The aim of this project is to test the hypothesis that the rate of renal blood flow remains constant under normal physiological conditions. Methods were developed for long term studies of renal blood flow (using the Doppler flow probe) and blood pressure in the conscious unrestrained rat. Special care was taken to maintain and observe the rats under normal, physiological conditions. Since the Doppler flow probe measures blood velocity rather than flow, changes in renal arterial diameter will alter the output of the flow meter, even if flow is constant. In order to measure flow more accurately, chanqes in vessel diameter were prevented by a short length of silastic tubing inserted into the renal, ortery under the probe. Blood flow was expressed as a percentage of the maximal flow measured when the animal was completely relaxed. Using this preparation, renal blood flow was measured continuously under normal conditions in both the light and dark cycles. Studies were performed to assess the effects on renal blood flow of anaesthesia, surgery and stress, and to assess the effect on renal blood flow of blockade of the renal nerves. Renal blood flow was depressed approximately 50% by anaesthesia, due to the effects of the anaesthetic agent rather than the accompanying surgery. Renal blood flow did not fully recover for 3-4 days after anaesthesia with, or without surgery. Renal blood flow was depressed by stress, even though blood pressure was unchanged. A mild sudden auditory disturbance caused a transient 23% fall in renal blood flow. A continuous auditory disturbance, depressed renal blood flow by 16% for several minutes. Handling the rat resulted in a 33% fall in renal blood flow which lasted for over 30 minutes. When measured continuously, renal blood flow was found to vary considerably. When the rat was completely relaxed renal blood flow was highest (defined as 100%). Renal blood flow remained high when eating and drinking (79%), but fell when the rat became alert but completely still (74%). During general movement renal blood flow was low (73%) with flow lowest while grooming (64%). Flow was apparently related to the degree of alertness and to activity. The extent of variation of flow is such that the calculated mean daily value of renal blood flow was only 80% of the maximum flow observed during complete relaxation. The depression of renal blood flow seen during activity and disturbance was largely prevented during reversible blockade of the ipsilateral renal nerve, induced by slow infusion of xylocaine around the renal artery. It was completely abolished by bilateral renal nerve blockade with Xylocaine. The depressive effects of activity and disturbance on renal blood flow were restored an hour after the Xylocaine infusion ceased. These studies clearly show that renal nerve is involved in control of renal blood flow under physiological conditions. It is clear that renal blood flow varies markedly under normal physiological conditions and in response to the external environment. A constant renal blood flow is not a necessity for normal renal function.

Renal sympathetic nerve activity (RSNA) plays an important role in the control of renal hemodynamics and function. It achieves this control by changing the mean levels of activity and/or changing the power of the distinct frequencies that comprise this mean activity. My studies, presented in four parts, investigate how total renal blood flow (RBF) and intrarenal blood flow are controlled by these changes in RSNA. While RSNA and RBF both show oscillations at various frequencies, the functional significance and regulation of these oscillations is not well understood. To establish whether the strength of these oscillations is under differential control I measured the frequency spectrum of RSNA and RBF following volume expansion in conscious rabbits. At least 6 days prior to experiments animals underwent surgery to implant an electrode for recording renal nerve activity and a flow probe for recording RBF. Volume expansion resulted in a 25 ± 5% decrease in mean RSNA, paralleled by an increase in RBF. Renal denervated rabbits did not show an increase in RBF with volume expansion. Spectral analysis of the different frequencies in RSNA showed oscillations in RSNA between 0.2 to 0.4 Hz were selectively