AGING AND THE DYNORPHINERGIC SYSTEM: EVALUATION OF MEMORY AND MOTOR SYSTEMS IN PRODYNORPHIN KNOCKOUT MICE

Dynorphins, endogenous peptide neurotransmitters expressed in the central nervous system, have been implicated in diverse pathophysiological processes, including excitotoxicity, chronic inflammation, traumatic injury, cognitive impairment, and motor dysfunction, with significant changes with aging or age-related disease processes. This has led to the hypothesis that the suppression of dynorphin expression would produce beneficial effects on learning and memory and motor function. To assess the phenotypic manifestations of chronic suppression of endogenous dynorphin, knockout (KO) mice lacking the coding exons of the gene encoding the prodynorphin (Pdyn) precursor protein, were tested in a series of behavioral, biochemical, and molecular biological studies. Moderately aged Pdyn KO perform comparatively better than similarly aged wild-type (WT) mice in the water maze task, although no Pdyn effect was seen among young adult mice. In addition, young adult Pdyn KO mice show mildly improved performance on a passive avoidance task. Minimal baseline differences were noted in spontaneous locomotor activity in an open-field assay, but Pdyn deletion produced a relative sparing of motor dysfunction induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus, striatum, and frontal cortex of WT mice by quantitative radioimmunoassay. While aging produces progressive decline in Dyn B in striatum and frontal cortex, Dyn A shows an upward trend in frontal cortex without significant change in striatum. Systemic MPTP produces significant short-term elevations in dynorphin peptides that regress to below baseline by 7 days. HPLC analysis of striatal dopamine shows an age-dependent increase in basal dopamine levels in Pdyn KO mice, an effect that is abolished after MPTP. Western blotting experiments demonstrate that Pdyn deletion is associated with greater phosphorylation at the serine-40 site of tyrosine hydroxylase (TH) despite relatively less total TH immunoreactivity, suggesting a suppressive effect of dynorphins on dopamine synthesis. Microarray analysis of hippocampal tissue from young and aged WT and Pdyn KO mice reveals a number of functional groups of genes demonstrating altered expression. The results of this dissertation support a role of endogenous dynorphins in age-associated cognitive and motor dysfunction

