Type-1 diabetes and influenza cause significant illness and unnecessary medical costs despite the existence of insulin for maintenance of diabetes and a vaccine for prevention of influenza. This dissertation describes three studies on the development and clinical translation of microneedles to improve the administration of these biopharmaceuticals. The first study reports on a sharp-tipped hollow metal microneedle designed to reduce manufacturing costs, improve insertion into skin, and improve fluid flow compared to other hollow microneedles used for drug delivery. The results showed sharp-tipped metal microneedles could be fabricated using an inexpensive electroplating and sacrificial micromolding process. Single-microneedle devices made by this method achieved high flow rates and delivered model drugs into tissue. The second study reports on insulin delivery using microneedles in children with type-1 diabetes. The results showed microneedle insertion was less painful, which is a promising result for improving injection compliance in children. Additionally, microneedle delivery showed rapid onset of insulin action compared to subcutaneous catheter delivery, which may enable automatic closed-loop insulin therapy. This was the first study of drug delivery to children using microneedles. The last study reports on microneedle patches for self-vaccination against influenza. Human subjects were recruited from greater Atlanta, were asked to self-administer placebo microneedle patches, and were then given a dynamic questionnaire to determine their views and preferences regarding influenza vaccination using microneedles compared to conventional intramuscular injection. The results showed that microneedles were usable by the participants, the introduction of microneedles may improve vaccination coverage by approximately 20%, and self-administration of vaccines may significantly reduce vaccination costs for a healthcare payer. This was the first study to assess the ability of human subjects to self- administer a microneedle patch and the first study to determine the potential impact of self-vaccination against influenza using a microneedle patch on vaccination coverage and vaccination cost. Overall, the fabrication advances and positive findings from human subjects research support additional translation of microneedles for insulin delivery and self-vaccination toward clinical use.

Rationale and objectives: Alcohol and nicotine are the most commonly abused drugs and they are often taken together. To help address some of clinical issues regarding nicotine and alcohol co-dependence, a procedure in which rats self-administer nicotine intravenously and alcohol orally during the same operant session has been developed. Methods: Male Wistar rats were trained to self-administer alcohol (12%, w/v; 0.19 ml/delivery) or implanted with jugular catheters and trained to self-administer nicotine (30 μg/kg IV/infusion) by pressing a lever or were trained to self-administer both drugs, some with alcohol first, and others with nicotine first. Results: Animals readily coadministered alcohol and nicotine concurrently. Access to alcohol reduced nicotine selfadministration significantly. Conclusions: These results show that rats will self-administer relevant amounts of intravenous nicotine and oral alcohol concurrently. They also provide further support for the important relationship between nicotine and alcohol.

Rationale and objectives: Alcohol and nicotine are the most commonly abused drugs and they are often taken together. To help address some of clinical issues regarding nicotine and alcohol co-dependence, a procedure in which rats self-administer nicotine intravenously and alcohol orally during the same operant session has been developed. Methods: Male Wistar rats were trained to self-administer alcohol (12%, w/v; 0.19 ml/delivery) or implanted with jugular catheters and trained to self-administer nicotine (30 μg/kg IV/infusion) by pressing a lever or were trained to self-administer both drugs, some with alcohol first, and others with nicotine first. Results: Animals readily coadministered alcohol and nicotine concurrently. Access to alcohol reduced nicotine selfadministration significantly. Conclusions: These results show that rats will self-administer relevant amounts of intravenous nicotine and oral alcohol concurrently. They also provide further support for the important relationship between nicotine and alcohol.

