Steven A. Thomas - US grants

Alterations in adrenergic transmission occur in anxiety and depressive disorders, and in Alzheimer's disease. In addition, 6 human patients with autonomic failure have been found to be congenitally deficient in the enzyme dopamine (beta-hydroxylase (DBH). They are unable to synthesize the adrenal hormone epinephrine (E) and the adrenergic neurotransmitter norepinephrine (NE). Despite the absence of these transmitters in their brains, these patients have normal mood and mental function. This is surprising given the postulated roles for NE in learning and memory, arousal and attention, and fear and anxiety. Dopamine (DA), the precursor of NE, is stored in and released from the adrenergic terminals of these patients. A hypothesis that could account for normal CNS function in these patients is that their brains develop to utilized DA as the adrenergic transmitter, either by activating dopaminergic or adrenergic receptors. We propose to examine the mouse model (dbh-/-) of human DBH-deficiency to investigate mechanisms that may arise during postnatal development to compensate for the absence of NE. We will determine the number and location of adrenergic cell bodies and terminals in dbh-/- and control mice by several histochemical techniques. We will test for elevated DA receptor expression due to the release of DA in novel locations during development. Because NE is absent, we will also quantitate adrenergic receptor expression. We will characterize the formation of the cerebellum, which has been implicated as being dependent on NE for their proper development. Importantly, any changes we observe in the dbh-/- mice may be due to either the absence of NE or the presence of DA in the adrenergic vesicles. We will create a new mouse model (th-/- /dat-th+/-) to distinguish these etiologies, and to identify any developmental changes due to the loss of NE that are masked by the presence of DA in the dbh-/- mice. Finally we will determine the permanence of phenotypes due to the absence of NE by restoring NE in the mutant mice using amino acid precursors. Results from these studies will determine what are the critical roles of adrenergic signaling in vivo during postnatal neural development, and whether DA can substitute for NE in the CNS.

DESCRIPTION (provided by applicant): We propose to examine the role of adrenergic signaling in synaptic plasticity, learning and memory using mouse molecular genetics. Specifically, we have created mice that are unable to synthesize norepinephrine (NE) and epinephrine due to a targeted disruption of the dopamine B-hydroxylase (Dbh) gene. Homozygotes (Dbh-/-) completely lack NE; however NE can be restored rapidly in vivo and in vitro using the synthetic amino acid precursor of NE (DOPS). This model has several advantages over prior pharmacologic approaches, including completeness of effect, specificity for NE, and reversibility. Prior studies using various techniques have often generated conflicting results with regard to the roles of NE in synaptic plasticity, learning and memory. Some studies have suggested a role for NE in the formation of emotional (aversive) memories. To test this possibility, we have begun to characterize the ability of Dbh-/- mice to learn and remember an aversive event using fear conditioning. Preliminary results indicate a specific deficit in the consolidation of contextual but not cued memory, suggesting hippocampal function may be altered in the absence of NE. For this reason we have begun to examine synaptic plasticity in the hippocampus. Preliminary results from these studies suggest that the late phase of long-term potentiation in region CAl is deficient. Because other studies have suggested a critical role of synaptic plasticity in region CAl for learning and memory, we propose to examine whether intracellular signaling pathways implicated in learning and memory are altered in region CAl following stimuli that elicit the late phase of LTP in vitro, and following fear conditioning in vivo. Finally, we will test whether compensation for the absence of NE occurs during development, and whether dopamine released from the adrenergic terminals of Dbh-/- mice can substitute at least partially for NE. These goals will be achieved through the use of a second mouse model (Th-/-/Dat-Th+/-) that should lack DA as well as NE in the adrenergic neurons specifically. Some of these mice will be raised with NE present (by supplying L-DOPA pre- and postnatally). L-DOPA will then be withdrawn in half prior to using the mice in the above studies.