decreased following volume expansion (14 ± 3 to 6±1% of total power in RSNA at < 3Hz). A corresponding decrease in the strength of oscillations in RBF at this frequency was also seen (20 ± 6 to 8 ± 2%). In contrast the strength of respiratory (0.8 to 2.0 Hz) and cardiac (3 to 6 Hz) related rhythms did not change with volume expansion. These results show that selective changes in the different frequency components of RSNA can occur. While RSNA plays a significant role in the regulation of RBF, little is known about the role renal nerves may play in the control of regional kidney blood flows i.e. cortical and medullary blood flow (CBF and MBF respectively). This is an important question as the control of blood flow to the renal medulla has been shown to be critical in the long-term control of arterial pressure, chiefly through its influence on tubular reabsorption of salt and water. If renal nerves are involved in the control of medullary blood flow then they may also have a role in long-term arterial pressure regulation. My aim was to investigate the role RSNA may have in the regulation of both CBF and MBF. In a series of experiments, using pentobarbitone anaesthetized rabbits, I electrically stimulated the renal nerves whilst simultaneously recording RBF, CBF, and MBF. Three sequences of stimulation were applied, 1) varying the amplitude of stimulation, 2) varying the frequency of stimulation and 3) stimulation with a modulated sinusoidal pattern which allows determination of the frequency response characteristics of each flow. Increasing amplitude or frequency of stimulation progressively decreased all flow variables. RBF and CBF responded similarly, but MBF responded less. For example, 0.5 V stimulation decreased CBF by 20 ± 9% but MBF fell by only 4 ± 6%. The amplitude of oscillations in all flow variables was progressively reduced as the frequency of sinusoidal stimulation was increased. An increased amplitude of oscillation was observed at 0.12 and 0.32 Hz in MBF, and to a lesser extent RBF, but not CBF. MBF therefore appears to be less sensitive than CBF to the magnitude of RSNA, but more able to respond to these higher frequencies of neural stimulation. These results show that regional renal blood flows may be differentially regulated by RSNA and indicate a role for RSNA in the control of MBF. To further investigate the effects of RSNA on intrarenal blood flow I performed another series of experiments in which I reflexly increased RSNA while simultaneously recording CBF and MBF. I exposed pentobarbitone anesthetized, artificially ventilated rabbits to graded reductions in inspired 02 content. A separate group of animals with denervated kidneys underwent the same protocol. Graded hypoxia (16, 14, 12 and 10% inspired 02) progressively reduced arterial 02 partial pressure and increased RSNA (by 8 ± 3, 44 ± 25, 62 ± 21 and 76 ± 37% respectively compared with air) without affecting MAP. This was accompanied by progressive reductions in CBF (by 2 ± 1, 5 ± 2, 11 ± 3 and 14 ± 2% respectively) in intact but not denervated rabbits. MBF was unaffected by hypoxia in either group. Thus, reflex increases in RSNA cause renal cortical vasoconstriction, but not at vascular sites regulating MBF. Taken together results from both these studies provide strong evidence that MBF is less sensitive to the vasoconstrictor influence of RSNA than is CBF. However, larger, chronic increases in RSNA, such as occur in heart failure, could be associated with decreases in MBF. This may implicate RSNA in the long-term control of arterial pressure. The 0.3 Hz oscillation in RSNA, and thus MAP, is intermittent in its presence. In a final series of experiments I studied the underlying reasons for the presence or absence of the 0.3 Hz oscillation. Using conscious animals I studied the oscillation under a range of sympathetic stimulants. I was unable to induce an oscillation in MAP at 0.3 Hz and propose reasons for this. To allow further analysis, I performed a second set of experiments in which I induced the oscillation in RBF by electrical stimulation of the aortic depressor nerve. I found that large amplitude oscillations in baroreceptor stimulation were required to induce oscillations in RBF and MAP. I speculate on what this may tell us about the nature of the slow oscillation.

