Doctor of Philosophy(PhD) / This thesis addresses the neurochemistry and function of specific nuclei in the autonomic nervous system that are crucial mediators of cardiorespiratory regulation. The primary aim is to build on previous knowledge about muscarinic cholinergic mechanisms within cardiorespiratory nuclei located in the ventrolateral medulla oblongata. The general focus is characterisation of gene expression patterns of specific muscarinic receptor subtypes in central nuclei involved in blood pressure control and respiratory control in normal rats. The findings were subsequently extended by characterisation of muscarinic receptor gene expression patterns in 1) a rat model of abnormal blood pressure control (hypertension) (Chapter 3) 2) a rat model of cholinergic sensitivity (Chapter 5) 3) the rat ventral respiratory group (Chapter 6) The results of a series of related investigations that ensued from the initial aims more finely characterise the neurocircuitry of the ventrolateral medulla, from a specifically cholinoceptive approach. All five muscarinic receptor subtypes are globally expressed in the ventrolateral medulla but only the M2R mRNA was significantly elevated in the VLM of hypertensive animals compared to their normotensive controls and in the VLM of animals displaying cholinergic hypersensitivity compared to their resistant controls. Surprisingly, M2R mRNA is absent in catecholaminergic cell groups but abundant in certain respiratory nuclei. Two smaller projects involving gene expression of other neurotransmitter / neuromodulators expressed in cardiorespiratory nuclei were also completed during my candidature. Firstly, the neurochemical characterisation of enkephalinergic neurons in the RVLM, and their relationship with bulbospinal, catecholaminergic neurons in hypertensive compared to normotensive animals was carried out (Chapter 4). A substantial proportion of sympathoexcitatory neurons located in the RVLM were enkephalinergic in nature. However, there was no significant difference in preproenkephalin expression in the RVLM in hypertensive compared to normotensive animals. Secondly, the identification and distribution of components of the renin-angiotensin aldosterone system (RAAS) within the brainstem, and differences in gene expression levels between hypertensive and normotensive animals was also investigated. The RAAS data was not included in this thesis, since the topic digresses substantially from other chapters and since it is published (Kumar et al., 2006). The mRNA expression aldosterone synthase, mineralocorticoid receptor (MR1), 12-lipoxygenase (12-LO), serum- and glucocorticoid- inducible kinase and K-ras) were found to be present at all rostrocaudal levels of the ventrolateral medulla. Expression of MR1 mRNA was lower in the RVLM of SHR compared with WKY rats and 12-LO mRNA levels were lower in the CVLM in SHR compared with WKY rats. Otherwise, there was no difference in gene expression level, or the method of detection was not sensitive enough to detect differences in low copy transcripts between hypertensive and normotensive animals.

The hypothalamic paraventricular nucleus (PVN) is known to be a major integrative region within the forebrain. It is composed of functionally different subgroups of neurons, including the parvocellular neurons that project to important autonomic targets in the brainstem e.g. the rostral ventrolateral medulla (RVLM) and the intermediolateral cell column (IML) of the spinal cord, where the sympathetic preganglionic motor-neurons are located. These regions are critical in cardiovascular regulation; hence, these projections are likely to mediate the effects of the PVN on sympathetic nerve activity and hence may contribute to the cardiovascular changes induced by physiological stimuli such as elevations in body temperature. The neurotransmitter such as nitric oxide (NO) is important in cardiovascular regulation and it is now emerging as a major focus of investigation in thermoregulation. One of the most striking accumulations of NO containing-neurons is in the PVN where it appears to be playing an important role in cardiovascular regulation and body fluid homeostasis. The results of the work show; 1. That spinally-projecting and nitrergic neurons in the PVN may contribute to the central pathways activated by exposure to a hot environment. 2. Suggests that nitrergic neurons and spinally- projecting neurons in the brainstem may make a small contribution to the central pathways mediating the reflex responses initiated by hyperthermia. 3. The present study also illustrates that these PVN neurons projecting to the RVLM may make a smaller contribution than the spinal-projecting neurons in the PVN to the cardiovascular responses initiated by heat. 4. The results of my studies showed that the microinjection of muscimol to inhibit the neuronal activity in the PVN abolished the reflex decrease in renal blood flow following an elevation of core body temperature. In addition, this effect was specific to the PVN, since microinjections of muscimol into areas outside the PVN were not effective. These findings demonstrate that the PVN is critical for this reflex cardiovascular response initiated by hyperthermia. In conclusion, PVN is critical for the reflex decrease in renal blood flow during elevations in core body temperature. We hypothesise that projections from the PVN to the spinal cord and the RVLM contribute to the reflex cardiovascular responses. Additionally, nitrergic neurons in the PVN may contribute but the physiological role of those neurons in the reflex responses elicited by hyperthermia needs to be investigated.