Novel perspectives in the physio-pharmacological regulation of opioid receptors

The opioid peptide receptors encompass three major “classical” subclasses, namely, μ, δ and κ (MOP, DOP and KOP receptors respectively) and a more recently identified subclass, the NOP receptor. The endogenous opioid and dopaminergic systems are known to interact in the modulation of motor functions under physio-pathological conditions. In particular, degeneration of dopamine (DA) neurons and loss of DA innervation in Parkinson’s disease (PD) is known to trigger adaptive changes in opioid systems in the basal ganglia (BG), which may be viewed either as pathogenic or compensatory. In the present thesis we investigated the role of DA, and specifically the role of D2 receptor-mediated transmission, in the motor effects exerted by DOP and NOP receptors ligands. Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, exerts a dual control over motor function, low doses being facilitating and high ones being inhibitory. Since the same dual response has been observed after administration of NOP receptor antagonists, we hypothesized that the motor effects exerted by NOP receptors ligands might involve different DA receptor subtypes. To test our hypothesis we used both pharmacological (i.e. DA receptor selective ligands) and genetic (i.e. mice lacking the D2 receptor (D2R-/-) or its long isoform (D2L-/-)) tools. The D2/D3 antagonist raclopride prevented the motor facilitation induced by low doses of N/OFQ or the NOP antagonist J-113397, but was ineffective in preventing the motor impairment induced by high doses of J-113397. These effects suggest that motor facilitation and motor inhibition might rely on different D2 receptor subpopulations. Therefore to dissect out the contribution of pre- and post-synaptic D2 receptors, D2 knockout mice were used. We observed that the motor facilitation induced by low doses of N/OFQ or J-113397 was lost in D2R-/- and D2L-/- mice. The inhibitory effect of high doses of J-113397 was retained in D2L-/- mice, but disappeared in D2R-/- mice, whereas motor inhibition induced by high doses of N/OFQ occurs even in the absence of D2 receptors. Our results demonstrate that motor stimulation induced by low doses of N/OFQ or J-113397 is mediated by D2L receptors whereas motor inhibition induced by J-113397 involves D2S, possibly pre-synaptic, receptors. N/OFQ-induced hypolocomotion is independent from D2 receptors, and possibly due to direct inhibition of nigral DA cells. The same combined approach was followed to study the role of D2 transmission in DOP receptor-mediated motor effects. Evidence that DOP receptor is an attractive target for the treatment of neuropsychiatric disorders, among which PD, has been growing in recent years. The DOP receptor agonist SNC-80 did not increase motor activity in C57BL naïve mice. However, it restored motor activity in mice rendered hypokinetic with raclopride, or emiparkinsonian 6-OHDA lesioning. This finding corroborates the hypothesis that the DOP receptor system plays a compensatory role under conditions where DA transmission and, consequently, motor function is impaired. To further confirm the involvement of D2 receptors in the motor action exerted by DOP receptor agonist, SNC-80 was administered to D2R-/- and D2L-/- mice, and wild-type controls. SNC-80 was ineffective in D2R+/+ and D2L+/+ mice but improved motor function in D2R-/- and D2L-/- mice. These results show that the absence of post-synaptic D2 receptors discloses a motor promoting action of DOP receptor ligands, suggesting the existence of a D2/DOP receptor interaction both at the membrane and network level. Receptor signalling is typically accompanied by desensitization, a complex feedback regulatory process whereby receptor responsiveness decreases on continued agonist stimulation. Signaling of opioid receptors is regulated negatively by regulators of G protein signalling (RGS) proteins and in vitro studies provided evidence for a DOP receptor-specific effect of RGS4. Small molecule inhibitors of RGS-box-Gα interactions should serve to enhance G-protein signals and act as adjuvants or “potentiators” of GPCR agonists. Therefore, we tested the hypothesis that the coapplication of SNC-80 and CCG-203769, a small molecule inhibitor of RGS4, developed by R. Neubig at the University of Michigan, could potentiate the effects of SNC-80 on motor activity, both under physiological and parkinsonian conditions. We showed that the coapplication of sub-threshold doses of CCG-203769 disclosed a mild, but significant, effect of SNC-80 on motor activity in naïve mice. In addition, it synergized with SNC-80 in restoring motor activity in raclopride-treated, or 6-OHDA hemilesioned rats. These data show that RGS4 contribute to extinguish DOP-triggered motor responses, opening the way to the use of RGS4 inhibitors as adjuncts to DOP agonists in PD. Overall, the data provide evidence for an opposite role of DA transmission in the modulation of N/OFQergic and enkephalinergic opioid systems. Moreover, these results demonstrate that endogenous RGS4 exerts a regulatory role on DOP receptor signalling and that the inhibition of RGS4 might be beneficial in modulating DOP transmission in pathological conditions. Finally, this study proposes new possible targets to regulate motor activity, which may translate into new drugs useful in the treatment of PD and other brain disorders

Non-nociceptive roles of opioids in the CNS: opioids' effects on neurogenesis, learning, memory and affect.

Mortality due to opioid use has grown to the point where, for the first time in history, opioid-related deaths exceed those caused by car accidents in many states in the United States. Changes in the prescribing of opioids for pain and the illicit use of fentanyl (and derivatives) have contributed to the current epidemic. Less known is the impact of opioids on hippocampal neurogenesis, the functional manipulation of which may improve the deleterious effects of opioid use. We provide new insights into how the dysregulation of neurogenesis by opioids can modify learning and affect, mood and emotions, processes that have been well accepted to motivate addictive behaviours

Kappa opioid receptor signaling in the brain: Circuitry and implications for treatment

Kappa opioid receptors (KORs) in the central nervous system have been known to be important regulators of a variety of psychiatry illnesses, including anxiety and addiction, but their precise involvement in these behaviors is complex and has yet to be fully elucidated. Here, we briefly review the pharmacology of KORs in the brain, including KOR's involvement in anxiety, depression, and alcohol addiction. We also review the known neuronal circuitry impacted by KOR signaling, and interactions with corticotrophin-releasing factor (CRF), another key peptide in anxiety-related illnesses, as well as the role of glucocorticoids. We suggest that KORs are a promising therapeutic target for a host of neuropsychiatric conditions

COCAINE-BASED SIGNALING CHANGES IN THE NUCLEUS ACCUMBENS, LATERAL HABENULA, AND THALAMUS

The brain is an extraordinarily complex and organized system. Environmental information reaches the brain via the sensory systems, and this information is processed to interpret and make sense of the world. The mechanisms used to transmit information between neurons are also involved in directing and modifying the strength of these connections. Thus, the brain is always in a plastic state and has the ability to both interpret neural information and be shaped by it. Cocaine addiction is a progressive condition highlighted by maladaptive and compulsive behavior that develops after exposure to cocaine. Thus, cocaine exposure changes neural processing in the brain in ways that lead to the addicted state. The work presented here examines how neural circuits in addiction-related brain regions, such as those involved in motivated behavior and translating emotion into action, change at the cellular and molecular levels in response to cocaine exposure. The results uncover a variety of novel cocaine-induced changes in neural circuitry and processing which likely contribute to the development and/or maintenance of addiction

