RESUMO
Ganglion cells are the output neurons of the retina and are known to remodel during the subtle plasticity changes that occur following the death of photoreceptors in inherited retinal degeneration. We examine the influence of retinal eccentricity on anatomical remodelling and ganglion cell morphology well after photoreceptor loss. Rd1 mice that have a mutation in the ß subunit of phosphodiesterase 6 were used as a model of retinal degeneration and gross remodelling events were examined by processing serial sections for immunocytochemistry. Retinal wholemounts from rd1-Thy1 and control Thy1 mice that contained a fluorescent protein labelling a subset of ganglion cells were processed for immunohistochemistry at 11 months of age. Ganglion cells were classified based on their soma size, dendritic field size and dendritic branching pattern and their dendritic fields were analysed for their length, area and quantity of branching points. Overall, more remodelling was found in the central compared with the peripheral retina. In addition, the size and complexity of A2, B1, C1 and D type ganglion cells located in the central region of the retina decreased. We propose that the changes in ganglion cell morphology are correlated with remodelling events in these regions and impact the function of retinal circuitry in the degenerated retina.
Assuntos
Células Fotorreceptoras/patologia , Degeneração Retiniana/patologia , Células Ganglionares da Retina/fisiologia , Animais , Plasticidade Celular/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Dendritos/fisiologia , Modelos Animais de Doenças , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Retinose Pigmentar/patologiaRESUMO
Retinitis Pigmentosa (RP) reflects a range of inherited retinal disorders which involve photoreceptor degeneration and retinal pigmented epithelium dysfunction. Despite the multitude of genetic mutations being associated with the RP phenotype, the clinical and functional manifestations of the disease remain the same: nyctalopia, visual field constriction (tunnel vision), photopsias and pigment proliferation. In this review, we describe the typical clinical phenotype of human RP and review the anatomical and functional remodelling which occurs in RP determined from studies in the rd/rd (rd1) mouse. We also review studies that report a slowing down or show an acceleration of retinal degeneration and finally we provide insights on the impact retinal remodelling may have in vision restoration strategies.
Assuntos
Terapia Genética/métodos , Retina/fisiopatologia , Epitélio Pigmentado da Retina/metabolismo , Retinose Pigmentar , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Retina/metabolismo , Retina/patologia , Epitélio Pigmentado da Retina/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/fisiopatologia , Retinose Pigmentar/terapiaRESUMO
Age-related macular degeneration (AMD) is a leading cause of vision loss, characterized by drusen deposits and thickened Bruch's membrane (BM). This study details the capacity of nanosecond laser treatment to reduce drusen and thin BM while maintaining retinal structure. Fifty patients with AMD had a single nanosecond laser treatment session and after 2 yr, change in drusen area was compared with an untreated cohort of patients. The retinal effect of the laser was determined in human and mouse eyes using immunohistochemistry and compared with untreated eyes. In a mouse with thickened BM (ApoEnull), the effect of laser treatment was quantified using electron microscopy and quantitative PCR. In patients with AMD, nanosecond laser treatment reduced drusen load at 2 yr. Retinal structure was not compromised in human and mouse retina after laser treatment, with only a discrete retinal pigment epithelium (RPE) injury, and limited mononuclear cell response observed. BM was thinned in the ApoEnull mouse 3 mo after treatment (ApoEnull treated 683 ± 38 nm, ApoEnull untreated 890 ± 60 nm, C57Bl6J 606 ± 43 nm), with the expression of matrix metalloproteinase-2 and -3 increased (>260%). Nanosecond laser resolved drusen independent of retinal damage and improved BM structure, suggesting this treatment has the potential to reduce AMD progression.