How is the brain capable of mediating pain relief via the mind alone? Placebo analgesia is just such a case, wherein an inert substance yields relief from a number of pain inducing stimuli. Scholars typically separate several factors thought to contribute to the placebo effect into psychological and neurobiological influences. Psychological mechanisms include expectation and conditioning of analgesic effects, while neurobiological mechanisms implicate the opioidergic descending pain system. The current paper proposes an integrative model in which the rostral anterior cingulate cortex (rACC), implicated in cognitive-affective modulation, receives goal-directed input (i.e., expected pain relief) from the prefrontal cortex. As the rACC processes the cognitive difference between expected and actual pain, it recruits a critical descending pain pathway by means of modulating the periaqueductal gray area (PAG). The PAG is a key relay station that connects to other endogenous subsystems of opioidergic pain relief. Whether the rACC and its connection to the PAG are necessary for the placebo effect is a question future research will have to address.

Morphine is the most widely prescribed opiate for alleviation of persistent pain; however, it is becoming increasingly clear that morphine is less potent in women compared to men. Morphine primarily binds mu opioid receptors, which are densely localized in the midbrain periaqueductal gray (PAG). Anatomical and physiological studies conducted in the 1960s identified the PAG, and its projections to the rostral ventromedial medulla (RVM) and spinal cord dorsal horn, as an essential neural circuit mediating opioid-based analgesia. Remarkably, the majority of studies since then were conducted in males with the implicit assumption that this circuit was the same in females; this is not the case. It is now well established that morphine produces greater analgesia in males compared to females in a wide range of vertebrates, however, the mechanism(s) driving this sex difference is not clear. Our recent studies indicate that two factors appear to be contributing to the sexually dimorphic effects of morphine. First, there are sex differences in the anatomy and physiology of the descending inhibitory pathway on which morphine acts to produce analgesia. Specifically, the projections from the PAG to the RVM are sexually dimorphic and activated to a greater degree by both inflammatory pain and systemic morphine in males. In the absence of pain, the PAG-RVM circuit is activated to a greater degree in males compared to females, while this activation steadily declines during the development of tolerance in males only. We also have evidence of a sexually dimorphic expression of mu opioid receptor within the PAG that appears to contribute to sex differences in morphine potency. Microinjection of morphine directly into the PAG produces significantly greater analgesia in males, indicating that the PAG is sufficient for eliciting this sexually dimorphic behavior. Furthermore, mu opioid receptor-expressing PAG neurons are necessary for eliciting a sexually dimorphic response to morphine as lesioning mu opioid receptor-expressing neurons attenuates analgesia in males only. Together, these data indicate that the PAG-RVM pathway and mu opioid receptor expression in the PAG is sexually dimorphic and provides a primary mechanism for sex differences in morphine potency.