DESCRIPTION (provided by applicant): In our desire to better understand the roles of central adrenergic signaling and their relation to neuropsychiatric conditions such as depression, we propose to create a system in mice for the inducible inactivation of genetically defined neurons. While generally applicable, this system will be applied to the study of adrenergic and dopaminergic neurons. Such a technique will provide a potent method for determining the functions of these neurons in vivo, either during development or in the adult. We propose to inactivate neurotransmitter release through proteolytic cleavage of one of the proteins (SNAREs) essential for synaptic vesicle fusion with the plasma membrane. We will achieve this by developing transgenic lines of mice that can express the proteolytically active light chain of either a C. tetani (TeNT) or C. botulinum (BoNT/E) neurotoxin. Expression of the mouse codon-optimized neurotoxin transgenes will be regulated by the use of an inducible promoter sensitive to the presence of a transactivation factor in combination with a small molecule inducer drug. Cell-specific expression will be achieved by targeting insertion of the codon-optimized transactivator gene and a mammalian internal ribosome entry site to the 3'-untranslated region of a gene that defines the neurons of interest. This approach should permit true expression of the transactivator without disrupting expression of the endogenous gene. Neurons would be inactivated following administration of the inducer drug. The strengths of this system are likely to be the specificity of the neurons inactivated (due to the targeting scheme), the completeness of inactivation (due to the potency of the neurotoxin), and the stability of the temporally controlled inactivation (due to the half-life of the neurotoxin's effects). Our proposed technique will be applicable to any set of neurons that can be genetically defined. We will test this system in dopamine (DA) neurons because their inactivation is predicted to result in the Parkinsonian phenotypes of hypomotility and hypophagia. Block of DA release will be assessed by microdialysis in vivo and cyclic voltammetry in vitro. The system will also be applied to the study of adrenergic neurons, using similar techniques to document inducible and reversible inactivation of neurotransmitter release. The approach will permit the study of animals before and after inactivation of these neurons, providing a valuable internal control. This technique will complement other techniques used to study the same neurons, such as their genetic ablation or the genetic elimination of a single neurotransmitter from those neurons. Use of this technique will provide a model for diseases such as Parkinson's or depression in which a neuronal population is hypothesized to become dysfunctional. In general, the development of this technique should provide a powerful tool for the dissection of how the brain operates.

DESCRIPTION (provided by applicant): The goal of this proposal is to better understand the molecular mechanisms by which the retrieval of declarative/episodic memory occurs. The proposal focuses on the time-limited role that norepinephrine (NE) and [unreadable]1-adrenergic signaling play in the hippocampus during memory retrieval. The first aim will determine in which hippocampal cell(s) [unreadable]1 signaling acts to promote memory retrieval. Contextual fear, which depends to NE and [unreadable]1 signaling, will be used to assess hippocampus-dependent memory. Cued (tone) fear, which does not depend on NE or [unreadable]1 signaling, will be used to test whether manipulations affect performance or memory. Imaging of immediate-early gene induction in mice will be employed as a marker of neuronal activity following exposure to salient and neutral contexts, [unreadable]1 signaling in the mice will be manipulated pharmacologically and genetically so that it is blocked or activated either, systemically, only in the dorsal hippocampus, or in specific subfields of the hippocampus. Because [unreadable]1 signaling activates the cAMP / protein kinase A pathway, the second aim will determine whether this pathway is required for memory retrieval. Dorsal hippocampal infusions of agents that block this pathway will be performed in control mice, and infusions of agents that stimulate this pathway will be performed in mutant mice that lack [unreadable]1 signaling. The relationship between NE and activation of extracellular signal-regulated kinase and phosphatidylinositol 3- kinase, which are also required for retrieval, will be determined. Further, one of the most prominent physiological effects of [unreadable]1 signaling in the hippocampus is reduction of the slow afterhyperpolarization (sAHP) that mediates accommodation of firing. The third aim will determine whether calcium influx through voltage-dependent calcium channels influences retrieval, and whether pharmacologic block of the sAHP rescues retrieval in mutant mice lacking [unreadable]1 signaling. This aim will also determine whether there is a transient reduction in the sAHP that can be observed in brain slices of mice after fear conditioning but not pseudoconditioning and, if so, whether the reduction depends on [unreadable]1 signaling. Finally, a key hypothesis explaining why NE is required for some but not all memory retrieval will be tested in the fourth aim. Relevance: Dysfunction of adrenergic signaling may contribute to symptoms of depression and post- traumatic stress disorder that include difficulties with memory retrieval in the former and unwanted, intrusive retrieval of traumatic memories in the latter. Results from this proposal may also be relevant to potential cognitive side effects that might arise when treating heart failure, hypertension and performance anxiety with drugs that block [unreadable] receptors. Finally, results should also be relevant to understanding how dysregulation of neuronal Ca++ homeostasis in the elderly may lead to memory deficits.