Assuntos
Terapia a Laser , Degeneração Macular/terapia , Retina/fisiopatologia , Doenças Retinianas/terapia , Idoso , Idoso de 80 Anos ou mais , Envelhecimento , Animais , Lâmina Basilar da Corioide/patologia , Feminino , Humanos , Imuno-Histoquímica , Degeneração Macular/fisiopatologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Projetos Piloto , Reação em Cadeia da Polimerase , Estudos Prospectivos , Doenças Retinianas/fisiopatologia , Epitélio Pigmentado da Retina/patologiaRESUMO
The vertebrate retina receives histaminergic input from the brain via retinopetal axons that originate from perikarya in the posterior hypothalamus. In the nervous system, histamine acts on three G-protein-coupled receptors, histamine receptor (HR) 1, HR2 and HR3. In order to look for potential cellular targets of histamine in the mouse retina, we have examined the retina for the expression of histamine and the presence of these three receptors. Consistent with studies of retina from other vertebrates, histamine was only found in retinopetal axons, which coursed extensively through the ganglion cell and inner plexiform layers. mRNA for all three receptors was expressed in the mouse retina, and immunohistochemical studies further localized HR1 and HR2. HR1 immunoreactivity was observed on dopaminergic amacrine cells, calretinin-positive ganglion cells and axon bundles in the ganglion cell layer. Furthermore, a distinct group of processes in the inner plexiform layer was labeled, which most likely represents the processes of cholinergic amacrine cells. HR2 immunoreactivity was observed on the processes and cell bodies of the primary glial cells of the mammalian retina, the Müller cells. This distribution of histamine and its receptors is consistent with a brain-derived source of histamine acting on diverse populations of cells in the retina, including both neurons and glia.
Assuntos
Encéfalo/fisiologia , Histamina/metabolismo , Retina/citologia , Retina/metabolismo , Animais , Calbindina 2 , Dopamina/metabolismo , Feminino , Histamina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nervo Óptico/fisiologia , RNA Mensageiro/metabolismo , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
Extracellular ATP acts as a neurotransmitter in the retina, via the activation of ionotropic P2X receptors and metabotropic P2Y receptors. The expression of various P2X and P2Y receptor subtypes has been demonstrated in the retina, but the localization of P2Y receptors and their role in retinal signaling remains ill defined. In this study, we were interested in determining the localization of the P2Y(4) receptor subtype in the rat retina, and using the electroretinogram (ERG) to assess whether activation of these receptors modulated visual transmission. Using light and electron microscopy, we demonstrated that P2Y(4) receptors were expressed pre-synaptically in rod bipolar cells and in processes postsynaptic to cone bipolar cells. Furthermore, we show that the expression of P2Y(4) receptors on rod bipolar cell axon terminals is reduced following dark adaptation, suggesting receptor expression may be dependent on retinal activity. Finally, using the electroretinogram, we show that intravitreal injection of uridine triphosphate, a P2Y receptor agonist, decreases the amplitude of the rod PII, supporting a role for P2Y receptors in altering inner retinal function. Taken together, these results suggest a role for P2Y(4) receptors in the modulation of inner retinal signaling.
Assuntos
Receptores Purinérgicos P2/metabolismo , Retina/fisiologia , Adaptação Ocular/fisiologia , Animais , Adaptação à Escuridão/fisiologia , Eletrorretinografia/métodos , Microscopia Imunoeletrônica/métodos , Proteínas do Tecido Nervoso/metabolismo , Análise de Componente Principal , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/ultraestrutura , Retina/efeitos dos fármacos , Retina/ultraestrutura , Uridina Trifosfato/farmacologiaRESUMO
P2X3 purinoceptors are involved in fast, excitatory neurotransmission in the nervous system, and are expressed predominantly within sensory neurons. In this study, we examined the cellular and synaptic localization of the P2X3 receptor subunit in the retina of the rat using immunofluorescence immunohistochemistry and pre-embedding immunoelectron microscopy. In addition, we investigated the activity of ecto-ATPases in the inner retina using an enzyme cytochemical method. The P2X3 receptor subunit was expressed in the soma of a subset of GABA immunoreactive amacrine cells, some of which also expressed protein kinase C-alpha. In addition, punctate immunoreactivity was observed within both the inner and outer plexiform layers of the retina. Double labeling studies showed that P2X3 receptor puncta were associated with both rod and cone bipolar cell axon terminals in the inner plexiform layer. Ultrastructural studies indicated that P2X3 receptor subunits were expressed on putative A17 amacrine cells at sites of reciprocal synaptic input to the rod bipolar cell axon terminal. Moreover, we observed P2X3 immunolabeling on amacrine cell processes that were associated with cone bipolar cell axon terminals and other conventional synapses. In the outer retina, P2X3 immunoreactivity was observed on specialized junctions made by putative interplexiform cells. Ecto-ATPase activity was localized to the inner plexiform layer on the extracellular side of all plasma membranes, but was not apparent in the ganglion cell layer or the inner nuclear layer, suggesting that ATP dephosphorylation occurs exclusively in synaptic regions of the inner retina. These data provide further evidence that purines participate in retinal transmission, particularly within the rod pathway.
