Calcium Activated Chloride Channels In Olfactory Transduction

Ca2+-activated Cl \u304 channels are an important component of olfactory transduction. Odorant binding to odorant receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca2+ concentration by Ca2+ entry through cyclic nucleotide-gated channels. Ca2+ activates a Cl \u304 channel that leads to an efflux of Cl \u304 from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl \u304 channel remains elusive. Recent evidences have indicated that bestrophins are able to form Ca2+-activated Cl \u304 channels channels in heterologous systems. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and co-localized with the main subunit of cyclic nucleotide-gated channels, CNGA2. We performed a functional comparison of the properties of Ca2+-activated Cl \u304 channels from native channels expressed in dendritic knob/cilia of mouse OSNs with those induced by heterologous expression of mBest2 in HEK-293 cells. Even if the two channels did not display identical characteristics, they have many similar features such as the same anion permeability, the Ca2+ sensitivity in micromolar range and the same side-specific blockage of the two Cl \u304 channel blockers commonly used to inhibit the odorant-induced Ca2+-activated Cl \u304 channels in OSNs, niflumic acid and 4-acetamido-4\u2019-isothiocyanato-stilben-2,2\u2019-disulfonate (SITS). However electroolfactogram recording from mBest2 null mice showed a normal sensitivity to odorant stimulation. Therefore mBest2 is a good candidate for being a molecular component of the olfactory Ca2+-activated Cl \u304 channels but its precise role in olfactory transduction remains to be clarified

Toward a mechanistic understanding of marine invertebrate behavior at elevated CO2

Elevated carbon dioxide (CO2) levels can alter ecologically important behaviors in a range of marine invertebrate taxa; however, a clear mechanistic understanding of these behavioral changes is lacking. The majority of mechanistic research on the behavioral effects of elevated CO2 has been done in fish, focusing on disrupted functioning of the GABAA receptor (a ligand-gated ion channel, LGIC). Yet, elevated CO2 could induce behavioral alterations through a range of mechanisms that disturb different components of the neurobiological pathway that produces behavior, including disrupted sensation, altered behavioral choices and disturbed LGIC-mediated neurotransmission. Here, we review the potential mechanisms by which elevated CO2 may affect marine invertebrate behaviors. Marine invertebrate acid–base physiology and pharmacology is discussed in relation to altered GABAA receptor functioning. Alternative mechanisms for behavioral change at elevated CO2 are considered and important topics for future research have been identified. A mechanistic understanding will be important to determine why there is variability in elevated CO2-induced behavioral alterations across marine invertebrate taxa, why some, but not other, behaviors are affected within a species and to identify which marine invertebrates will be most vulnerable to rising CO2 levels

The role of melatonin and the pineal gland in the photoperiodic control of reproduction and smoltification in Salmonid fish

The timing of seasonal events in salmonids is thought to be controlled by endogenous circannual rhythm(s) which are entrained by the seasonally-changing daylength. This thesis investigates the role of the pineal gland in the perception of the photoperiodic zeitgeber and the subsequent transmission of this information to the brain through neural or hormonal pathways. Melatonin biosynthesis by isolated rainbow trout pineal glands was shown to exhibit a differential response to graded photic or thermal stimuli. In vitro experiments were carried out at 10±0.50 C as this provided optimum melatonin levels for radioimmunoassay analysis together with a pineal longevity of up to 14 days. By incorporating a variety of light intensities into the light/dark cycle, the salmonid pineal gland was shown to synthesise significantly different levels of melatonin even when light levels varied by only 0.5 lux. Early work on the salmonid pineal suggested it was unresponsive to red light, having a spectral sensitivity which peaks between 500 and 550 nm, this study has revealed that the pineal is also capable of responding to wavelengths between 660 to 800 nm, at which pineal reception was previously thought to be severely limited. No endogenous rhythm of melatonin secretion was observed within the isolated rainbow' trout pineal gland. Both Atlantic salmon and Atlantic halibut pineals exhibited elevated levels- of melatonin in response to the dark phase, however, they also appeared capable of maintaining this rhythm in the absence of external stimuli. This provides the first evidence that the daily rhythm of melatonin production in these species is controlled by an endogenous circadian oscillator located within the pineal II gland. The pinealectomy technique developed during the course of this thesis successfully abolished the diel rhythm of melatonin secretion and, together with an enucleation procedure, enabled the pineal to be identified as the predominant source of the dark phase melatonin in Atlantic salmon and rainbow trout. However, the lateral eyes did contribute significantly to plasma melatonin levels in both species. Long term experiments, involving pinealectomy and/or implantation of melatonin, were used to investigate the role of the pineal gland in the timing of rainbow trout maturation and smoltification in Atlantic salmon. Pineal removal at the summer or winter solstices did not significantly alter the timing of smoltification. However, significantly higher blood serum osmolarities following seawater challenge tests were observed in smolts implanted with melatonin. This, together with a significant growth increase shown by salmon parr within 1 month of implantation, indicates that melatonin may directly affect the development of salmonids through either a physiological response or by influencing the entrainment of endogenous rhythms. The increased growth observed in the implanted parr is also thought to be responsible for the unimodal population distribution and high percentage of S1 smolts within this group. Investigations into the role of the pineal gland in the timing of spawning in rainbow trout found that pineal removal at the summer solstice caused a 6 week delay in spawning time compared to intact fish. However, no clear effects on spawning time were observed when pineal removal, with or without melatonin implantation, was performed to coincide with the change from long to short daylengths which is known to advance spawning times. Although no significant effect in spawning times was observed between groups, the 4 month spawning period of the pinealectomised group compared to 1 month in the shampinealectomised fish also suggested that pineal removal may have caused a desynchronisation in spawning time. Pinealectomy and/or implantation did not alter egg size or fecundity, but plasma calcium levels were shown to be significantly lower in the pinealectomised trout over the spawning period. To summarise, the pineal gland and melatonin play a significant role in salmonid development. It is suggested that melatonin can influence biological systems through a direct physiological action while the pineal gland may synchronise . circannual events through the photoneuroendocrine transduction of seasonal environmental information