Increases in intracellular Ca2+ play a central role in cochlear function. The ryanodine receptor (RyR) intracellular Ca2+ release channel, a ubiquitous element of Ca2+ signalling, has been implicated in the regulation of sound transduction and auditory neurotransmission. Despite this, the molecular basis underlying RyR-mediated Ca2+ signalling in the cochlea has been limited. This thesis investigates the molecular and functional characterisation of RyR expression in the cochlea. RT-PCR analysis showed expression of RyR1, RyR2 and RyR3 isoform mRNA transcripts in the rat cochlea and also in the spiral ganglion. Localisation of RyR protein revealed differential expression of these isoforms in the cochlea. Strong RyR immunolabelling for RyR1, RyR2 and RyR3 were detected in the spiral ganglion neuron (SGN) cell bodies. RyR3 labelling extended to the synaptic terminals innervating the inner and outer hair cells. RyR2 expression also occurred in the inner hair cells and supporting cells of the organ of Corti. Cells associated with ion homeostasis in the cochlea were also labelled, including RyR1 in spiral limbus interdental cells, and RyR2 and RyR3 in spiral prominence epithelial cells and stria vascularis basal cells. In the SGN cell bodies, confocal imaging of Ca2+ store release confirmed the presence of a functional RyR-gated Ca2+ store. Superfusion of glutamate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) evoked large Ca2+ responses in the SGN cell bodies that were dependent upon Ca2+ entry. However, subsequent depletion of SGN RyR-gated Ca2+ stores substantially reduced the glutamate- and AMPA-induced Ca2+ responses, demonstrating that the majority of the Ca2+ signal derived from RyR-gated Ca2+ stores via Ca2+ -induced Ca2+ release. Involvement of the AMPA/Kainate-type glutamate receptor was confirmed by elimination of glutamate- and AMPA-induced Ca2+ responses with an AMPA/Kainate receptor antagonist. These findings support a role for RyR in the regulation of auditory neurotransmission, sound transduction and cochlear electrochemical homeostasis. These data also demonstrate coupling between somatic AMPA-type glutamate receptors and RyR-gated Ca2+ stores, which is likely to influence auditory neurotransmission. / Whole document restricted, but available by request, use the feedback form to request access.

The primary function of the ocular lens is as a light-focusing structure. To this end the lens is avascular, with a highly ordered arrangement of cells that acts to reduce light scattering. However, the avascular nature of the lens presents the problem of supplying cells deep within the lens with nutrients and removing waste products of cellular metabolism, i.e. maintaining cellular homeostasis, To solve this problem the lens possesses intercellular channels, called gap junctions, that facilitate cell-to-cell communication and are regulated by changes in the subcellular distribution and age dependent processing of the gap junction protein subunits, called connexins. The lens expresses three types of connexins (Cx): Cx43 in the anterior surface epithelial cells and connexins 46 and 50 in the fibre cells. Therefore, to fully examine the role of GJs in lens homeostasis, regional differences in GJ structure and function need to be quantified. In this thesis I present the results of experiments designed to map the differentiation dependent changes to subcellular distribution and posttranslational processing of Cx46 and Cx50 in mouse and rat lenses. My results quantitatively show for the first time that Cx50 undergoes two discrete cleavage events in a similar manner to that previously shown for Cx46, and that qualitatively Cx46 does the same in the mouse lens' Such posttranslational processing has important effects on the proposed lens circulation model (Mathias et al., 1997) that accounts for lens homeostasis. In addition, I have examined the functional contribution of gap junctions to localized intercellular communication in different regions of wildtype and Cx46-KO mouse lenses, using highly localised uncaging of fluorescein with Two-Photon Excited Flash Photolysis (TPEFP) in half lenses, The results of these experiments have provided the first supporting evidence for a macromolecule-permeable pathway, previously suggested by Shestoplaov and Bassnett (2000, 2003), that acts in conjunction with gap junctions to facilitate intercellular communication in the lens core. My results show that this pathway acts at a localised level to confer cell-to-cell communication that is as effective as that mediated by gap junctions, and therefore a re-evaluation of current models of lens homeostasis is now required. / Whole document restricted, but available by request, use the feedback form to request access.

