type 2 BAC neuron reactivity to kainate indicate that the BAC neurons in the dorso-medial group, specifically type 1 and type 2, may overlap with the dorso-medial a-GluR5/6/7 immunoreactive group. Further characterization needs to be done both in dorso-medial BAC neurons and a-GluR5/6/7 IR cells, but an overlap may provide evidence to support a modulatory connection mechanism involving the dorso-medial BAC neurons, specifically type1 and type 2 BAC neurons.

Confirmation and Distribution of Cotransmitter Buccalin in the Central Nervous System of Two Closely Related Pond Snails, B. glabrata and H. trivolvis

Harita Dharaneeswaran (Neuroscience)

Advisor: Nancy Kleckner

Buccalin-related neuropeptides modulates feeding behavior in various marine mollusks such as Aplysia californica and Clione limacine, in addition to arthropods such as the crab, Cancer boreali. However, not much is known about buccalin-related peptides in the pond snails, Helisoma trivolvis and Biomphalaria glabrata. Buccalin-related peptides were predicted to exist in B. glabrata based on a search of the expressed sequences tags (EST) database from the National Center for Biotechnology Information (NCBI). These predicted peptides in B. glabrata from the EST database were homologous to known A. californica buccalin-related peptides. The purpose of this study was to validate the presence and to localize buccalin-related peptides in H. trivolvis and B. glabrata. This was done using two techniques: (1) immunocyctochemistry , which was used to localize the buccalin-related peptides using rabbit-serum derived from injection of A. californica anti-buccalin A sera and (2) a  MALDI-FTMS analysis, which was conducted to indentify the presence of putative buccalin-related peptides in the pedal ganglia that contained the largest clusters of immunolabelled neurons from the immunocytochemical surveys. Both the immunocytochemical surveys and the MALDI-spectral analysis, indicated the presence of buccalin-related peptides in all ganglia in the central nervous system of B. glabrata and H. trivolvis. Differences in the distribution of buccalin-related peptides in the CNS between the two species, B. glabrata and H. trivolvis indicated that buccalin peptides have multifaceted roles among different species. Widespread distribution of buccalin-related peptides were observed in the CNS within each species suggesting that buccalin peptides do not only have multifaceted roles between different species but also with a single species. Also, some unidentified neurons projecting in the PBNs, LBNs and VBNs suggests the possibility that buccalin may play a role in modulation at neuromuscular junctions. A neuron B4, is known to innervate the salivary glands was found to be immunoreactive to buccalin, indicating that buccalin had modulatory functions at the neuroglandular junctions. Given that several identical buccalin-related peptides were identified in the pedal ganglia using MALDI-FTMS, in two phylogenetically closely-related species, B. glabrata and  H. trivolvis, the data suggests that closely-related species have more sequence homology between their buccalin peptides. Ultimately, this study in a small scale furthers our understanding of the function of buccalin neuropeptides and their physiological roles in B. glabrata and H. trivolvis and in relation to other mollusks.

The Indiscriminate Application of Cognitive Control in utilitarian and Non-Utilitarian Moral Decision Making

Emily Kuehn (Neuroscience)

Advisor: Nancy Koven

Current dual process theories concerning moral decision making combine rational and intuitive thinking styles as competitive processes, whereby cognitive control overrides prepotent emotional responses in the formation of a utilitarian decision (Greene, 2008). It is the purpose of this study to further classify the role of cognitive control in moral decision making and to examine whether or not superior response inhibition and working memory selectively facilitate utilitarian judgment. Participants in this study were grouped according to utilitarian and non-utilitarian judgment styles, and scores for response inhibition tasks (CPT-II and D-KEFS Color-Word Interference Test) as well as from verbal and spatial working memory tasks (WMS-III Verbal and Spatial Span Tests) were compared using MANCOVA, controlling for current mood state. Results raise doubts about the role of cognitive control in moral dual process theories posited by Greene (2008), for enhanced selective attention was observed in the non-utilitarian group, which may suggest that cognitive control contributes selectively to the formation of a non-utilitarian judgment. Not all aspects of cognitive control contribute to moral judgment style, for there were no observed effects of working memory, but this may indicate an inherent dissociation between neural mechanisms involved in attributing moral significance to a response. Overall, cognitive control and emotional components of moral decision making are likely integrated into both utilitarian and non-utilitarian decisions instead of acting as mutually competitive.