The role of the kappa opioid receptor system in forebrain dependent associative learning

The opioid receptor system has been shown, through various lines of evidence, to be involved in pain processing, addiction, and learning and memory. It has been previously established that the mu opioid receptor (MOR) is intimately involved in the acquisition and consolidation of memories. While these studies have provided valuable insight into the role of MOR in learning, researchers have recently begun to elucidate a role for the kappa opioid receptor (KOR) with learning. Previous reports have demonstrated that KOR and its ligands are capable of modifying complex learned behaviors in paradigms such as water maze and fear conditioning. While these studies have established an important foundation suggesting that KOR plays a role in learning and memory, clear evidence for, and an understanding of the mechanistic role of KOR in learning is still lacking. To explore the role of KOR in learning, we have used the associative learning paradigm whisker trace eyeblink (WTEB) conditioning. With WTEB conditioning animals are trained to associate a neutral conditioned stimulus (CS - whisker stimulation), following a stimulus free trace interval with a salient unconditioned stimulus (US – periorbital shock). With successive CS-US paired presentations, the subject begins to elicit a conditioned response (CR - eyeblink) to the CS, prior to US onset. Acquisition for this paradigm has been demonstrated to be forebrain dependent as removal of either the hippocampus or the neocortex can either hinder, or entirely block acquisition of the association (Solomon et al., 1986, Moyer et al., 1990, Kim et al., 1995, Weiss et al., 1999, Takehara et al., 2002, Galvez et al., 2007). Using this paradigm, the following thesis outlines a series of experiments designed to examine opioid and specifically KOR involvement in forebrain dependent learning mechanisms. To do this we first demonstrated in Chapter 2 that a global opioid antagonist, naloxone, administered intraperitoneally effectively blocks acquisition of the WTEB association. Further examining this relationship, in Chapter 3, systemic and local somatosensory neocortical injections of the KOR specific antagonist, NorBNI, demonstrate that blocking KOR results in a similar impaired acquisition. Following this pathway downstream, Chapter 4 outlines a phasic property of KOR in WTEB conditioning, in that early-phase antagonism causes deficits in acquisition of the association, while late-phase antagonism causes deficits in memory consolidation. Lastly, Chapter 5 demonstrated a significant increase in the amount of the dynorphin precursor peptide prodynorphin (PD) in the somatosensory cortex during and immediately following WTEB training. Collectively, the findings presented in this dissertation outline a novel role for KOR in the acquisition and consolidation of forebrain dependent associative memories while, providing additional insight into the underlying neocortical mechanism mediating our ability to learn and consolidate information

Involvement of the opioid system and BDNF in neuroplastic alterations occurring in neuropathic pain conditions

Chronic pain affects one in five adults, reducing quality of life and increasing risk of developing co-morbidities such as depression. Neuropathic pain results by lesions to the nervous system that alter its structure and function leading to spontaneous pain and amplified responses to noxious and innocuous stimuli. The Opioid System is probably the most important system involved in control of nociceptive transmission. Dynorphin and nociceptin systems have been suggested key mediators of some neuropathic pain aspects. An important role also for BDNF has been recently suggested since its involvement in the peripheral and central sensitization phenomena is known. We studied neuroplastic alterations occurring in chronic pain in mice subjected to the chronic constriction injury (CCI). We investigated gene expression alterations of both BDNF and Opioid System at spinal level at different intervals of time. A transient upregulation of pBDNF and pDYN was observed in spinal cord, while increasing upregulation of ppN/OFQ was found in the DRGs of injured mice. Development of neuropathic behavioral signs has been observed in ICR/CD-1 and BDNF+/+ mice, subjected to CCI. A different development of these signs was observed in BDNF+/-. We also studied gene expression changes of investigated systems in different brain areas fourteen days after surgery. We found pBDNF, pDYN, pKOP, ppN/OFQ and pNOP gene expression alterations in several areas of CCI mice. In the same brain regions we also determined bioactive nociceptin peptide levels, and elevated N/OFQ levels were observed in the amygdala area. Histone modifications studies have been performed in BDNF and DYN gene promoters of CCI animal spinal cord showing selected alterations in pDYN gene promoter. In addition, a preliminary characterization of the innovative NOP-EGFP mice was performed. Overall, our results could be useful to understand which and how neuropeptidergic systems are involved in neuroplastic mechanism occurring in neuropathic pain