An individual who has sustained either irreversible cessation of circulatory and respiratory functions, or irreversible cessation of all functions of the entire brain, including the brain stem is dead. Brain death is currently the legal definition of death in many countries. Many people confuse brain death with vegetative states. Patients in a vegetative state are unaware of themselves or their environment. Both patients with brain death and those in a vegetative state are unconscious following severe brain injury. Unlike the brain death, vegetative patient¡¦s vital vegetative functions, such as cardiac action, respiration, and maintenance of blood pressure are preserved. The rostral ventrolateral medulla (RVLM) is the origin of a ¡§life-and-death¡¨ signal identified from systemic arterial blood pressure spectrum and intimately related to brain death. Based on the animal models of brain death, the observations that the power density of the vasomotor components of SAP signals undergoes both augmentation and reduction during the progression towards death strongly suggest that both ¡¥¡¥pro-life¡¦¡¦ and ¡¥¡¥pro-death¡¦¡¦ programs are present in the RVLM. A number of those ¡¥¡¥pro-life¡¦¡¦ and ¡¥¡¥pro-death¡¦¡¦ programs in the RVLM has now been identified along with their cellular and molecular mechanisms. As the neural substrate that is intimately related to brain death, one unresolved question is whether the proteome expressed in RVLM is unique. To address the issue, we used the cerebral cortex, which is defunct under persistent vegetative state for comparison. 2-DE electrophoresis, MALTI-TOF MS and peptide mass fingerprinting were used for investigation the proteomic difference between the rat RVLM and cerebral cortex. Quantitative analysis on silver-stained 2-DE electrophoresis gels revealed highly comparable distribution patterns of these protein spots for both brain regions, with 85.9 ¡Ó 2.3 % of protein spots from RVLM matched those from cerebral cortex. According to the protein function, these proteins were classed into binding activity, chaperone, antioxidant, oxidoreductase, ubiquitin- proteasome system, cell cycle, catalytic activity, glycolysis, tricarboxylic acid cycle, electron transport chain, endocytosis and exocytosis, structural molecular function, apoptosis, transport, differentiation and neurogenesis, protein biosynthesis, cell junction, and others. We found that a group of antioxidant proteins, including members of the peroxiredoxin (Prx) family (Prx-1, Prx-2, Prx-5, and Prx-6), thioredoxin and mitochondrial manganese superoxide dismutase exhibited significantly higher protein and mRNA expression levels in RVLM when compared to cerebral cortex. Tissue oxygen, ATP contents and ATP synthase subunits alpha and beta in RVLM were also significantly elevated. On the other hand, protein and mRNA levels of members of the ubiquitin-proteasome system, including proteasome subunit alpha type-1, ubiquitin, uniquitin-conjugating enzyme E2 N, ubiquitin carboxyl-terminal hydrolase isozyme L1 and L3, were comparable in both brain regions. The presence of higher levels of tissue oxygen and ATP synthase subunits in RVLM, leading to augmented ATP production, provides a cellular safeguard mechanism to reduce the possibility of irreversible reduction in intracellular ATP contents that precipitate brain death. By manifesting an augmented tissue oxygen and metabolic energy production, RVLM is more prone to oxidative stress. We conclude that a significantly elevated level of antioxidant proteins and mRNA in RVLM is consistent with the exhibition of higher tissue oxygen tension and metabolic energy production in this neural substrate, which together constitute a safeguard mechanism against brain death.