PCP-induced behavioral changes in a hole-board apparatus as a rat model of schizophrenia

Rebecca H. Lange (Psychology)

Advisor: John Kelsey

The glutamate NMDA receptor antagonist phencyclidine (PCP) has been shown to induce all three symptom sets of schizophrenia – positive, negative, and cognitive – in healthy individuals, and is often used to reproduce these symptoms in animal models of the disorder.  In our lab, acute injections of PCP (4 mg/kg) induce hyperlocomotion and decrease head-dipping in rats within a hole-board apparatus (HBA), two behaviors presumed to reflect the positive and negative/cognitive symptoms of schizophrenia, respectively.  Consistent with this hypothesis, in the current study, the typical antipsychotic haloperidol attenuated the PCP-induced hyperlocomotion, but had no effect on the PCP-induced decrease in head-dipping, and the atypical antipsychotic olanzapine reversed both the PCP-induced effects, reflective of their clinical efficacies.  In addition, the metabotropic glutamate 5 receptor (mGluR5) antagonist MPEP had no effect on the PCP-induced increase in activity, but potentiated the PCP-induced decrease in head-dipping.  The first set of data further suggest that the HBA is a valid model for assessing the antipsychotic efficacy of various drugs, and the second set of data provide support for the hypothesis that activation of the mGluR5 receptor may provide a novel approach for the treatment of at least some of the symptoms of schizophrenia.

Differential Expression of Glutamate Receptor Subtypes in Helisoma trivolvis Central Nervous System

Jing Qin (Neuroscience, Economics, Biochemistry- Honors)

Advisor: Nancy Kleckner

Glutamate is a ubiquitous amino acid that serves as a neurotransmitter in both the vertebrate and invertebrate central nervous systems (CNS). In the pond snail, Helisoma trivolvis, glutamate is particularly vital to the neural network that controls feeding behavior, in which a modifiable central pattern generator (CPG) is responsible for the rhythmic activation and inhibition of different subunits of neurons (S1-S3), resulting in distinct phases of feeding. Glutamate receptor (GluR) subtypes in vertebrates include AMPA (GluR1-4), kainate (GluR5-7), NMDA, and metabotropic receptors. Previous immunocytochemical studies using a-GluR5/6/7 and a-GluR6/7 antibodies indicate that the GluRs of the known feeding CPG and motor neurons are not kainate-like. Furthermore, both genes of AMPA-like and kainate-like GluRs have been cloned from the Helisoma trivolvis CNS. This study uses immunocytochemistry and electrophysiology of immunoreactive CPG neurons through intracellular recording to identify and characterize the different GluRs expressed in the CNS. A S2 motor neuron, B27, and a S3 motor neuron of the feeding CPG, B19, express AMPA-like glutamate receptor subtypes that consist of GluR2/3-like receptor subunits. The findings of this project suggest that carboxyl termini of AMPA GluRs are likely conserved throughout evolution, indicating the importance of their regulation through phosphorylation of terminal residues.

A Look into the Unknown: Viral, Autoimmune, and Nonspecific Encephalitis

Dan Rosenzsweig (Psychology)

Advisor: Nancy Koven

Encephalitis is a neurological condition in which the parenchyma of the brain swell as an immune system response to a perceived infection, just as other tissues might. Unlike other bodily tissues, edema in the brain produces intracranial pressure, which, if unsuccessfully treated, can cause permanent damage to other regions in the brain and sometimes death. The high proportion of encephalitis cases reported as having nonspecific etiology is often assumed to be viral in nature. However, there may be evidence in the form of patterns of symptoms and recovery that may uncover a subset within the nonspecific group. This group is likely to have increased recovery rates as well as more moderate symptoms, and may represent a milder form of encephalitis. The current study attempts to isolate this subgroup with cluster analysis.

Longitudinal Investigation of Cancer Treatment-induced Changes in Executive Function and Neuroanatomical Correlate Morphometry

Lindsey Scherer (Neuroscience)

