Evidence from previous studies on the multiple memory systems model suggests that specific brain regions cooperate and compete to mediate the navigational strategies used to locate a goal in a spatial environment. Specifically, the cholinergic system within these discrete brain regions plays a key role in balancing this mediation such that acetylcholine release, genomic changes, and receptor regulation at cholinergic synapses are altered following learning and subsequent memory consolidation. Based on previous findings, we proposed to test learning-induced changes in muscarinic receptor binding expression in adult male rats following training on a water maze task guided either by a cue proximal to the escape platform (stimulus-response strategy), by cues surrounding the maze (place strategy) or by alternating between the two strategies (strategy-switching). The primary findings of the current study demonstrate that adult male rats that navigated to an escape platform guided by cues surrounding a water maze (place-trained) learned the task at a significantly slower rate than males that were guided by a cue proximal to the platform (stimulus-response-trained) or males that were required to switch strategies on alternating days. Additionally, males that were required to switch strategies over alternating days expressed higher ratios of muscarinic binding in the hippocampus relative to the striatum compared to place-trained rats, stimulus-response-trained rats, and swim-only controls. These results indicate that the use of a place learning strategy slows acquisition of a water maze task while the requirement to switch strategies as the demands of the task change over days engages the cholinergic system in the hippocampus most heavily. Taken together, the results from the current study further confirm the involvement of cholinergic function in regulating the balance between multiple memory systems. / acase@tulane.edu

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

We investigated the role of muscarinic receptor subtypes at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone, in mevinphos (Mev) intoxication. Adult Sprague-Dawley rats anesthetized by pentobarbital (45 mg/kg) and maintained by propofol (30 mg/kg/h) were used. Co-microinjection bilaterally of Mev (10 nmol) and artificial cerebrospinal fluid (aCSF) into the RVLM resulted in an increase (Phase I) followed by a decrease (Phase II) in the power density of the vasomotor components of systemic arterial pressure spectrum, our experimental index for sympathetic vasomotor tone. These changes in sympathetic vasomotor outflow in both phases of Mev intoxication were significantly and dose-dependently reduced on co-microinjection of Mev and the M2 subtype of muscarinic receptor (M2R) antagonist methoctramine (0.5 or 1 nmol) or M4R antagonist tropicamide (0.5 or 1 nmol). On the other hand, the M1R antagonist pirenzepine (0.5 or 1 nmol) or M3R antagonist 4-DAMP (0.5 or 1 nmol) was ineffective. Western blot analysis further revealed that the increase in NOS I protein levels at the RVLM during Phase I Mev intoxication or the augmented level of NOS II during both phases were significantly blunted on co-microinjection bilaterally of Mev and methoctramine (1 nmol) or tropicamide (1 nmol) into the RVLM. Pirenzepine (1 nmol) or 4-DMAP (1 nmol) was again ineffective. We conclude that both M2R and M4R subtypes in the RVLM may be involved in Mev intoxication. Whereas the prevalence of NOS I over NOS II at the RVLM during Phase I results in sympathoexcitation, sympathoinhibition induced by NO from NOS II in the RVLM is primarily involved in Phase II Mev intoxication.

Knockdown of the M5 acetylcholine muscarinic receptor in the ventral tegmental area (VTA) reduces brain-stimulation reward sensitivity in rats. Knockout (KO) of the M5 receptor in mice reduces morphine-induced dopamine efflux, locomotion, conditionedplace- preference, and mating-induced 30-110 kHz ultrasonic vocalizations (USVs). The GFP-labeled M5 receptor gene was transfected using a Herpes simplex virus either into the VTA or 0.2-0.7 mm posterior in the medial tegmentum (MT) of male M5 KO mice. HSV-M5-GFP transfection in VTA fully restored mating-induced USVs and augmented morphine-induced locomotion and stereotypy consistent with activation of DA neurons by M5 receptors. HSV-M5-GFP transfection sites in the MT inhibited USVs and morphine-induced locomotion presumably through inhibition of DA neurons. Putative transfection of M5 in GABA neurons of the rostromedial tegmental nucleus (RMTg) or 5HT neurons of the median raphe (mR) may explain this inhibition. Therefore, HSV-M5- GFP transfection in the VTA enhances DA-mediated behaviours while MT transfections inhibits these behaviours.

Knockdown of the M5 acetylcholine muscarinic receptor in the ventral tegmental area (VTA) reduces brain-stimulation reward sensitivity in rats. Knockout (KO) of the M5 receptor in mice reduces morphine-induced dopamine efflux, locomotion, conditionedplace- preference, and mating-induced 30-110 kHz ultrasonic vocalizations (USVs). The GFP-labeled M5 receptor gene was transfected using a Herpes simplex virus either into the VTA or 0.2-0.7 mm posterior in the medial tegmentum (MT) of male M5 KO mice. HSV-M5-GFP transfection in VTA fully restored mating-induced USVs and augmented morphine-induced locomotion and stereotypy consistent with activation of DA neurons by M5 receptors. HSV-M5-GFP transfection sites in the MT inhibited USVs and morphine-induced locomotion presumably through inhibition of DA neurons. Putative transfection of M5 in GABA neurons of the rostromedial tegmental nucleus (RMTg) or 5HT neurons of the median raphe (mR) may explain this inhibition. Therefore, HSV-M5- GFP transfection in the VTA enhances DA-mediated behaviours while MT transfections inhibits these behaviours.

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D.)--Tufts University, 1998. / Adviser: Barry A. Trimmer. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 92-105). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.