Hypertensive heart failure has long been associated with diminished cardiac contractile function, yet the underlying cellular mechanisms are not well understood. The aim of this Thesis was to investigate the relationship between intracellular calcium ([Ca2+]i) and isometric force during the relatively narrow time frame in which long-standing compensated hypertrophy progresses to decompensated end-stage heart failure in an animal model of human essential hypertension. In order to carry out this aim, left ventricular trabeculae were utilized from failing hearts of spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto (WKY) controls. At a physiological stimulation frequency (5 Hz), and temperature (37 °C), the peak stress of SHR trabeculae was significantly reduced compared to WKY, although no differences in the time-course of the twitch were detected. Measurements using fura-2/AM as an index of intracellular [Ca2+] showed that, for SHR, both the peak of the Ca2+ transient and the resting [Ca2+]i were increased and the decay of the Ca2+ transient was prolonged compared to WKY. This unexpected result, i.e. depression of twitch force despite an increased Ca2+ transient, was investigated further by utilizing experimental protocols known to affect [Ca2+]i and force. Varying extracellular calcium ([Ca2+]o) between 0.5 and 5 mM showed that the reduction of force development by SHR trabeculae was not associated with reduced myofilament Ca2+ sensitivity, since, although peak [Ca2+]i continued to increase with increasing [Ca2+]o, peak stress reached a plateau. Investigation of the force-frequency response between 0.2 and 10 Hz showed that the mismatch in peak Ca2+ and peak force was apparent across all frequencies for SHR. A consistent finding of studies that have made measurement of [Ca2+]i in failing myocardium is that the decay of intracellular Ca2+ following SR release is prolonged. Additionally, expression levels of the SR Ca2+ -ATPase have been reported as reduced, in conjunction with increased expression of the sarcolemmal Na+/Ca2+ exchanger. Although the decay of fluorescence was slower for SHR in this study, no experimental evidence was found to suggest that sarcolemmal Ca2+ extrusion was increased in SHR in comparison to WKY. The re-circulation fraction of activator Ca2+ during recovery from potentiation was not different between rat strains, indicating that SL Ca2+ extrusion was not increased in SHR. Additionally, the decay of fluorescence remained slower for SHR even when the SR Ca2+ -ATPase contribution was functionally removed. Inhibition of the SL Ca2+ -ATPase, together with the functional removal of the SR, removed the differences in the decay of fluorescence between rat strains. A decrease in the sarcolemmal extrusion of [Ca2+]i by the Ca2+ -.ATPase might therefore explain the observed differences in the resting [Ca2+]i and in the amplitude of the Ca2+ transient between rat strains. In summary, this study has provided the first measurements of [Ca2+]i and isometric force carried out at physiological temperature and stimulation frequency in LV trabeculae from failing SHR hearts and their age-matched, normotensive, WKY controls. Most importantly, for this animal model the contractile dysfunction typical of heart failure is not associated with reduced availabilty of [Ca2+]i. Instead it is suggested that contractile function is compromised in these LV trabeculae by the increased collagen, and its three-dimensional organisation.

Previous investigations1,2 have shown that the capacity of rats and rabbits reared at an elevated environmental temperature to thermoregulate in the cold is impaired. This impairment appears permanent and is associated with other thermoregulatory dysfunction (altered febrile response and altered response to intrahypothalamic noradrenaline injection). The present study investigated the role of the thermal environment in the development of thermoregulatory function by studying the performance of the intact thermoregulatory system and components thereof at different levels of processing of thermal information (thermoreceptive, early integrative and effector levels). The present study confirmed the impaired capacity of heat-reared rats to maintain colonic temperature in the cold and future indicated that this was likely to be attributable to active rather than passive components of the thermoregulatory system. Exposure to an environmental temperature of 20°C for 20 days was able to prevent thermoregulatory dysfunction (as assessed by intrahypothalamic noradrenaline injection) if it occurred after about 14 days of age. Exposure to 20°C was progressively less effective in averting thermoregulatory dysfunction after about 60-80 days of age. The experimental design identified these effects as being of environmental rather than genetic origin. Thermal information processing at the level of the caudal trigeminal nucleus resembled that described at thermoreceptors. The static and dynamic responses of thermoreceptive neurones at this level were quantified and found not to differ between 20°C – and 30°C-reared rats. The distribution of facial thermal receptive fields and the projections of these at the caudal trigeminal nucleus were mapped and also found not to differ. Thermal information originating from truncal skin was intercepted at a midline midbrain level. Data presented here suggested that the sites at which this