abstract: 5-HT2A receptor (R) antagonists and 5-HT2CR agonists attenuate reinstatement of cocaine-seeking behavior (i.e., incentive motivation). 5-HT2Rs are distributed throughout the brain, primarily in regions involved in reward circuitry, including the prefrontal cortex (PFC), caudate putamen (CPu), and basolateral (BlA) and central (CeA) amygdala. Using animal models, we tested our hypotheses that 5-HT2ARs in the medial (m) PFC mediate the incentive motivational effects of cocaine and cocaine-paired cues; 5-HT2ARs and 5-HT2CRs interact to attenuate cocaine hyperlocomotion and functional neuronal activation (i.e, Fos protein); and 5-HT2CRs in the BlA mediate the incentive motivational effects of cocaine-paired cues and anxiety-like behavior, while 5-HT2CRs in the CeA mediate the incentive motivational effects of cocaine. In chapter 2, we infused M100907, a selective 5-HT2AR antagonist, directly into the mPFC and examined its effects on reinstatement of cocaine-seeking behavior. We found that M100907 in the mPFC dose- dependently attenuated cue-primed reinstatement, without affecting cocaine-primed reinstatement, cue-primed reinstatement of sucrose-seeking behavior, or locomotor activity. In chapter 3, we used subthreshold doses of M100907 and MK212, a 5-HT2CR agonist, to investigate whether these compounds interact to attenuate cocaine hyperlocomotion and Fos protein expression. Only the drug combination attenuated cocaine hyperlocomotion and cocaine-induced Fos expression in the CPu, but had no effect on spontaneous locomotion. Finally, in chapter 4 we investigated the effects of a 5- HT2CR agonist in the BlA and CeA on cocaine-seeking behavior and anxiety-like behavior. We found that CP809101, a selective 5-HT2CR agonist, infused into the BlA increased anxiety-like behavior on the elevated plus maze (EPM), but failed to alter cocaine-seeking behavior. CP809101 infused into the CeA attenuated cocaine-primed reinstatement and this effect was blocked by co-administration of a 5-HT2CR antagonist. Together, these results suggest that 5-HT2ARs in the mPFC are involved in cue-primed reinstatement, 5-HT2A and 5-HT2CRs may interact in the nigrostriatal pathway to attenuate cocaine hyperlocomotion and Fos expression, and 5-HT2CRs are involved in anxiety-like behavior in the BlA and cocaine-primed reinstatement in the CeA. Our findings add to the literature on the localization of 5-HT2AR antagonist and 5-HT2CR agonist effects, and suggest a potential treatment mechanism via concurrent 5-HT2AR antagonism and 5-HT2CR agonism. / Dissertation/Thesis / Ph.D. Psychology 2013

Indiana University-Purdue University Indianapolis (IUPUI) / Adolescent alcohol abuse is a global problem that initiates lifelong addiction. Alcohol use during adolescence is associated with subsequent Meth dependence in humans. Specifically, female adolescents are particularly vulnerable to serial alcohol and Meth use. However, it is unknown if prior voluntary alcohol drinking impacts subsequent Meth-taking in female adolescent rats. Both alcohol and Meth increase the prostaglandin synthesis enzyme cyclooxygenase-2 (COX-2) in the brain but the effect of serial exposure to alcohol and Meth on COX-2 has not been determined. The first study uses a novel method of serial voluntary alcohol drinking and Meth self-administration in female adolescent rats to model human patterns of co-abuse. Prior alcohol drinking did not affect subsequent Meth self-administration, but it reduced the cue-primed reinstatement of Methseeking after abstinence from Meth. Rats with a history of adolescent alcohol drinking also had increased COX-2 in the dorsal striatum, regardless of subsequent Meth selfadministration. These findings demonstrate that a history of adolescent alcohol drinking does not alter Meth self-administration but persistently reduces cue-primed Meth seeking and increases COX-2 after prolonged abstinence from alcohol. To further examine the role of COX-2 in alcohol drinking, the second study found that adolescent alcohol drinking not only increased COX-2 after four weeks of alcohol abstinence, but also increased endothelin-1 (ET-1) and prostaglandin E2 (PGE2) in the dorsal striatum. Furthermore, adolescent alcohol drinking increased alcohol drinking after abstinence, and this increase was attenuated by treatment with the COX-2 inhibitor nimesulide during abstinence. Antagonism of the interaction between PGE2 and its receptor 1 (EP1) also attenuated the increase in relapse drinking and restored alcohol drinking to the rate of alcohol naïve rats. Overall, these experiments identified a prostaglandin-mediated mechanism that is a putative target for the treatment of alcohol relapse following abstinence in individuals with a history of adolescent alcohol abuse.