Functional Organization of the Gustatory System in the Brains of Ictalurid Catfish: a Combined Electrophysiological and Neuroanatomical Study (Taste, Viscerotopic, Sensory Maps, Forebrain).

The present study utilizes electrophysiological and neuroanatomical techniques to investigate the functional organization of the gustatory system in the brainstem and the forebrain of the channel catfish, Ictalurus punctatus. Neuroanatomical studies indicate an overlapping, segmental pattern of projection of glossopharyngeal-vagal branches in the vagal lobe. The vagal nerve complex is divisible into an interoceptive input (consisting of general visceral fibers) from abdominal viscera and an exteroceptive-branchial component (consisting of special and general visceral fibers) innervating the oro-pharyngeal region. The interoceptive-visceral input converges onto the exteroceptive, oro-pharyngeal input in the nucleus intermedius of the vagal lobe (nIV). In addition, extra-oral and oral gustatory information converges onto the nucleus intermedius of the facial lobe (nIF) and sensory inputs from separate regions of the oropharynx converge onto separate halves of the dorsal cap of the vagal lobe. Overlapping taste and tactile sensory maps of the oropharynx are present in the vagal lobe of the catfish. The representation of the oropharynx is less well defined than the somatotopic map in the facial lobe except for the bilaterally mapped extra-oral surface. Gustatory information reaches the area dorsalis pars medialis of the telencephalon and several nuclei in the ventral diencephalon of the catfish. The central gustatory pathway ascends from the medulla to the level of the diencephalon via the secondary gustatory nucleus as well as to the telencephalon via small neurons in the diencephalic lobo-bulbar nucleus. Neurons in the gustatory region of the telencephalon descend to the diencephalic level primarily via the medial forebrain bundle

STUDIES ON THE STRUCTURE AND FUNCTION OF THE TELEOST PSEUDOBRANCH

Four main types of pseudobranch were distinguished on the basis of their epithelial covering. The terms 'free', ‘semi-free’, 'covered' and 'buried' are used to describe these types. All pseudobranchs possess specific ‘pseudobranch type’ cells, characterised by an orderly arrangement of tubules around closely packed mitochondria. They also contain vacuoles which may play a role in osmoreception by causing changes in the size and shape of the cells. Another specialised cell type, similar to the gill 'chloride' cell was found in 'free' and 'semi-free' pseudobranchs of salt water fish. They are associated with smaller 'accessory' cells from which they are separated by 'leaky' junctions which may provide a structural basis for the proposed ion secretory nature of 'chloride type' cells. Ultrastructural changes in 'chloride type' and 'pseudobranch type' cells were noted under osmotic stress but the cells still remained distinguishable from each other, giving no reason to suggest that they were different forms of the same cell. The two other main cell types found in the pseudobranch epithelium were mucous and rodlet cells. The epithelial surface possesses numerous microridges which are thought to aid anchorage of mucous. The vascular system of the pseudobranch shows close similarities to that of the gill and contains a well developed arterio-venous pathway as well as an arterio-arterial system. Arterio-venous anastomoses were found between the efferent filament artery and the central venous sinus of the bass pseudobranch. The pseudobranch innervation is extremely complex. Morphological and denervation studies suggest an autonomic innervation and physiological evidence indicates the presence of at least two and possibly four types of receptor. The results of this study indicate that the pseudobranch has a number of inter-related functions associated with the development of specific cell types and a complex innervation linked directly or indirectly to the vascular system.THE MARINE BIOLOGICAL ASSOCIATIO

Magnetic Resonance Imaging of the Rat Retina

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2 emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed high blood-volume in the choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layer-specific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway

Magnetic Resonance Imaging of the Rat Retina: a Dissertation

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed a superfluous choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layerspecific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway