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Michael Kalloniatis

Publications and source records attributed to Michael Kalloniatis.

At least 19 recordsLinked to original sources
PubMed2006

Mapping glutamate responses in immunocytochemically identified neurons of the mouse retina.

The mammalian retina contains as many as 50-60 unique cell types, many of which have been identified using various neurochemical markers. Retinal neurons express N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and kainic acid (KA) receptor subunits in various mixtures, densities, and spatial distributions. Ionotropic glutamatergic drive in retinal neurons can be mapped using a cation channel permeant guanidinium analog called agmatine (1-amino-4-guanidobutane; AGB). This alternative approach to physiologically characterize neurons in the retina was introduced by Marc (1999, J Comp Neurol 407:47-64, 407:65-76), and allows the simultaneous mapping of responses of glutamate receptor-gated channels from an entire population of neurons. Unlike previous AGB studies, we colocalized AGB with various macromolecular markers using direct and indirect immunofluorescence to characterize the glutamate agonist sensitivities of specific cell types. Activation with NMDA, AMPA, and KA resulted in AGB entry into neurons in a dose-dependent manner and was consistent with previous receptor subunit localization studies. Consistent with the various morphological phenotypes encompassed by the calbindin and calretinin immunoreactive cells, we observed various functional phenotypes revealed by AGB labeling. Not all calbindin or calretinin immunoreactive cells showed ligand-evoked AGB permeation. A small proportion either did not possess functional glutamate receptors, required higher activation thresholds, or express functional channels impermeable to AGB. AMPA and KA activation of bipolar cells resulted in AGB permeation into the hyperpolarizing variety only. We also studied the glutamate ligand-gating properties of 3[alpha1-3]-fucosyl-N-acetyl-lactosamine (CD15) immunoreactive cells and show functional responses consistent with receptor subunit gene expression patterns. CD15-immunoreactive bipolar cells only responded to AMPA but not KA. The CD15 immunoreactive amacrine cells demonstrated an identical selectivity to AMPA activation, but were also responsive to NMDA. Finally, localization of AGB secondary to glutamate receptor activation was visualized with a permanent reaction product.

Agmatine↗
PubMed2006

Layer positioning of late-born cortical interneurons is dependent on Reelin but not p35 signaling.

We tested the response of interneurons to the absence of Reelin signaling or p35 in the mouse neocortex. We provide three independent strands of evidence to demonstrate that layering of late-born (but not early-born) interneurons is regulated by Reelin signaling. First, early-born and late-born interneurons behaved differently in mice lacking Reelin or disabled 1 (Dab1). Early-born interneurons showed layer inversion, whereas late-born interneurons did not demonstrate layer inversion but were randomly distributed across the cortex. Second, in p35 mutant brains (in which Reelin signaling is intact), late-born interneurons are appropriately positioned in the upper layers despite the malpositioning of all other cortical neurons in these mice. Third, transplanted late-born interneuron precursors (wild type) into Dab1(-/-) cortices showed appropriate upper layer segregation. Together, these results indicate that, in the absence of Reelin signaling, late-born interneurons fail to laminate properly, and this is restored in an environment in which Reelin signaling is intact. These studies suggest different mechanisms for the stratification of cortical interneurons. Whereas the early-born interneurons appear to be associated with projection neuron layering, late-born interneurons rely on Reelin signaling for their correct lamination.

Animals↗
PubMed2005

Early markers of retinal degeneration in rd/rd mice.