thermal information could be intercepted included but probably were not restricted to the raphe nuclci. Both warm- and cold-responsive units were present at the midbrain. The abundance of thermoresponsive units was the same in 20°C- and 30°C-reared rat as was the ratio of warm:cold-responsive units. The stimulus configurations by which responses of midbrain thermoresponsive units could be characterised were more complex than at the caudal trigeminal nuclcus. Thermal responses were principally static with little response to temperature change. The pooled static responses of warm- and cold-responsive units over the 5-45°C range were the same in 20°C- and 30°C-reared rats. The cutaneous receptive fields at the midbrain level were diffuse, covering most of the truncal surface. There was an almost universal and consistent interaction with noxious input at midbrain thermoresponsive units. Warm-responsive units were inhibited by simultaneous noxious stimulation at almost any body site while the activity of cold-responsive units was augmented by such stimulation. At the thermoeffector level, noradrenaline-induced thermogenesis was equal in 20°C- and 30°C- reared rats. Heat-rearing was not observed to produce identifiable effects on the peripheral processing of thermal information, either at the afferent or efferent limbs of the thermoregulatory system. Heat-rearing does however produce changes in thermophysiological responses that are primarily mediated via a central mechanism (responses to intrahypothalamic noradrenaline, febrile response). It is therefore surmised that alterations in thermoregulatory function following heat-rearing are attributable to changes in central rather than peripheral processing of thermal information. 1 Cooper et al., (1980) J Physiol [ Lond ] 303: 165-172 2 Ferguson et al., (1981) Can J Physiol Pharmacol 59: 91-95

The role oxygen plays in the developing ovarian follicle is of interest not only to the field of developmental biology but also to in-vitro fertilisation (IVF) technologists, as oxygenation of the oocyte is considered to be a potential determinant of oocyte competence. Oxygen transport through the developing ovarian follicle, and practical aspects of the analysis of oxygen in human follicular fluid were investigated in this work. Mathematical modelling of oxygen transport in the pre-antral, and antrallpreovulatory follicle revealed a number of interesting findings, Contrary to previous conclusions (Gosden & Byatt-Smith, 1986), oxygen can reach the oocyte in the small pre-antral follicle. Improved estimates of diffusion coefficients through the granulosa cell layer and the inclusion of fluid voidage in this layer showed that oxygen can also reach the oocyte in large pre-antral follicles. The amount of oxygen that reaches the oocyte in the pre-antral follicle is a function of its size and degree of vascularisation. Symmetrically distributed vascularisation is superior in achieving a well oxygenated follicle. However, the large pre-antral follicle will eventually reach a size beyond which it cannot grow without anoxic regions developing. The size at which this occurs is consistent with the size at which antrum formation is observed in human follicles. The model predicts that the follicle can avoid an anoxic state through antrum formation, and shows that the follicle develops in a way that is consistent with overcoming mass transport limitations. The oxygen status of the follicle during the antrallpre-ovulatory phase of growth requires that the volume of granulosa cells be balanced by the volume of follicular fluid. Further predictions suggest that oocyte respiration becomes sub-maximal at follicular fluid volumes below approximately 4m1, vascularisation levels below 38%, or fluid i dissolved oxygen levels below 5.1 ~01%. These values are consistent with observations in the literature. It was also shown that the measurement of follicular fluid dissolved oxygen levels could provide a simple measure of the respiratory status of the oocyte, and this may be superior to the measurement of follicular vascularisation which requires knowledge of more parameters. Methodology for the analysis of follicular fluid oxygen solubility and diffusivity was developed using a Clark oxygen electrode. Analysis of these parameters showed that they are similar to human plasma, and allowed the predictive uncertainty of the model to be reduced. Experimental studies into the effects of IVF aspiration on follicular fluid were carried out. Aspiration results in significant changes in the properties of follicular fluid. Dissolved oxygen levels rose 5 * 2 vol%, pH increased by 0.04 * 0.01 pH units, and temperature dropped by 7.7 * 1.3 "C. Mathematical modelling of blood contaminated follicular fluid also showed that contamination results in significant changes in the dissolved oxygen of the fluid. This suggests that if the composition of follicular fluid is to be determined (particularly dissolved oxygen), sampling andlor measurement of fluid must take place before the collection vial of the aspiration kit, and blood contamination must be eliminated. Based on this result, the design and testing of devices capable of reliable sampling andlor rneasurement of oxygen levels of follicular fluid was considered. This presents a continuing challenge, including the integration of routine follicular fluid oxygen measurement into clinical practice.