Assuntos
Expressão Gênica/fisiologia , Neurônios/metabolismo , Receptores Purinérgicos P2/metabolismo , Retina/citologia , Adenosina Trifosfatases/metabolismo , Animais , Microscopia Imunoeletrônica/métodos , Neurônios/ultraestrutura , Parvalbuminas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteína Quinase C-alfa/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X3 , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Postembedding immunocytochemistry was used to determine the cellular localization of the amino acid neurotransmitters glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine in the avian retina. The through retinal pathway was glutamatergic, with all photoreceptors, bipolar cells, and ganglion cells being immunoreactive for glutamate. Bipolar cells displayed the highest level of glutamate immunoreactivity, with the cell bodies terminating just below the middle of the inner nuclear layer. All lateral elements, horizontal cells, amacrine cells, and interplexiform cells were immunoreactive for glycine or GABA. The GABAergic neurons consisted of two classes of horizontal cells and amacrine cells located in the lower part of the inner nuclear layer. GABA was also localized in displaced amacrine cells in the ganglion cell layer, and a population of ganglion cells that co-localize glutamate and GABA. Both the horizontal cells and GABAergic amacrine cells had high levels of glutamate immunoreactivity, which probably reflects a metabolic pool. At least two types of horizontal cells in the avian retina could be discriminated on the basis of the presence of aspartate immunoreactivity in the H2 horizontal cells. Glycine was contained in a subclass of amacrine cells, with their cell bodies located between the bipolar cells and GABAergic amacrine cells, two subclasses of bipolar cells, displaced amacrine cells in the ganglion cell layer, and ganglion cells that colocalize glutamate and glycine. Glycinergic amacrine cells had low levels of glutamate. We have also identified a new class of glycinergic interplexiform cell, with its stellate cell body located in the middle of the inner nuclear layer among the cell bodies of bipolar cells. Neurochemical signatures obtained by analyzing data from serial sections allowed the classification of subclasses of horizontal cells, bipolar cells, amacrine cells, and ganglion cells.
Assuntos
Aminoácidos/metabolismo , Neurotransmissores/metabolismo , Retina/metabolismo , Aminoácidos/imunologia , Animais , Ácido Aspártico/imunologia , Ácido Aspártico/metabolismo , Galinhas , Glutamatos/imunologia , Glutamatos/metabolismo , Glicina/imunologia , Glicina/metabolismo , Imuno-Histoquímica , Neurotransmissores/imunologia , Retina/anatomia & histologia , Células Ganglionares da Retina/metabolismo , Ácido gama-Aminobutírico/imunologia , Ácido gama-Aminobutírico/metabolismoRESUMO
We used postembedding immunocytochemistry to determine the localisation of the amino acid neurotransmitters glutamate, gamma-aminobutyrate (GABA), and glycine, potential neurotransmitter precursors (aspartate and glutamine), and taurine in the rat retina during postnatal development. All amino acids investigated were present at birth; however, only the inhibitory neurotransmitters GABA and glycine displayed neuronal localisation. GABA was localised in a sparse population of amacrine cells, and glycine immunoreactivity was found in cells within the ventricular zone that appeared to migrate through the neuroblastic layer. Glutamate labelling was diffuse across the retina until postnatal day (PND) 8. Localisation of glutamine was evident within Müller's cells by PND 6, in agreement with the known age of onset of glutamine synthetase activity. Based on the findings of uptake of radiolabelled glutamate and GABA by PND 8 and changes in immunoreactivity, we propose that Müller's cells evolve at PND 6-8 from their precursor cells, the radial glial cells. Evidence for differences in glutamate turnover in the infant retina was seen on examination of aspartate and glutamine immunoreactivity. Aspartate labelling was weak until PND 11, when ganglion cells and some amacrine cells were labelled. Unlike the mature retina, a large number of amacrine cells were glutamine immunoreactive in the PND 6 retina. One reason for the observed differences in precursor pooling may be a lack of neuronal neurotransmitter release and overall low metabolic activity. We also investigated the response of the developing retina to ischaemic insult to test the physiological hypoxia model of vascular development. Our findings are consistent with the hypothesis that the developing retina has increased tolerance to ischaemic insult. Our findings suggest that, although the retina is morphologically adult like by PND 8, there are differences in neurotransmitter turnover in the immature rat retina.