DESCRIPTION (provided by investigator): The goal of this proposal is to advance our ability to inducibly manipulate gene expression within subsets of neurons. Four neurotransmitter systems have been chosen for this proposal based on their having been implicated in numerous neurophysiologic and behavioral processes, as well as disorders of those processes. The four are the adrenergic (norepinephrine and epinephrine), dopaminergic, serotonergic and orexinergic systems. Because of the unique ability to create gene-targeted mice among mammals, mice will be the model organism employed. One goal is to inducibly activate, inactivate or modify endogenous genes that have been endowed with unique loxP sites that are recognized by bacteriophage P1 Cre recombinase (Cre). The main approach for achieving this will be the neurotransmitter-specific expression of the tamoxifen-inducible Cre fusion protein, CreERT2, that is fused to a modified estrogen receptor ligand-binding domain (ERT2). Because there may be occasions when using tamoxifen should be avoided due to effects on endogenous estrogen receptor signaling, a second approach for achieving inducible Cre activity will also be pursued. For this approach, the tetracycline-inducible transactivator (tTA2) and reverse tTA (rtTA2S-M2) will be expressed specifically in each neurotransmitter system. When crossed with previously characterized tetO-Cre mice, the transactivator (TA) mice will induce the neurotransmitter-specific expression of Cre either in the absence (tTA) or in the presence (rtTA) of antibiotic. In addition, the TA mice will permit the induction of any other Ptet-cDNA transgene in a neurotransmitter-specific fashion. To obtain neurotransmitter-specific expression, genes that uniquely define these neurotransmitter systems will be utilized: for the adrenergic system - dopamine 2- hydroxylase, for the dopaminergic system - dopamine transporter, for the serotonergic system - tryptophan hydroxylase 2, and for the orexinergic system - orexin. CreERT2 and the TAs will be targeted to the 3'- untranslated region of each gene via homologous recombination in embryonic stem cells. To achieve bicistronic expression, CreERT2 and the TAs will be preceded by an internal ribosome entry site. Reporter mice for Cre activity will be used to correlate temporal and spatial expression patterns of Cre with those for the targeted endogenous gene. Finally, the utility of the CreERT2 and TA mice will be demonstrated by crossing them with mice harboring a floxed tyrosine hydroxylase gene for inducible transmitter depletion. PUBLIC HEALTH RELEVANCE The study of these four neurotransmitter systems via inducible changes in gene expression that are specific to each system is highly relevant to the understanding of fundamental neurophysiological and behavioral processes such as sleep, arousal and attention, motivation and reward, and learning and memory, as well as disorders related to these processes that include narcolepsy, drug abuse, anxiety, depression and post- traumatic stress disorder. It is expected that the proposed mouse models will permit significant insights into the etiology, identification and treatment of these disorders.