Brain stem cardiovascular regulatory dysfunction during brain stem death is underpinned by an upregulation of nitric oxide synthase II (NOS II) in rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from blood pressure of comatose patients that disappears before brain stem death ensues. At the same time, the ubiquitin-proteasome system (UPS) is involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain stem death by engaging in both synthesis and degradation of NOS II in RVLM. In a clinically relevant experimental model of brain stem death using Sprague-Dawley rats, pretreatment by microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) antagonized the hypotension and reduction in the life-and-death signal elicited by intravenous administration of Escherichia coli lipopolysaccharide (LPS). On the other hand, pretreatment with an inhibitor of ubiquitin-recycling or UCH-L1 potentiated the elicited hypotension and blunted the prevalence of the life-and-death signal. Real-time polymerase chain reaction, Western blot, electrophoresis mobility shift assay, chromatin immunoprecipitation and co-immunoprecipitation experiments further showed that the proteasome inhibitors antagonized the augmented nuclear presence of NF-£eB or binding between NF-£eB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated I£eB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-£eB in RVLM. In animals pretreated with UCH-L1 inhibitor and died before significant increase in nos II mRNA occurred, NOS II protein expression in RVLM was considerably elevated. We conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain stem death by engaging in both synthesis and degradation of NOS II at RVLM. Our results provide information on new therapeutic initiatives against this fatal eventuality.

In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. Ubiquitin is best known for its role in targeting proteins for degradation by the proteasome. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is found specifically in central and peripheral neurons, and is responsible for the removal of small peptide fragments from the ubiquitin chain and for co-translational processing of ubiquitin gene products to generate free monomeric ubiquitin. In response to extreme conditions, cells exhibit an up-regulation of heat shock protein (HSP) expression, which contributes to repair and protective mechanisms. Within the HSP family, HSP70 is the major inducible member that protects against cell death. Based on the pharmacologic property of organophosphates as an inhibitor of cholinesterase, it is generally contended that manifestations of organophosphate poisoning, including secretion and muscle fasciculation, stupor, cardiopulmonary collapse, respiratory failure, coma or death, result from accumulation of, and over-stimulation by acetylcholine at peripheral of central synapses. One approach in furthering our understanding on organophosphate poisoning is delineation of its potential protective mechanisms. In this regard, the information on the cellular and molecular mechanisms that underlie organophosphate poisoning has received attention. Our laboratory demonstrated previously that a crucial brain site via which mevinphos (Mev), an organophosphate insecticide of the P=O type, acts is the rostral ventrolateral medulla (RVLM), the medullary origin of premotor sympathetic neurons that are responsible for the maintenance of vasomotor tone. The phasic changes in cardiovascular events over the course of acute Mev intoxication also parallel fluctuations of the ¡§life-and-death¡¨ signals that emanate form the RVLM. Based on a rat model of organophosphate poisoning that provides continuous information on cellular and molecular mechanisms in the RVLM, the present study was undertaken to evaluate whether changes in protein level of UCH-L1 or HSP70 are associated with death arising from Mev intoxication. We also evaluated the efficacy of both of them in the neuroprotection against fatality during Mev intoxication. The first part of this study investigated whether UCH-L1 plays a neuroprotective role at the RVLM, where Mev acts to elicit cardiovascular toxicity. In Sprague-Dawley rats maintained under propofol anesthesia, Mev (960 µg/kg, i.v.) induced a parallel and progressive augmentation in UCH-L1 or ubiquitin expression at the ventrolateral medulla during the course of Mev intoxication. The increase in UCH-L1 level was significantly blunted on pretreatment with microinjection bilaterally into the RVLM of a transcription inhibitor, actinomycin D (5 nmol) or a translation inhibitor, cycloheximide (20 nmol). Compared to artificial cerebrospinal fluid (aCSF) or sense uch-L1 oligonucleotide (100 pmol) pretreatment, microinjection of an antisense uch-L1 oligonucleotide (100 pmol) bilaterally into the RVLM significantly increased mortality, reduced the duration of the phase I (¡§pro- life¡¨ phase), blunted the increase in ubiquitin expression in ventrolateral medulla, and augmented the induced hypotension in rats that received Mev. The second part of this study investigated whether HSP70 plays a neuroprotective role at the RVLM. Intravenous administration of Mev (960