Assuntos
Aminoácidos/metabolismo , Animais Recém-Nascidos/metabolismo , Neurotransmissores/metabolismo , Retina/crescimento & desenvolvimento , Retina/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Autorradiografia , Imuno-Histoquímica , RatosRESUMO
The Royal College of Surgeons' (RCS) rat is an experimental model for a group of hereditary retinal diseases commonly called retinitis pigmentosa. We used postembedding immunocytochemistry to determine the localisation of glutamate, gamma-aminobutyric acid (GABA), glycine, aspartate, glutamine, taurine, and arginine in the RCS rat retina during postnatal development. In addition, we evaluated the uptake characteristics for the three dominant amino acid neurotransmitters, glutamate, GABA, and glycine. Whereas, cellular localisation of all amino acids was similar to control retinas, there were major changes in the level of immunoreactivity, even before eye opening, and well before the onset of visibly detectable photoreceptor degeneration. Two major patterns emerged. First, neurochemical changes evident before degeneration, involving the amino acids glutamate, GABA, aspartate, glutamine, and arginine. Second, neurochemical changes that become evident during photoreceptor degeneration involving the amino acids taurine and glycine. Anomalies in uptake characteristics also become evident during the degeneration phase and are likely to reflect changes in cellular function as a consequence of the degeneration process. Neurochemical changes evident before photoreceptor degeneration involve both glutamate and GABA manufacturing pathways. Müller's cells displayed elevated levels of glutamine and arginine from an early age, and the neuroblastic layer in the RCS retina showed high glutamate levels. Modified aspartate immunoreactivity began at postnatal day 11 and is consistent with altered metabolic activity. These results suggest that amino acid neurochemistry is different in the RCS rat retina from an early age, which may indicate an underlying metabolic defect affecting multiple cell classes.
Assuntos
Envelhecimento/fisiologia , Aminoácidos/metabolismo , Neurotransmissores/metabolismo , Retina/citologia , Retina/metabolismo , Degeneração Retiniana/metabolismo , Animais , Animais Recém-Nascidos , Arginina/metabolismo , Ácido Aspártico/metabolismo , Glutamina/metabolismo , Glicina/metabolismo , Imuno-Histoquímica , Ratos , Ratos Endogâmicos BUF , Ratos Mutantes , Retina/patologia , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Taurina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
We used post-embedding immunocytochemistry to determine the cellular localization of glutamate, gamma-amino butyric acid (GABA), glycine, aspartate, glutamine, arginine, and taurine in the normal and degenerating rat retina. Müller's cell function was also evaluated by determining the uptake and degradation characteristics for glutamate. Immunocytochemical localization of amino acids in adult Royal College of Surgeons (RCS) and control rat retinas were similar with respect to cell classes. Differences in the intensity of labelling for glutamate, aspartate, glutamine, and glycine were observed in several classes of neurons, but the most prominent differences were shown by bipolar cells of the adult RCS rat retina. In addition, glutamine labelling within Müller's cells was higher in the RCS rat than the control. These changes may have occurred because of alterations in the glutamate production or degradation pathways. We tested this hypothesis by determining Müller's cells glutamate uptake and degradation characteristics in adult and postnatal day 16 RCS retinas. High affinity uptake of 3[H]-glutamate revealed an accumulation of grains over Müller's cell bodies in the adult RCS retina implying glutamate degradation anomalies. We confirmed anomalies in glutamate metabolism in RCS Müller's cells by showing that exogenously applied glutamate was degraded over a longer time course in postnatal day 16 RCS retinas, compared to control retinas. Differences in arginine immunoreactivity in adult and immature RCS retinas conform to the presumed dysfunction of Müller's cells in these degenerating retinas. The anomalies of amino acid localization, uptake and degradation lead us to conclude that Müller's cells in the RCS retina show abnormal function by postnatal day 16; an earlier time to previously reported anatomical and functional changes in this animal model of retinal degeneration.
Assuntos
Degeneração Neural/fisiologia , Células Fotorreceptoras/química , Células Fotorreceptoras/patologia , Ratos Endogâmicos/fisiologia , Aminoácidos/análise , Animais , Especificidade de Anticorpos , Arginina/análise , Arginina/imunologia , Ácido Aspártico/análise , Ácido Aspártico/imunologia , Autorradiografia , Ácido Glutâmico/imunologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacocinética , Glicina/análise , Glicina/imunologia , Imuno-Histoquímica , Neurotransmissores/análise , Células Fotorreceptoras/efeitos dos fármacos , Epitélio Pigmentado Ocular/patologia , Ratos , Taurina/análise , Taurina/imunologia , Trítio , Ácido gama-Aminobutírico/análise , Ácido gama-Aminobutírico/imunologiaRESUMO
gamma-Aminobutyric acid (GABA), is a main source of inhibitory modulation of the rod pathway in the mammalian retina. The authors previously showed that rod bipolar cells express at least three types of ionotropic GABA receptors. Here, the authors sought to determine which neurons are the presynaptic partners at these synapses in the rabbit retina. Indoleamine-accumulating amacrine cells (IACs) were immunolabeled with an antiserum against serotonin (5HT) in vertical sections and wholemounts of rabbit retinae that had been preloaded with 5HT. The tissue was double labeled for the rho subunits of the GABA(C) receptor or the alpha3 subunit of the GABA(A) receptor. Punctate immunofluorescence was observed for both receptor subunits and was found to coincide with the dendrites and varicosities of IACs. The localization of rho subunits was examined at the ultrastructural level by using postembedding techniques on slam-frozen, cryosubstituted tissue. Double labeling at the electron microscopic level revealed that 5HT-immunoreactive processes were presynaptic to rod bipolar cells through GABA(C) receptors. Intracellular injection of the two morphologic subclasses of IAC amacrine cells, S1 and S2, with Lucifer yellow followed by immunolabeling for the alpha3 or rho subunits revealed that varicosities on the dendrites of both cell types were in register with alpha3- and rho-immunoreactive puncta. Taken together, these results suggest that IACs are presynaptic to rod bipolar cells through GABA(C) receptors and possibly through GABA(A) receptors.
Assuntos
Indóis/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de GABA/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Serotonina/metabolismo , Animais , Coelhos , Células Fotorreceptoras Retinianas Bastonetes/citologiaRESUMO
Rod bipolar (RB) cells of mammalian retinae receive synapses from different gamma-aminobutyric acid (GABAergic) amacrine cells in the inner plexiform layer (IPL). We addressed the question whether RB cells of the rabbit and of the rat retina express different types of GABA receptors at these synapses. RB cells were immunolabeled in vertical sections of rat retinae with an antibody against protein kinase C (PKC). The sections were double-labeled for the alpha 1, alpha 2, alpha 3, or gamma 2 subunits of the GABAA receptor. Punctate immunofluorescence, which represents synaptic localization, was found for all four subunits. Many of the alpha 1-, alpha 3-, or gamma 2-immunoreactive puncta coincided with the axon terminals of the PKC-immunolabeled RB cells. Sections and wholemounts of rabbit retinae were also double labeled for PKC and the rho subunits of the GABAC receptor. Rabbit RB cells were decorated by many rho-immunoreactive puncta, which were shown by electron microscopy to represent synaptic localization. Previous work from our laboratory has shown that the alpha 1, alpha 2, alpha 3, and rho subunits are not found within the same synapse but are expressed at different synaptic sites. Taken together, these results suggest that RB cells of mammalian retinae express at least three different types of GABA receptors at synaptic sites in the IPL: GABAC receptors, GABAA receptors containing the alpha 1 subunit, and GABAA receptors containing the alpha 3 subunit.