Caffeine is widely consumed for its psychoactive effects worldwide. No pre-clinical study has established reliable caffeine self-administration, but we found that caffeine can enhance the reinforcing effects of non-drug rewards. The goal of the present studies was to determine if this effect of caffeine could result in reliable caffeine self-administration. In 2 experiments rats could make an operant response for caffeine delivered in conjunction with an oral ‘vehicle’ including saccharin (0.2% w/v) as a primary reinforcer. In Experiment 1, intravenous (IV) caffeine infusions were delivered in conjunction with oral saccharin for meeting the schedule of reinforcement. In control conditions, oral saccharin alone or presentations of IV caffeine alone served as the reinforcer. In Experiment 2, access to caffeine was provided in an oral vehicle containing water, decaffeinated instant coffee (0.5% w/v), or decaffeinated coffee and saccharin (0.2%). The concentration of oral caffeine was then manipulated across testing sessions. Oral and IV caffeine robustly increased responding for saccharin in a manner that was repeatable, reliable, and systematically related to unit IV dose. However, the relationship between oral caffeine dose and operant behavior was less systematic; the rats appeared to titrate their caffeine intake by reducing the consummatory response (drinking) rather than the appetitive response (lever pressing). These studies establish reliable volitional caffeine self-administration in rats. The reinforcement enhancing effects of caffeine may help to explain widespread caffeine use by humans, who ingest caffeine in complex vehicles with reinforcing properties.

No description available.

Caffeine is the most consumed psychoactive substance in the world, and most caffeine consumption in coffee and energy drinks is intended to produce a psychoactive effect. However, caffeine is not a primary reinforcer in preclinical paradigms – non-human species do not reliably take the drug to produce a psychoactive effect. However, caffeine is a ‘reinforcement enhancer’ in preclinical models; the effects of caffeine increase the motivation to obtain other non-drug reinforcers. The overall goal of this project was to determine if these reinforcement enhancing effects of caffeine could promote caffeine self-administration and to subsequently investigate the behavioral and neurochemical underpinnings of this effect. We hypothesized reliable caffeine self-administration would occur by adventitious pairing of caffeine with saccharin, a primary reinforcer. Second, we hypothesized that caffeine enhances reinforcement by increasing the salience of incentive stimuli, which are stimuli that come to evoke approach behaviors through associative learning (e.g., Pavlovian conditioning). Finally, incentive salience is moderated by dopamine release in the nucleus accumbens (NAc), an area highly involved in reward-learning and substance dependence. Therefore, we hypothesized that if caffeine enhanced control of approach behavior by incentives, then it would increase the ability of incentive stimuli to evoke dopamine in the NAc. These studies show that intravenous delivery of caffeine with oral saccharin increases operant relative to control groups responding for intravenous caffeine or oral saccharin. The effect was also dose-dependent, confirming that the psychoactive effects of caffeine increased behavior. We also extended this effect to an oral model of caffeine self-administration, which included a simple sweetener (saccharin) or a complex oral vehicle (saccharin with decaffeinated coffee) to mask the bitter taste of caffeine. Presenting caffeine with oral saccharin promoted self-administration, relative to saccharin alone and did not depend on the nature of the complexity of the vehicle. Caffeine also dose-dependently increased approach to an incentive stimulus and this effect was associated with increased extracellular dopamine in the NAc. These findings suggest caffeine enhances incentive motivation and that this effect may result from increases in CS-evoked striatal dopamine.