PURPOSE: In the rd/rd mouse, the cell death of rod photoreceptors has been correlated to abnormal levels of the cyclic nucleotide cGMP within photoreceptors. Given that cGMP is required for opening of the cationic channels, there is the possibility that a high cGMP concentration would maintain these channels open, at a high energy cost for the retina. METHODS: We investigated whether cation channels were maintained in an open state in the rd/rd mouse retina by determining the labeling pattern of an organic cationic probe (agmatine, AGB) which selectively enters cells through open cationic channels. The metabolic activity of the rd/rd mice was measured by assaying lactate dehydrogenase (LDH) activity in several tissues and Na+/K+ ATPase activity was measured as a function of development and degeneration of the retina. RESULTS: AGB neuronal labeling showed a systematic increase consistent with the known neuronal functional maturation in the normal retina. There was a significant higher AGB labeling of photoreceptors in the rd/rd mouse retina from P6 supporting the possibility of open cationic channels from an early age. There were no changes in the LDH activity of tissues that contain PDE6 or that have a similar LDH distribution as the retina. However, LDH activity was significantly higher in the rd/rd mouse retina than in those of control mice from birth to P6, and it dramatically decreased from P9 as the photoreceptors degenerated. The predominant LDH isoenzyme changes and loss after degeneration appeared to be LDH5. ATPase activity increased with age, reaching adult levels by P16. Unlike LDH activity, there was no significant difference in Na+/K+ ATPase activity between control and rd/rd mice at any age examined. CONCLUSIONS: We conclude that AGB is a useful marker of photoreceptors destined to degenerate. We discard the possibility of a generalized metabolic effect in the rd/rd mice. However, the elevated LDH activity present before photoreceptor differentiation indicated altered retinal metabolic activity that could not be associated with open cationic channels alone. Therefore, altered metabolic activity as indicated by LDH measurements in the retina appeared to be the earliest sensitive sign of future photoreceptor dysfunction in the rd/rd mice.

Aging↗
PubMed2005

Excitation mapping with the organic cation AGB2+.

Excitation mapping is a method of visualizing the signaling history of neurons with permeant organic cations. It is compatible with high-resolution imaging, allowing concurrent visualization of all neuronal classes and their glutamate-gated excitation histories. Excitation mapping documents the stability and precision of neuronal signaling within a given neuronal class, arguing that single unit electrophysiological sampling accurately reflects neuronal diversity. We here review the theory of excitation mapping, provide methods and protocol links; outline imaging concepts; provide parametric data on the temporal range and physiological sensitivity of excitation mapping; and show that immunocytochemical methods for macromolecules are compatible with excitation mapping.

Agmatine↗
PubMed2005

Creatine transporter localization in developing and adult retina: importance of creatine to retinal function.

Creatine and phosphocreatine are required to maintain ATP needed for normal retinal function and development. The aim of the present study was to determine the distribution of the creatine transporter (CRT) to gain insight to how creatine is transported into the retina. An affinity-purified antibody raised against the CRT was applied to adult vertebrate retinas and to mouse retina during development. Confocal microscopy was used to identify the localization pattern as well as co-localization patterns with a range of retinal neurochemical markers. Strong labeling of the CRT was seen in the photoreceptor inner segments in all species studied and labeling of a variety of inner neuronal cells (amacrine, bipolar, and ganglion cells), the retinal nerve fibers and sites of creatine transport into the retina (retinal pigment epithelium, inner retinal blood vessels, and perivascular astrocytes). The CRT was not expressed in Müller cells of any of the species studied. The lack of labeling of Müller cells suggests that neurons are independent of this glial cell in accumulating creatine. During mouse retinal development, expression of the CRT progressively increased throughout the retina until approximately postnatal day 10, with a subsequent decrease. Comparison of the distribution patterns of the CRT in vascular and avascular vertebrate retinas and studies of the mouse retina during development indicate that creatine and phosphocreatine are important for ATP homeostasis.

Animals↗
PubMed2005

Visual function: the problem with eccentricity.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. With an ageing population, the prevalence of such a condition has resulted in a large proportion of the population relying on peripheral vision to undertake activities of daily living. Peripheral vision is not a scaled-down version of the fovea, simply requiring larger print or increased contrast for detection of objects or reading text. Even when print size is scaled and eye movements are minimised, the peripheral retina cannot perform at the level of the foveal region. Understanding how and why reading performance is limited as a function of eccentricity has important implications for how we approach rehabilitation of patients with central visual loss. This brief review of the extensive literature on reading with peripheral vision and the research aimed at better reading rehabilitation for low vision patients focuses on why many of the problems associated with the reduced reading capability of peripheral vision cannot be completely solved with magnification, reducing eye movements or modifying print.