Assuntos
Receptores de GABA-A/análise , Receptores de GABA/análise , Células Fotorreceptoras Retinianas Bastonetes/citologia , Sinapses/ultraestrutura , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Substâncias Macromoleculares , Mamíferos , Microscopia Imunoeletrônica , Proteína Quinase C/análise , Coelhos , Ratos , Receptores de GABA/biossíntese , Receptores de GABA-A/biossíntese , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismoRESUMO
The distribution and synaptic clustering of N-methyl-D-aspartate (NMDA) receptors were studied in the rat retina by using subunit specific antisera. A punctate immunofluorescence was observed in the inner plexiform layer (IPL) for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent labeling of receptors clustered at postsynaptic sites. Double labeling of sections revealed that NMDA receptor clusters within the IPL are composed of different subunit combinations: NR1/NR2A, NR1/NR2B, and in a small number of synapses NR1/NR2A/NR2B. The majority of NMDA receptor clusters were colocalized with the postsynaptic density proteins PSD-95, PSD-93, and SAP 102. Double labeling of the NMDA receptor subunit specific antisera with protein kinase C (PKC), a marker of rod bipolar cells, revealed very little colocalization at the rod bipolar cell axon terminal. This suggests that NMDA receptors are important in mediating neurotransmission within the cone bipolar cell pathways of the IPL. The postsynaptic neurons are a subset of amacrine cells and most ganglion cells. Usually only one of the two postsynaptic processes at the bipolar cell ribbon synapses expressed NMDA receptors. In the outer plexiform layer (OPL), punctate immunofluoresence was observed for the NR1C2; subunit, which was shown by electron microscopy to be localized presynaptically within both rod and cone photoreceptor terminals.
Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismo , Animais , Proteína 4 Homóloga a Disks-Large , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas do Tecido Nervoso/metabolismo , Ratos , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Sinapses/ultraestruturaRESUMO
The effects of GABA in the CNS are mediated by three different GABA receptors: GABA(A), GABA(B) and GABA(C) receptors. GABA(A) and GABA(B) receptors, but not yet GABA(C) receptors, have been demonstrated in the enteric nervous system, where GABA has been proposed to be a transmitter. The purpose of this study was to determine whether GABA(C) receptors are present and thus may play a role in mediating the effects of GABA in the myenteric plexus of the rat gastrointestinal tract. We examined the expression of the three known GABA(C) receptor subunits, rho1, rho2 and rho3, in the rat duodenum, ileum and colon using the reverse transcriptase-polymerase chain reaction. We determined the localization of GABA(C) receptors in the myenteric plexus of these regions using two different antisera directed against GABA(C) receptor subunits. The polymerase chain reaction revealed that all three subunits were expressed in the gastrointestinal tract. When the layers of the intestine were separated and the layer containing myenteric neurons was assayed, the rho3 subunit was found in the ileum and colon, whereas rho1 was expressed in the duodenum and weakly in the colon and rho2 was expressed in the ileum. Immunocytochemistry revealed numerous labeled neurons in the myenteric plexus of each region. Colocalization showed that a large proportion of calbindin plus calretinin immunoreactive neurons (intrinsic primary afferent neurons) were immunoreactive for the GABA(C) receptor, and that 56% of nitric oxide synthase immunoreactive neurons (inhibitory motor neurons) exhibited the receptor. These results indicate that GABA(C) receptors of differing subunit compositions are expressed by neurons in the rat gastrointestinal tract. The effects of GABA on intrinsic sensory and on inhibitory motor neurons are likely to be mediated in part through GABA(C) receptors.