DESCRIPTION (provided by applicant): Stress is an important factor in either eliciting or exacerbating many neuropsychiatric disorders. The goal of this proposal is to better understand the molecular mechanisms by which stress affects CNS physiology and behavior. Such knowledge will ultimately lead to more effective measures for preventing the deleterious effects of stress on behavior and health. A prominent mediator of stress is the neuroendocrine system that includes glucocorticoid signaling. Classic glucocorticoid signaling modulates gene expression via steroid hormone (glucocorticoid and mineralocorticoid) receptor transcription factors. This genomic mechanism is partly responsible for stress effects observed over hours to days. Acute stress effects occurring over minutes or longer can also be mediated by glucocorticoids. These effects of stress are thought to occur through non- genomic mechanisms, however, the identity of these mechanisms remains largely unknown. In a recent study examining the acute effects of stress on hippocampus-dependent memory retrieval, we identified the ¿22-adrenergic receptor (¿22AR) as a critical mediator of the impairing effects of stress and glucocorticoids. Based on these and other observations, we now hypothesize that there is a specific interaction between either the glucocorticoid receptor (GR) or its ligand (cort) and ¿22AR, and that many of the acute effects of glucocorticoids depend on this interaction and the downstream signaling activated by ¿22AR. Here we propose to identify additional systems in which this interaction is relevant to the stress response, characterize the interaction betwee cort and ¿22AR at the molecular level, and define the downstream signaling events that mediate this coincident signaling. Results from these aims will identify specific molecular mechanisms for the non-genomic effects of glucocorticoids and the acute effects of stress that can persist under conditions of chronic stress. Because stress can impair normal CNS physiology and behavior, as well as exacerbate many neurologic and psychiatric disorders, understanding the mechanisms that underlie stress effects may aid in their prevention.

Project Summary: Convergent evidence from clinical and preclinical studies have highlighted the deleterious effects of aberrant accumulation of the amyloid beta (A?42) peptide, from neuronal dysfunction to behavioral and psychological manifestations of disease. Compelling evidence from recent clinical studies reveal that elevated levels of A?42 peptides are associated with anxiety and depression symptoms in middle-aged and older non-demented adults, as well as those with mild cognitive impairment (MCI) or in early stages of Alzheimer's disease (AD). Stress is a risk factor for psychiatric disease, as well as for developing AD. Further, it has been demonstrated that amplification of the stress system disrupts cellular and molecular processes at the synapse, promoting the production and secretion of A?42 peptides. The norepinephrine (NE)- locus coeruleus (LC) system is a stress-responsive neurocircuit implicated in stress-related psychiatric disorders, and in the etiology and progression of AD. Numerous studies in the literature support a role for NE as a regulator of A?42 peptide levels, as there are cellular mechanisms by which NE can influence both the production and the degradation and clearance of A?42 peptides. However, there exist significant gaps in knowledge regarding how NE regulates A?42 peptide levels. Early dysregulation of the NE system is thought to underlie the behavioral and psychiatric symptoms of dementia (BPSD), which are often the first symptoms observed in MCI patients that later progress to AD. Because NE can exert profound effects on the production and clearance of A?42 peptides, the dysregulation of NE under conditions of chronic stress, psychiatric disease, or LC degeneration may directly contribute to aberrant accumulation of A?42 peptides. Thus, targeting the LC-NE system may be a novel avenue to modulate A?42 levels in early stages to slow or halt the progression of disease. The proposed studies aim to investigate the role of NE in regulating amyloid beta A?42 peptide levels. We will build on our recent published work showing anatomical localization of A?42 peptides to LC somatodendritic processes and to noradrenergic axon terminals of the naïve male and female rat medial prefrontal cortex (mPFC), a region important for the integration of the stress response and a major projection site of LC neurons. We also examined consequences of NE depletion on A?42 peptides using genetic deletion of D?H, the NE synthesizing enzyme. Results showed that NE depletion significantly decreased levels of A?42 peptides as measured by ELISA. Moreover, in a model that utilizes increased excitatory input to augment LC activity, simulating chronic stress, there is increased localization of A?42 to somatodendritic processes of the LC. These data have informed our current approach to examine dynamic regulation of A?42 levels following LDOPS administration in D?H knockout mice and in D?H-CRE x floxed Adra2a mice, a more direct model of increased NE transmission. Both males and females will be examined to probe for potential sex differences in mechanism of action of NE on A?42 peptide levels.