Blindness↗
PubMed2005

Short- and long-term enzymatic regulation secondary to metabolic insult in the rat retina.

Changes in oxygen and/or glucose availability may result in altered levels of ATP production and amino acid levels, and alteration in lactic acid production. However, under certain metabolic insults, the retina demonstrates considerable resilience and maintains ATP production, and/or retinal function. We wanted to investigate whether this resilience would be reflected in alterations in the activity of key enzymes of retinal metabolism, or enzymes associated with amino acid production that may supply their carbon skeleton for energy production. Enzymatic assays were conducted to determine the activity of key retinal metabolic enzymes total ATPase and Na(+)/K(+)-ATPase, aspartate aminotransferase and lactate dehydrogenase. In vitro anoxia led to an increase in retinal lactate dehydrogenase activity and to a decrease in retinal aspartate aminotransferase activity, without significant changes in Na(+)/K(+)-ATPase activity. In vivo inhibition of glutamine synthetase resulted in a short-term significant decrease in retinal aspartate aminotransferase activity. An increase in retinal aspartate aminotransferase and lactate dehydrogenase activities was accompanied by altered levels of amino acids in neurons and glia after partial inhibition of glial metabolism, implying that short- and long-term up- and down-regulation of key metabolic enzymes occurs to supply carbon skeletons for retinal metabolism. ATPase activity does not appear to fluctuate under the metabolic stresses employed in our experimental procedures.

Adaptation, Physiological↗
PubMed2004

Quantification of amino acid neurochemistry secondary to NMDA or betaxolol application.

BACKGROUND: Alterations in retinal amino acid neurochemistry are an indicator of metabolic function. Glutamate is the primary excitatory amino acid neurotransmitter within the retina, and excessive levels of glutamate can potentially cause excitotoxicity, in particular, through the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. Anomalies in NMDA receptor function have been implicated as causing many neurodegenerative disorders, and overactivation leads to neuronal death secondary to metabolic insult. Several pharmaceutical agents have been proposed as potential neuroprotective agents against excitotoxicity (e.g. betaxolol), yet any effects such drugs have on retinal neurochemistry have not been determined. Therefore, the aim of this study was to quantify the changes in retinal amino acid neurochemistry secondary to the application of NMDA with and without betaxolol. METHODS: Functional NMDA channel activation was confirmed in both amacrine and ganglion cells by quantifying the entry into these neurones of a channel permeable probe (agmatine: 1-amino-4-guanidobutane [AGB]). By probing serial thin sections with immunoglobulins targeting AGB, glutamate, gamma-aminobutyric acid (GABA) and glycine, it was possible to simultaneously study the neurochemical characteristic as well as the NMDA-evoked AGB responses of different neurochemical populations of inner retinal neurones. RESULTS: The authors have previously shown no accumulation of glutamate or GABA within Muller cells following NMDA application. Herein they report altered GABA and glycine immunoreactivity, but not glutamate immunoreactivity within neurones of the amacrine and ganglion cell layers following NMDA application. Finally, the addition of betaxolol did not significantly alter the normal neurochemistry of the retina. CONCLUSION: The retina possesses intrinsic mechanisms that allow it to maintain metabolic integrity during short periods of high NMDA application.

Adrenergic beta-Antagonists↗
PubMed2004

Retinitis pigmentosa: understanding the clinical presentation, mechanisms and treatment options.

Retinitis pigmentosa (RP) is a leading cause of human blindness due to degeneration of retinal photoreceptor cells. Causes of retinal degeneration include defects in the visual pigment, defects in the proteins important for photoreceptor function or in enzymes involved in initiating visual transduction. Despite the diversity of genetic mutations identified in inherited forms of retinal dystrophy, there is a common end result of photoreceptor death and functional blindness. In this review, pertinent anatomical and physiological pathways involved in RP and the underlying genetic mutations are outlined, including a discussion on the inheritance patterns revealed by advances in molecular biological techniques. Characteristics of progression rates of visual field loss and current management options will provide useful clinical guidelines for the management of patients with RP.