Dendritic spines are fundamental units of information processing within the nervous system, responsible for independent modulation of synaptic input to neurons. Filopodia, often morphologically indistinguishable from spines, are involved in formation of synapses during neuronal development. Despite the importance of these structures for neuronal function, no detailed study of their presence on motoneurons has yet been made. Here, the presence of spines on hypoglossal motoneurons (HMs) is described at three developmental stages: at P0–2 and P9–11, spines are present at an average density of ~0.1 spines/micron, but at P19 spine density becomes negligible. In P0–2 and P9–11, spines are nonuniformly distributed, occuring in clusters, and at lower density in the most proximal and distal regions to the soma than at intermediate regions. HM spines coincide with a decrease in cell input resistance, which reduces excitability during development. Thus one may speculate that these spines are involved in the formation of new synapses required to maintain adequate excitatory drive. A major difficulty for the study of spines is their small size, which complicates measurement using optical methods. Here, I present a novel method for reconstructing spine morphology using geometric models based on a priori knowledge of spine structure. Tests of the technique using simulated data indicate that it has a resolving capability of up to 40 nm (limited by noise). The technique has been used to measure dendritic spines on HMs, showing that these structures have necks as small as 0.22 micron. For purely passive modulation of synaptic strength, spine necks need to be <~ 0.15 micron. These data suggest that if modulation of synaptic input occurs, biochemical and/or active electrical processes are needed. The methods developed in this Thesis, which have here been applied to HMs, are generally applicable to the study of spine morphology, and its effect on synaptic processing, in all classes of neurons.

Dendritic spines are fundamental units of information processing within the nervous system, responsible for independent modulation of synaptic input to neurons. Filopodia, often morphologically indistinguishable from spines, are involved in formation of synapses during neuronal development. Despite the importance of these structures for neuronal function, no detailed study of their presence on motoneurons has yet been made. Here, the presence of spines on hypoglossal motoneurons (HMs) is described at three developmental stages: at P0–2 and P9–11, spines are present at an average density of ~0.1 spines/micron, but at P19 spine density becomes negligible. In P0–2 and P9–11, spines are nonuniformly distributed, occuring in clusters, and at lower density in the most proximal and distal regions to the soma than at intermediate regions. HM spines coincide with a decrease in cell input resistance, which reduces excitability during development. Thus one may speculate that these spines are involved in the formation of new synapses required to maintain adequate excitatory drive. A major difficulty for the study of spines is their small size, which complicates measurement using optical methods. Here, I present a novel method for reconstructing spine morphology using geometric models based on a priori knowledge of spine structure. Tests of the technique using simulated data indicate that it has a resolving capability of up to 40 nm (limited by noise). The technique has been used to measure dendritic spines on HMs, showing that these structures have necks as small as 0.22 micron. For purely passive modulation of synaptic strength, spine necks need to be <~ 0.15 micron. These data suggest that if modulation of synaptic input occurs, biochemical and/or active electrical processes are needed. The methods developed in this Thesis, which have here been applied to HMs, are generally applicable to the study of spine morphology, and its effect on synaptic processing, in all classes of neurons.

Dendritic spines are fundamental units of information processing within the nervous system, responsible for independent modulation of synaptic input to neurons. Filopodia, often morphologically indistinguishable from spines, are involved in formation of synapses during neuronal development. Despite the importance of these structures for neuronal function, no detailed study of their presence on motoneurons has yet been made. Here, the presence of spines on hypoglossal motoneurons (HMs) is described at three developmental stages: at P0–2 and P9–11, spines are present at an average density of ~0.1 spines/micron, but at P19 spine density becomes negligible. In P0–2 and P9–11, spines are nonuniformly distributed, occuring in clusters, and at lower density in the most proximal and distal regions to the soma than at intermediate regions. HM spines coincide with a decrease in cell input resistance, which reduces excitability during development. Thus one may speculate that these spines are involved in the formation of new synapses required to maintain adequate excitatory drive. A major difficulty for the study of spines is their small size, which complicates measurement using optical methods. Here, I present a novel method for reconstructing spine morphology using geometric models based on a priori knowledge of spine structure. Tests of the technique using simulated data indicate that it has a resolving capability of up to 40 nm (limited by noise). The technique has been used to measure dendritic spines on HMs, showing that these structures have necks as small as 0.22 micron. For purely passive modulation of synaptic strength, spine necks need to be <~ 0.15 micron. These data suggest that if modulation of synaptic input occurs, biochemical and/or active electrical processes are needed. The methods developed in this Thesis, which have here been applied to HMs, are generally applicable to the study of spine morphology, and its effect on synaptic processing, in all classes of neurons.