Advisor: Nancy Koven

Since the early 1970s, cancer patients treated with chemotherapy have expressed concern over loss of cognitive abilities experienced during and after chemotherapy.  Recent studies assessing cognition with neuropsychological testing have indicated subtle, but recognizable differences in executive function and memory.  Brain imaging techniques assessing structure, volume, and activation patterns during executive function tasks have implicated abnormalities largely in the prefrontal region.  The purpose of this study was to conduct a longitudinal investigation of breast cancer (BC) patients? executive dysfunction, as it may relate to frontal lobe structural changes over time. The sample includes 17 female BC patients treated with chemotherapy (CTX+), 12 BC patients treated without chemotherapy (CTX-), and 18 healthy controls (HC).  All subjects were evaluated using structural MRI, neuropsychological testing, and mood evaluation at each of three time points: baseline (BL; post-surgery but prior to radiation, chemotherapy, and/or anti-estrogen treatment), one month post-chemotherapy (M1), and one year (Y1).  It was hypothesized individuals would not differ by diagnostic status at baseline in DLPFC volume or cognitive function.  However, at M1, both BC groups would demonstrate cognitive and volumetric decline relative to the HC group and relative to their respective BL scores but would experience partial recovery of volume and function at Y1.  This study stands alone in it employment of a prospective, longitudinal approach to report decline in processes necessary for executive function, such as fine motor and attention skills, planning, cognitive flexibility, and abstract thinking, and changes in the respective frontal lobe neurocorrelates using FreeSurfer software.  The diagnosis stage requiring chemotherapy and chemotherapy treatment seem primarily responsible for inducing the neuropsychological and neuroanatomical alterations.

The Effects of D-Cycloserine on a Conditioned Place Aversion and Stress-Induced Resinstatement in Morphine-Dependent Rats

Lauren P. Shapiro (Neuroscience)

Advisor: John Kelsey

Although it is relatively easy to get drug addicts through withdrawal, it has proven difficult to prevent subsequent relapse. Relapse can be induced by environmental cues that were once associated with drug use or withdrawal, such as a particular context, that can trigger cravings and relapse. As a result, extinction therapy, in which the cue is repeatedly presented in the absence of the drug or withdrawal, has become a treatment for addiction. However, even following such extinction, relapse can be induced by re-exposure to the drug, other drug cues, or stress. D-cycloserine (DCS) is a glutamate NMDA receptor co-agonist that has been found to improve the rate and effectiveness of extinction of drug- and withdrawal-related cues. Although DCS has been shown to enhance extinction of a naloxone-induced conditioned place aversion (CPA) in morphine-dependent rats (Myers & Carlezon, 2010a), it is not clear if that enhancement has any effect on relapse. Consequently, I sought to determine the effect of DCS on stress-induced relapse.  Surprisingly, DCS was observed to have the opposite effect as 15 mg/kg DCS given during extinction sessions slowed the rate of extinction of the CPA in morphine-dependent rats and potentiated reinstatement induced by 30 min of restraint stress. I interpret this opposite effect as a DCS-induced enhancement of reconsolidation. Future studies are needed to determine the circumstances under which exposure to the CS results in extinction vs. reinstatement in order to improve the effectiveness of addiction treatments that seek to prevent the rate of relapse.

The Endogenous Opiate Dynorphin Does Not Appear to Mediate Morphine Withdrawal in Rats

Lauren E. Slipp (Neuroscience)

Advisor: John Kelsey

Dynorphin is an endogenous opioid peptide that has been implicated as part of a compensatory system that serves to reduce the rewarding effects of drugs of abuse, and, thus, may mediate drug withdrawal and subsequent stress-induced reinstatement of drug-seeking behaviors (Shippenberg et al., 2007). Although blocking the kappa opiate receptors (KORs) on which dynorphin acts should therefore decrease opiate withdrawal, previous studies are split on this issue. In general, blocking KORs prior to induced withdrawal decreases the severity of withdrawal symptoms and aversiveness (McClung, Nestler, & Zachariou, 2005; Sawyer, 2007; Schierberl, 2008), whereas blocking KORs prior to initiation of drug dependence has the opposite effect (Spanagel et al., 1994; Suzuki, Narita, Takahashi, Misawa, & Nagase, 1992; Zachariou et al., 2006). In the current experiments, rats were made morphine-dependent via escalating (10-50 mg/kg) intraperitoneal (i.p.) injections of morphine over 5.5 days, subjected to naltrexone-induced (4 mg/kg, i.p.) withdrawal, and tested for a conditioned place aversion (CPA) 48 hr later. Blocking KORs with the antagonist nor-BNI (20 mg/kg, i.p.) 16 hr prior to initiation of morphine dependence had no effect on the prevalence of classic withdrawal symptoms observed during the 30-min withdrawal session. More critically, KOR blockade at this time point also had no effect on the magnitude of CPA, an indicator of the aversiveness of the withdrawal experience. Consistent with previous work from our laboratory (Sawyer, 2007), KOR blockade 30 min prior to precipitated withdrawal decreased weight loss and feces excretion in morphine-dependent rats, but, in contrast to the previous study, did not reduce the magnitude of CPA. Thus, the role of dynorphin in mediating opiate withdrawal remains unclear. In a third experiment designed to investigate the effect of KOR blockade prior to stress-induced reinstatement, I extinguished the withdrawal-induced CPA, but failed to reinstate it by 7-15 min of forced swim. Therefore, I was not able to determine the role of dynorphin in mediating stress-induced reinstatement.