Adult↗
PubMed2004

Monocarboxylate transport inhibition alters retinal function and cellular amino acid levels.

We assessed the effect of the in vivo application of monocarboxylate transport inhibitors on retinal function and amino acid immunocytochemistry. We wanted to determine the impact that altered aerobic metabolite availability has on retinal function and the characteristics of amino acid shunting into metabolic pools. Electroretinograms were collected from anaesthetized rats at various times after intravitreal injection of the monocarboxylate transport inhibitors alpha-cyano-4-hydroxycinnamate (4-CIN; 2 micro L, 0.1-10 mm) or p-(dipropylsulphamoyl)benzoic acid (probenecid; 1-10 mm). Changes in retinal function were compared with quantitative amino acid immunocytochemical changes in retinas harvested 20 and 40 min after either 4-CIN or vehicle treatment. The injection of 4-CIN resulted in a dose-dependent reduction of the ON-bipolar cell P2 wave amplitude (20-80%) and delay in its implicit time. The phototransduction sensitivity was mildly reduced whereas the ON-bipolar cell P2 sensitivity was unaffected. Probenecid induced functional changes similar to those observed with 4-CIN. We also mapped the amino acid alterations within specific cell classes induced by 4-CIN application. All neurones displayed a reduced glutamate content averaging 48%; reduced GABA (31%) and glycine (28%) were found within amacrine cells and glutamine was reduced in all cell classes except photoreceptor and Müller cells. All cell classes in the retina demonstrated increases in aspartate (57%), whereas leucine (24%) and ornithine (21%) were only significantly increased in photoreceptor and bipolar cells. The reduction in glutamate immunolabelling in specific retinal cell classes was mirrored by an increase in aspartate levels at these locations. In addition, attenuated glutamine immunolabelling also closely matched the spatial pattern observed for glutamate. Our immunocytochemical analysis provides evidence that monocarboxylate transport inhibition induces a shift in the equilibrium of glutamate transamination reactions involving aspartate throughout the retina whereas photoreceptor and bipolar cells also use glutamate transamination reactions involving ornithine and leucine. The distribution pattern of glutamine secondary to monocarboxylate inhibition suggests that this amino acid is a major precursor for glutamate throughout the retina.

Amino Acids↗
PubMed2004

Retinal function loss after monocarboxylate transport inhibition.

PURPOSE: To test the proposal that inhibiting monocarboxylate transport in the rat retina results in altered retinal function measured using the electroretinogram (ERG) and to evaluate the efficacy of exogenous metabolic substrates to restore any functional deficit. METHODS: Full-field white-flash ERGs were measured after monocarboxylate transport inhibition with intravitreal injection of alpha-cyano-4-hydroxycinnamic acid (4-CIN, 10 mM), and functional recovery was assessed after the introduction of various exogenous metabolic substrates (10 mM): lactate, pyruvate, alpha-ketoglutarate, alanine, succinate, and glutamine. The efficacy of glutamine as a metabolic substrate was also considered in the presence of phosphate-activated glutaminase inhibition (6-diazo-5-oxo-norleucin, 10 mM) or aminotransferase inhibition (aminooxyacetic acid, 10 mM). Pyruvate and alanine recovery was also assessed after aminooxyacetic acid application. RESULTS: 4-CIN application resulted in an increased phototransduction amplitude but a mild reduction of gain. A greater reduction of postreceptoral b-wave and oscillatory potential amplitudes (80%) was observed, along with delayed implicit times (35 ms). Partial recovery of b-wave amplitudes was achieved with exogenous lactate (24%), pyruvate (27%), alpha-ketoglutarate (27%), alanine (25%), and succinate (26%), whereas glutamine provided 62% recovery. However, none of the substrates improved phototransduction gain. Both 6-diazo-5-oxo-norleucin and aminooxyacetic acid completely suppressed the glutamine-induced b-wave recovery. Aminooxyacetic acid also abolished the b-wave recovery from 4-CIN afforded by pyruvate and alanine. CONCLUSIONS: The greater loss of the b-wave and oscillatory potentials may reflect preferential routing of amino acid carbon skeletons to oxidative metabolic pathways, which in turn reduces glutamate availability for neurotransmission between photoreceptors and ON-bipolar cells. The reduction in log S provides evidence that inhibition of monocarboxylate transport produced some metabolic dysfunction in the rat.