Assuntos
Sistema Digestório/inervação , Sistema Nervoso Entérico/metabolismo , Expressão Gênica/fisiologia , Inibição Neural/fisiologia , Neurônios/metabolismo , Receptores de GABA/genética , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Sistema Digestório/citologia , Sistema Digestório/metabolismo , Sistema Nervoso Entérico/citologia , Imuno-Histoquímica , Masculino , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Neurônios/citologia , Neurônios Aferentes/citologia , Neurônios Aferentes/metabolismo , Óxido Nítrico Sintase/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transmissão Sináptica/fisiologia , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
PURPOSE: To determine the role of smooth or rough lipopolysaccharide on adherence of Pseudomonas aeruginosa bacteria to the rat cornea in vitro and on contact lenses of differing types. METHODS: Adherence of a smooth (AK957) and isogenic rough strain (AK1012) of P. aeruginosa bacteria to rat corneas that were either normal, traumatized using a 20-gauge needle or treated for 15 min with 0.1N sodium hydrochloric acid was assessed by homogenization and viable counting. Adherence of these organisms to 43 unworn contact lenses representing the four Food and Drug Administration lens groups was also assessed using viable counts. RESULTS: Attachment to contact lenses was greater for the smooth strain for all four lens types (P < 0.001). No variation in adherence to the different lens types was observed. Smooth bacteria also adhered to the cornea to a greater extent than the rough strain, regardless of trauma type (P < 0.001). Adherence to traumatized corneas was greater than to nontraumatized corneas for both strains of P. aeruginosa bacteria (P < 0.01). Measurement of surface hydrophobicity of the two bacterial strains revealed that the smooth strain was more hydrophobic than the rough strain (P < 0.001), perhaps accounting for the adherence pattern. CONCLUSIONS: These results indicate that bacterial surface characteristics may be important determinants of adherence and could explain the propensity of certain bacterial strains to infect the cornea.
Assuntos
Aderência Bacteriana , Lentes de Contato , Lipopolissacarídeos , Pseudomonas aeruginosa/fisiologia , Animais , Contagem de Colônia Microbiana , Córnea/microbiologia , Úlcera da Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Feminino , Fenótipo , Ratos , Ratos Endogâmicos BUFRESUMO
Retinitis pigmentosa refers to a family of hereditary retinal degenerations that lead to photoreceptor death and vision loss. The underlying cause(s) are not known. In recent years there has been accumulating evidence of neurochemical changes during degeneration. In particular, the amino acids glutamate, GABA, and glycine show alterations in labelling intensity in subsets of neurons. Furthermore, there are differences in the labelling of the precursors, glutamine and aspartate, prior to, during, and following loss of photoreceptors, suggesting that the metabolic pathways involved in neurotransmitter formation and degradation may be abnormal. In addition, there is an elevation in glutamine and arginine content within Müller cells prior to the onset of photoreceptor death. Investigations evaluating Müller cell function indicate that formation and degradation of glutamate, in particular, is abnormal in the degenerating retina from an early age. These studies suggest that even though the primary genetic defect of the RCS rat is within the retinal pigment epithelium, Müller cells develop abnormally, and may contribute to the observed photoreceptor loss.