Rationale. Alcohol use disorders (AUDs) and tobacco dependence are frequently identified as co-morbid. Although less than 20% of the general population smokes, over 80% of people with AUDs are considered daily smokers. In fact, people with AUDs are more likely to die from smoking-related health issues, than from alcohol related health issues. Surprisingly, there is very little evidence that alcohol and nicotine are concurrently self-administered in pre-clinical models. We hypothesized that low doses of nicotine that enhancing responding for other rewards would be self-administered and enhance self-administration of alcohol. Objective. The goal of this study was to determine if low-doses of nicotine, typically not self-administered alone, would promote alcohol self-administration in a concurrent access paradigm. Method. Alcohol preferring rats (females) were requested from the University of Indiana Medical School breeding facility. They were randomly assigned to one of three groups – NIC-Alone, ALC-Alone, or ALC+NIC. All rats were fluid restricted and shaped to lick for water at two sipper tubes that could record lick responses and deliver aliquiots of fluid into the sipper tube via a solenoid valve. After shaping, rats were instrumented for IV self-administration. During self-administration tests, rats in the ALC-Alone received access to oral ethanol (15% v/v) for meeting the schedule of reinforcement at one sipper tube (e.g., right) and saline infusions for meeting the schedule of reinforcement at the other sipper tube (e.g., left). The NIC-Alone group received IV nicotine infusions (15 ug/kg/inf) and oral licorice (1%) for meeting the schedule of reinforcement at one sipper tube (e.g., left) and oral water for meeting the schedule of reinforcement at the other sipper tube (e.g., right). The ALC+NIC group received IV nicotine and oral licorice for meeting the schedule of reinforcement on the left sipper, and oral ethanol for meeting the schedule of reinforcement on the right sipper. Price manipulations for nicotine were performed by adjusting the schedule of reinforcement on the sipper associated with nicotine infusions. Results. During acquisition, nicotine did not enhance alcohol self-administration – alcohol intake was comparable between ALC-Alone and ALC+NIC rats. In addition, alcohol did not enhance nicotine self-administration as responding for NIC was comparable between ALC+NIC and NIC-Alone rats. However, when the price of nicotine was manipulated, alcohol created a greater demand for nicotine, as indicated by higher rates of nicotine consumption with increases in price. Manipulating the price of nicotine did not alter demand for alcohol. Conclusion. The interaction between alcohol and nicotine reinforcers may depend on changes in demand for nicotine. Future studies should investigate whether demand for alcohol is altered by concurrently available nicotine infusions. *the first and second authors contributed equally to this project

Background: Studies with experimental administration of alcohol offer inconsistent approaches and interpretations in which ways an acute alcohol exposure affects the HPA-system and the cortisol secretion. So far published alcohol experiments differ in alcohol application, the possibility of alcohol self-administration at the subjects own discretion as well as the age of the participants. Question: Is cortisol secretion modified by gender during alcohol infusion? Do men and women show different cortisol levels under alcohol exposure when compared to the baseline? Is there a dose-response relationship between cortisol secretion and acute alcohol exposure? Have family history, smoking habits and alcohol induced side effects like nausea an influence on the cortisol secretion under alcohol exposure? Materials and methods: 48 18 year old subjects participated in two identical sessions in which they were able to regulate their maximum blood alcohol concentration up to a safety limit of 1.2 ‰ (i.e., 0.12%). The experiment was conducted by using a special software for self-infusion of ethanol (CASE) which guided the participants through a two and a half hours long experiment. CASE is founded on a validated physiologically-based pharmacokinetic model and involves calculating the infusion volume needed to increase the blood alcohol concentration in a linear manner. The BAC increases after each alcohol request by 0,075 ‰ (i.e., 0.0075%) within in two and a half minutes. If the subject infuses no alcohol the blood alcohol concentration will decrease by 0.01 ‰ (i.e., 0.001%) per minute. Through the precise calculation of the infusion rate, individual differences can be eliminated. Cortisol levels were measured at five time points: an initial baseline measurement and four measurements at fixed time points during the alcohol self-administration of subjects with two completed alcohol experiments. As an appropriate measure to examine the effect of alcohol self-administration on cortisol secretion, the maximum blood alcohol concentration was determined. In addition the day of experiment, gender and family history were observed as well as exploratory maximum nausea and smoking habits. Results: In conclusion, the results confirmed that women have higher cortisol levels than men at baseline and under alcohol influence. Blood alcohol concentration as examined influencing variable was shown to have different effects on the HPA system on day one and two. On the first day of experimentation there was no effect of blood alcohol concentration on the HPA system. On the second day a dose-response relationship could be identified between cortisol secretion and acute alcohol exposure. Individually higher blood alcohol concentrations attenuated cortisol stronger in comparison to subjects with lower blood alcohol concentrations. Family history, smoking habits and unpleasant side effects (nausea) did not affect the cortisol secretion under alcohol exposure in this series of experiments. Conclusions: Current data suggests that alcohol experiments affect the cortisol secretion in young social drinkers. These findings could be detected for the first time. Up to this point there has not been an experimental study that investigated and evaluated the dose-effect relationship between cortisol secretion and alcohol in a study design which uses intravenous alcohol self-administration. It can be theorized that the first day of experimentation is suitable as a settling-in phase due to unspecific confounding factors, whereas the second day can be considered, in an identical setting, apt for hypothesis testing. The increased cortisol level in women when compared to men is consistent with previous studies and there was no indication that family history, smoking habits and alcohol induced unpleasant side effects have an influence on cortisol secretion.