Alanine↗
PubMed2003

Inner retinal neurons display differential responses to N-methyl-D-aspartate receptor activation.

The N-methyl-D-aspartate (NMDA) responses of neurons from within the inner rabbit retina were mapped using a channel permeable cation, 1-amino-4-guanidobutane (agmatine, AGB). Serial sections were subsequently probed with immunoglobulins targeting AGB, glutamate, gamma-aminobutyric acid (GABA), and glycine to visualize the NMDA responses of neurochemical subpopulations of neurons. Most inner retinal subpopulations of neurons demonstrated an NMDA concentration-dependent increase in activation. This NMDA-induced activation displayed a distinct pattern, with the most sensitive class to least sensitive class ranking being GC > GABA cAC > GABA/Gly cAC > Gly cAC > GABA dAC (GC, ganglion cells; AC, amacrine cells; c, conventional; d, displaced; Gly, glycine). The variable NMDA response may reflect differences in NMDA receptor subunit disposition or differences in receptor density. In addition to the variable NMDA activation pattern, we found that virtually all ganglion cells (87%) showed NMDA-gated AGB entry, compared with only 58% of amacrine cells. We conclude that a large cohort of amacrine cells do not possess functional NMDA receptors. In addition to most ganglion cells being activated by NMDA, a large subpopulation displayed the highest sensitivity to NMDA application. The functional significance of this finding is that the ganglion cell population will be the first neuronal class to be susceptible to glutamate-induced neurotoxicity mediated through the NMDA receptor. The addition of betaxolol significantly reduced NMDA-mediated AGB entry into most neuronal groups (ganglion cells, GABA, and glycine amacrine cells), with the greatest effect being on ganglion cells. Betaxolol had no significant effect on NMDA-gated entry of AGB on the GABA/Gly amacrine cell population.

Adrenergic beta-Antagonists↗
PubMed2003

Correlating retinal function and amino acid immunocytochemistry following post-mortem ischemia.

We wanted to determine the characteristics associated with electrophysiological and neurochemical changes secondary to ischemic insult as well as correlate these electrophysiological and neurochemical changes. A Ganzfeld source was used to elicit electroretinograms in anesthetized adult Sprague-Dawley rats. Following baseline recordings, one eye was removed for control quantitative amino acid immunocytochemistry, and ischemic insult was induced by cervical dislocation. Following the induction of ischemia, a single electroretinogram signal was collected at 1, 2, 4, 6, 8, 16, 32 or 64 min, after which the eye was removed for immunocytochemistry. The post-receptoral b-wave was undetectable after 1 min post-ischemia, whereas phototransduction declined more gradually and persisted for up to 16 min post-mortem. Both phototransduction saturated amplitude and sensitivity decayed with a similar time course (tc=3.06 (2.73, 3.48) versus 3.29 (2.61, 4.62)min). Significant elevation of amino acid neurotransmitter levels was not observed until 6 min post-mortem. Between 8 and 16 min post-ischemia, glutamate and GABA were significantly accumulated in neurons and Müller cells (p<0.05). Beyond 16 min, the neurotransmitter elevation in neurons and Müller cells was relatively attenuated. Aspartate immunoreactivity was significantly elevated at 4 and 6 min post-ischemia in neurons, prior to a change in any other amino acid. Moreover, of the amino acids assessed the post-ischemic change in aspartate immunoreactivity showed the best correlation with phototransduction decay (r2=0.68). Our findings show that complete impairment of phototransduction coincides with the accumulation of amino acid neurotransmitter. The correlation of aspartate immunoreactivity and phototransduction provides evidence of heightened glutamate oxidation during ischemic insult.