Levodopa-Induced Dyskinesias: Development and Treatment in a Unilateral 6-OHDA Rat Model of Parkinson’s Disease

Helena S. Turner (Neuroscience)

Advisor: John Kelsey

Parkinson’s Disease (PD) is characterized by tremors, rigidity, and difficulty initiating movement. First line treatment consists of levodopa (L-DOPA), which controls these symptoms for a time, but prolonged use provokes debilitating excess movements called dyskinesias. A number of drugs have shown promise in treating PD or LID, but rarely both as they reflect the relative absence and excess of dopamine, respectively. To examine the role played by a variety of neurotransmitter systems, a unilateral 6-OHDA rat model of PD was used to asses the impact of acute administration of 0.1 mg/kg SCH 23390 (D1 receptor antagonits), 0.2 mg/kg 8-O-DPAT (5-HT1A agonist), 15 mg/kg caffeine (adenosine antagonist), 0.05 mg/kg rimonabant (CB1 antagonist), and 10 mg/kg naltrexone (nonspecific opiate antagonist) on stepping and dyskinesias of animals treated with either chronic 10 mg/kg L-DOPA or acute 35 mg/kg L-DOPA. SCH 23390 decreased stepping and dyskinesias, whereas 8-OH-DPAT increased stepping while decreasing dyskinesias. Caffeine and rimonabant increased stepping and had no effect on dyskinesias in all animals, and altrexone had no clear effects on stepping or dyskinesias. Based on the results of this and other studies, it appears that caffeine and rimonabant may be a good adjunctive therapy to L-DOPA, as they show the potential to reduce PD without exacerbating LID. 8-OH-DPAT appears to have tremendous potential in treating humans with PD who have also developed LID, given its ability to reduce symptoms of each.

Further characterization of the buccal A cluster network and its connectivity to the feeding central pattern generator in the buccal ganglia of the pond snail, Helisoma trivolvis

Alexia Y. Zhang (Neuroscience)

Advisor: Nancy Kleckner

The feeding central pattern generator (CPG) in the pond snail, Helisoma trivolvis, controls a triphasic feeding pattern. The snail feeds by scraping its toothed radula over the substrate in protraction, retraction and hyperretraction pattern, commonly known as phase 1,2 and 3, respectively. The CPG that controls the feeding pattern exists entirely in the buccal ganglia and consists of interneurons that correspond with each phase of feeding. The interneurons innervate motorneurons that control the muscle movement in the buccal cavity. The feeding CPG can be modulated by a group of neurons termed the buccal A cluster (BAC) network. The BAC network switches the feeding pattern from a 1-2-3 to a 1-2 pattern that induces regurgitation. This occurs under the influence of aversive stimuli or during reproductive behaviors when substrate cleaning occurs. Evoking the BAC neurons produces action potential that induce additional non-phase 2 hyperpolarizations in phase 3 motorneurons in a one to one relationship. A second network of 22-28 cells that are immunoreactive for aGluR 5/6/7 antibodies (GR neurons) was discovered and is thought to also modulate the feeding CPG. Recently, it was discovered that some GR neurons show BAC-like morphology, indicating that these two networks may overlap. The goal of this study is to determine the degree of overlap between these two networks, the connection to the feeding CPG, as well as to further characterize the BAC network. Simultaneous electrophysiological recordings from a BAC neuron and the phase 3 motorneuron B19 was conducted to determine the connectivity between the BAC neuron and neuron B19. Dopamine, GABA, neuropeptide phenyalanine were perfused to evoke the BAC neurons. Each BAC neuron was injected with Neurobiotin to characterize the morphology of the BAC neuron and processed with α-GluR5/6/7 antibodies to determine whether the recorded neuron contain GluR 5/6/7 subunits. Neuropeptide phenylalanine, GABA and current injections were all able to evoke the BAC neuron to fire action potentials that produced additional non-phase 2 hyperpolarizations in neuron B19 in a one to one relationship. Dopamine was not able to cause the BAC neuron to fire action potentials, but it was able to depolarize the BAC neuron membrane potential and inhibit neuron B19 activity. This study shows that there is significant overlap between the BAC neurons and the α-GluR5/6/7-IR cells and this population of BAC/GR neurons also showed a direct connectivity to the phase 3 motorneuron B19.