In utero and Postnatal Oxycodone Exposure: Implications for Intergenerational Effects

Prescription opioid abuse during and after pregnancy is a rising public health concern. Adding a layer of complexity is the role of heredity in the overall development of these exposed offspring. The present work uses a preclinical rat model mimicking oxycodone (oxy) exposure in utero (IUO) and postnatally (PNO) to investigate comparative and intergenerational effects in the two different exposure groups. To understand the direct effects of IUO and PNO exposure on the F1 generation, we employed a systems biology approach encompassing proton magnetic resonance spectroscopy (1H-MRS), electrophysiology RNA-sequencing, and pain assessment to elucidate molecular and behavioral changes in these offspring. 1H-MRS studies revealed significant changes in brain metabolites that were corroborated with changes in synaptic currents. RNA-sequencing of the prefrontal cortex further revealed alterations in the expression of key genes associated with synaptic transmission, neurodevelopment, mood disorders, and addiction. Von Frey testing showed lower pain thresholds in both oxy-exposed groups. Further, because addictive drugs produce significant and persistent changes in the synapse, we investigated the synaptic vesicle (SV) contents of the PNO and IUO groups. To that end, we found that the expression levels of key SV proteins associated with functional pathways and neurological disease were altered in oxy-exposed groups. While our earlier studies characterized the effects PNO and IUO exposure have on the F1 generation, we next sought to compare the overall development between F1 offspring and their progeny, the F2 generation. We observed significant differences in phenotypic attributes of both generations in each treatment group, and RNA-sequencing of the nucleus accumbens revealed alterations in the expression of key synaptic genes in both generations. Post-validation of these genes using RT-PCR highlighted the differential expression of several neuropeptides associated with the hypocretin system, a system recently implicated in addiction. Further, behavior studies revealed anxiety-like behaviors and social deficits in both treatment groups that persisted into the F2 generation. Collectively, our studies reveal a new line of investigation on the potential risks associated with oxy use during and after pregnancy, specifically the disruption of neurodevelopment and the intergenerational impact on behavior

Kappa opioid receptor modulation of neurotransmission in the amygdala

Kappa opioid receptors (KORs) and their endogenous ligand, dynorphin, have been implicated in a variety of neuropsychiatric disorders including anxiety and alcohol addiction. Here, we demonstrate the function and role of KORs in the bed nucleus of the stria terminalis (BNST), a key brain region involved in these diseases. In the first series of experiments, we show that KORs in the BNST inhibit glutamate release via a presynaptic, p38- and calcium- dependent mechanism. This synaptic inhibition is specific to basolateral amygdala (BLA) inputs, a previously identified key pathway in rodent models of anxiety-related behaviors. Additionally, we identified a frequency-dependent, light-evoked, local dynorphin-induced heterosynaptic plasticity of glutamate inputs to the BNST, allowing for optogenetic control of peptidergic transmission. We found differential KOR modulation of the BLA-BNST input based on the postsynaptic neurochemical identity. Collectively, these results demonstrate a local dynorphin- and KOR- dependent mechanism of inhibiting an anxiolytic pathway, providing a discrete therapeutic target for treatment of anxiety disorders. In the second series of experiments, we show that following chronic intermittent iv ethanol exposure (CIE), a model of alcohol exposure, KORs differentially modulate glutamate and GABA in the BNST. KOR inhibition of electrically-evoked glutamate inputs is decreased, while KOR inhibition of electrically-evoked GABA inputs is increased, despite overall properties of glutamatergic and GABAergic transmission remaining intact. This change in synaptic physiology is complementary to a KORdependent behavioral change: mice exposed to ethanol show decreased social preference as compared to air exposed, an effect which is partially rescued by systemic pre-administration of the KOR antagonist JDTic. Taken together, these experiments demonstrate KOR-dependent alterations of synaptic transmission in the BNST following CIE, making the BNST a potential site of action for KOR targeted therapies related to alcohol and anxiety. Jointly, these experiments expand our understanding of how key peptidergic transmission in the extended amygdala can play a role in anxiety and addiction related diseases.Doctor of Philosoph