Animals↗
PubMed2003

The contribution of glycolytic and oxidative pathways to retinal photoreceptor function.

PURPOSE: To consider how aerobic and anaerobic metabolic processes limit posthypoxemic decay in retinal function, measured by electroretinogram (ERG). METHODS: The hypothesis that lowering metabolic demand would prolong endogenous metabolic stores was tested by comparing the rate of ERG decay in rats in dark- (n = 5) versus light-adapted (15 minutes, 112 cd/m(2), n = 5) conditions and with serial versus single (n = 5 at each of seven time points) light stimulation. Postmortem hypoxemia was induced by cervical dislocation. Glucose (10 and 100 mM) and glutamine or lactate (100 mM) were injected into the vitreous 10 minutes before hypoxemic insult, to consider glycolytic-oxidative versus oxidative metabolism, respectively. RESULTS: Lowering the metabolic drain by light adaptation or serial stimulation significantly improved the photoreceptoral saturated amplitude during the first 5 to 7.5 minutes after postmortem hypoxemia. Increasing substrate availability with exogenous glucose preloading delayed the loss of the photoreceptoral response, thereby extending the delay constant from 4.8 to 10.9 minutes. Postreceptoral amplitudes were not improved by any exogenous substrate. Providing glucose at 5 minutes after hypoxemia provided no benefits. Similar to glucose, glutamine and lactate loading significantly delayed photoreceptoral decay over the first 7.5 minutes, after which time glucose was the more effective substrate. CONCLUSIONS: The postmortem decay of photoreceptoral function reflects depletion of both endogenous oxygen and carbon substrate reserves. The findings provide evidence that a transition between aerobic and anaerobic metabolism occurs after approximately 8 minutes of complete hypoxemia.

Aerobiosis↗
PubMed2002

Cellular dispersion patterns and phenotypes in the developing mouse superior colliculus.

The mammalian superior colliculus is structurally and functionally divided into two entities: superficial visual and deep multimodal motor. To discover the role, if any, of developmental processes in establishing separate tectal compartments, we have used highly unbalanced mouse chimaeras to mark cell dispersion pathways and trace cell lineages. Two forms of cell dispersion were detected: radial and tangential. Neither radial nor tangential forms of cell dispersion were found to exist on their own in any group of labeled cells. Radial cell dispersion was the predominant form of cell movement from the germinal zones and primarily associated with the differentiation of glutamatergic neurons. In contrast, tangential cell dispersion involved a minority of tectal cells, concentrated chiefly in the superficial layers and often associated with the upper aspects of radial columns. More scattered cells expressed gamma-aminobutyric acid (GABA) compared to columnar cells. Taken together, these results indicate separate developmental constraints for the development of glutamatergic and GABAergic neurons in the superior colliculus.

Animals↗
PubMed2002

Left--right word recognition asymmetries in central and peripheral vision.

Word recognition for Western languages shows an increased probability of a correct response when words are presented to the right of fixation. We considered whether this right bias was consistent at eccentricities superior and inferior to fixation and whether this bias can be altered by different presentation strategies. A right bias of up to approximately 0.9 degrees to the right of fixation was found when words were presented along one horizontal meridian. The eccentricities tested extended up to 8 degrees above and below the point of fixation. However, the right bias was reduced for stimulus conditions where the word was randomly presented within a mosaic containing all possible presentation locations. We have therefore demonstrated that reading habit (right bias) can be manipulated based upon experimental paradigm, strongly supporting the proposition that the left-right asymmetry is a consequence of attending to a particular area of visual space as part of the normal reading habit, rather than an innate superiority for word recognition of the right visual field or reduced visual performance.

Fixation, Ocular↗