The rostral ventrolateral medulla (RVLM) regulates vasomotor activity via sympathoexcitation and sympathoinhibition to maintain blood pressure. Nitric oxide synthesized by nitric oxide synthase (NOS) I and NOS II within RVLM is responsible for sympathoexcitation and sympathoinhibition respectively. In our previously study, under physiological condition RVLM neurons contain both NOS I and NOS II protein, and NOS III protein is expressed mainly on blood vessels. Under Mevinphos (Mev) intoxication, our previously study demonstrates that the expression of RVLM NOS I and II mRNA or protein are both increased under Mev intoxication phase I, and NOSII mRNA or protein are further increased under Mev intoxication phase II. On the other hand, in rat central nervous system, about 65% of total cells are glial cells, including astrocytes, microglia and oligodendrocytes. However, the expressions of NOS isoforms in RVLM glial cells still need to be determined. We used double immunofluorescence staining and confocal microscopy to investigate the distributions of NOS isoforms protein in RVLM neurons and glial cells under physiological condition and under pathological condition using Mev intoxication as our model. We further compared the distributions of NOS isoforms in RVLM neurons and glial cells under physiological or pathological conditions. The confocal images indicate that NOS I protein reactivity co-localized with neurons and microglia in the RVLM. NOS II protein reactivity co-localized with neurons, astrocytes and microglia. NOS III protein reactivity co-localized with blood vessels and microglia. The distributions of NOS isoforms protein reactivity in RVLM neurons and glial cells under Mev intoxication are the same as under physiological condition. Furthermore, the expressions of NOS I protein within neurons or microglia and NOS II in neurons, astrocytes or microglia are progressively increased under Mev intoxication. On the other hand, the expression of NOS III within microglia under Mev intoxication was similar to physiological condition. The population of NOS I-positive neurons or microglia, and NOS II-positive neurons, astrocytes or microglia increased under Mev intoxication. However the population of NOS III-positive microglia decreased under Mev intoxication. These results indicate that within RVLM, the distributions of NOS I are in neurons and microglia; NOS II are in neurons, astrocytes and microglia; NOS III are in blood vessels and microglia. We suggest that under Mev intoxication, the source of up-regulated NOS I protein includes neurons and microglia; and the up-regulated NOS II protein comes from neurons, astrocytes and microglia.

Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia Sepsis is a complex pathophysiologic state resulting from an exaggerated whole-body inflammatory response to infection or injury. Metabolic disturbances, abnormal regulation of blood flow and diminished utilization of oxygen at the cellular level may account for tissue damage and lead to multiple organ failure and death. As the primary site of cellular energy generation is the mitochondrion, it presents itself as an important target for the septic cascade. In this regard, the notion that bioenergetic failure due to mitochondrial dysfunction contributes to organ failure during sepsis has received attention. We established the low frequency fluctuations in the systemic arterial pressure signals are related to the sympathetic neurogenic vasomotor tone, and reflect the functional integrity of the brain stem. Their origin is subsequently traced to the premotor sympathetic neurons at the rostral ventrolateral medulla (RVLM), whose neuronal activity is intimately related to the ¡§life-and-death¡¨ process. Based on a rat model of experimental endotoxemia that provides continuous information on changes in neuronal activity in the RVLM, the present study was undertaken to evaluate whether changes in mitochondrial respiratory functions are associated with death arising from sepsis. We also evaluated the efficacy of a new water-soluble coenzyme Q10 (CoQ10, ubiquinone) formula in the protection against fatality during endotoxemia by microinjection into bilateral RVLM. Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia in the Rat We investigated the functional changes in mitochondrial respiratory chain at the RVLM in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. Experiments were carried out in adult male Sprague-Dawley rats that were maintained under propofol anesthesia. Intravenous administration of E. coli lipopolysaccharide (LPS; 30 mg/kg) induced progressive hypotension, with death ensued within 4 hours. The sequence of cardiovascular events during this LPS-induced endotoxemia can be divided into a reduction (Phase I), followed by an augmentation (Phase II; ¡§pro-life¡¨ phase) and a secondary decrease (Phase III; ¡§pro-death¡¨ phase) in the power density of the vasomotor components (0-0.8 Hz) of systemic arterial pressure (SAP) signals. Enzyme assay revealed significant decrease of the activity of NADH cytochrome c reductase (Complex I+III) and cytochrome c oxidase (Complex IV) in the RVLM during all 3 phases of endotoxemia. On the other hand, the activity of succinate cytochrome c reductase (Complex II+III) remained unaltered. Neuroprotective Effects of Coenzyme Q10 at Rostral ventrolateral Medulla Against Fatality During Experimental Endotoxemia in the Rat CoQ10 is a highly mobile electron carrier in the mitochondrial respiratory chain that also acts as an antioxidant. We evaluated the neuroprotective efficacy of CoQ10 against fatality in an experimental model of endotoxemia, using a novel water-soluble formulation of this quinone derivative. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of E. coli LPS (30 mg/kg) induced experimental endotoxemia. Pretreatment by microinjection bilaterally of CoQ10 (1 or 2 mg) into RVLM significantly diminished mortality, prolonged survival time, and reduced the slope or magnitude of the LPS-induced hypotension. CoQ10 pretreatment also significantly prolonged the duration of Phase II endotoxemia and augmented the total power density of the vasomotor components of SAP signals in Phase II endotoxemia. The increase in superoxide anion production induced by LPS at the RVLM during Phases II and III endotoxemia was also significantly blunted. Conclusion The present study revealed that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM is closely associated with fatal endotoxemia. CoQ10 provides neuroprotection against fatality during endotoxemia by acting on the RVLM. We further found that a reduction in superoxide anion produced during endotoxemia at the RVLM may be one of the mechanisms that underlie the elicited neuroprotection of CoQ10. These findings therefore open a new direction for future development of therapeutic strategy in this critical, complicated and highly fatal condition known as sepsis.

Heat shock proteins (HSPs) are abundantly produced in cells that are under stress or injury by acting as a chaperone or promoting folding, unfolding, packing, degradation or denaturing of proteins or peptides. This study evaluated the role of HSP60, HSP70 or HSP90 in the rostral ventrolateral medulla (RVLM), in experimental endotoxemia in the rat. Adult, male Sprague-Dawley rats maintained by i.v. infusion propofol (25 mg/kg/h) were used. During experimental endotoxemia induced by intravenous administration of E. coli lipopolysaccharide (LPS, 30 mg/kg; serotype 0111:B4), the power density of the vasomotor component of systemic arterial pressure (SAP) spectrum underwent a decrease (Phase I), followed by an increase (Phase II; ¡§pro-life¡¨) and a secondary decrease (Phase III; ¡§pro-death¡¨). Western blot analysis revealed that HSP60 expression in the RVLM was significantly increased during Phase II and Phase III endotoxemia; and HSP70 expression was maximally increased during Phase II. HSP90 protein expression in the RVLM was not significantly changed during endotoxemia. We further studied the role of HSP60, HSP70 or HSP90 at the RVLM in experimental endotoxemia by pretreating animals with bilaterally microinjection of an anti-HSP serum (HSPAb, 1:20), normal mouse serum, antisense oligodeoxynucleotide (hsp AODN, 50 pmol), sense oligodeoxynucleotide (hsp SODN) or scrambled AODN (hsp SC). Pretreatment with HSP60Ab or hsp60 AODN resulted in significantly higher mortality, shorter survival time and shorter Phase II duration. In addition, the augmented power density of the vasomotor component of SAP signals during Phase II endotoxemia was significantly reduced. Even more detrimental effects were obtained on local application of HSP70Ab or hsp70 AODN into the RVLM. Pretreatment with HSP90Ab or hsp90 AODN was ineffective. We conclude that the expression of HSP60 and HSP70 in the RVLM may play a ¡§pro-life¡¨ role in fatal experimental endotoxemia; and HSP90 may not be involved.