Assuntos
Neurotransmissores/análise , Degeneração Retiniana/metabolismo , Animais , Animais Recém-Nascidos , Ácido Aspártico/análise , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Glutamina/análise , Glicina/análise , Humanos , Imuno-Histoquímica , Neurotransmissores/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Retina/crescimento & desenvolvimento , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Retinose Pigmentar/metabolismo , Fatores de Tempo , Ácido gama-Aminobutírico/análiseRESUMO
Molecular cloning has introduced an unexpected diversity of neurotransmitter receptors. In this study we review the types, the localization and possible synaptic function of the inhibitory neurotransmitter receptors in the mammalian retina. Glycine receptors (GlyRs) and their localization in the mammalian retina were analyzed immunocytochemically. Specific antibodies against the alpha 1 subunit of the GlyR (mAb2b) and against all subunits of the GlyR (mAb4a) were used. Both antibodies produced a punctate immunofluorescence, which was shown by electron microscopy to represent clustering of GlyRs at synaptic sites. Synapses expressing the alpha 1 subunit of the GlyR were found on ganglion cell dendrites and on bipolar cell axons. GlyRs were also investigated in the oscillator mutant mouse. The complete loss of the alpha 1 subunit was compensated for by an apparent upregulation of the other subunits of the GlyR. GABAA receptors (GABAARs) and their retinal distribution were studied with specific antibodies that recognize the alpha 1, alpha 2, alpha 3, beta 1, beta 2, beta 3, gamma 2 and delta subunits. Most antibodies produced a punctate immunofluorescence in the inner plexiform layer (IPL) which was shown by electron microscopy to represent synaptic clustering of GABAARs. The density of puncta varied across the IPL and different subunits were found in characteristic strata. This stratification pattern was analyzed with respect to the ramification of cholinergic amacrine cells. Using intracellular injection with Lucifer yellow followed by immunofluorescence, we found that GABAARs composed of different subunits were expressed by the same ganglion cell, however, they were clustered at different synaptic sites. The distribution of GABAC receptors was studied in the mouse and in the rabbit retina using an antiserum that recognizes the rho 1, rho 2 and rho 3 subunits. GABAC receptors were found to be clustered at postsynaptic sites. Most, if not all of the synapses were found on rod and cone bipolar axon terminals. In conclusion we find a great diversity of glycine and GABA receptors in the mammalian retina, which might match the plethora of morphological types of amacrine cells. This may also point to subtle differences in synaptic function still to be elucidated.
Assuntos
Receptores de GABA/análise , Receptores de Glicina/análise , Retina/química , Animais , Axônios/química , Dendritos/química , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Mutação , Coelhos , Ratos , Receptores de Glicina/genética , Retina/ultraestruturaRESUMO
Contamination of contact lenses is thought to increase the risk of infectious keratitis, yet factors promoting attachment of bacteria to contact lenses are not fully understood. It has been suggested that strains of Pseudomonas aeruginosa attach to mucosal surfaces via pili which are appendages found on some strains. This study investigated the role of pili and the effect of incubation time on the attachment of P. aeruginosa to 20 unworn hydrogel lenses representative of each of the four FDA categories. Ten lenses were incubated for 15 minutes and another ten for 180 minutes. Lenses were incubated with either PAK + P. aeruginosa which possessed pili or its isogenic mutant pair, PAK-, which was genetically similar except for the absence of pili. Bacteria were quantified, following homogenization of the contact lens, by viable counts. Non-piliated bacteria were significantly more likely to adhere to the lenses (p < 0.001). A significant interaction between lens type and incubation time was observed (p < 0.05); thus it is difficult to generalize about either of these effects in isolation. These results show that surface characteristics may confer an attachment advantage to bacteria.
Assuntos
Aderência Bacteriana , Lentes de Contato , Fímbrias Bacterianas/fisiologia , Pseudomonas aeruginosa/fisiologia , Contagem de Colônia Microbiana , Hidrogel de Polietilenoglicol-Dimetacrilato , Técnicas Microbiológicas , Polietilenoglicóis , Fatores de TempoRESUMO
Simple Doppler was compared with routine arteriography in assessing patency of the arteries at the ankle and the pedal arch. Fifty-six limbs were evaluated--29 with rest pain or trophic lesions, 20 with claudication, and 7 without symptoms. On Doppler examination 177 of 220 (80%) vessels were assessed patent, compared with 127 of 220 (58%) on arteriography. Of the 49 judged occluded on arteriograms, 59% were patent by Doppler and of 44 in which arteriograms were inadequate 82% had Doppler signals. Doppler signals could be heard in 20 of 177 (11%) vessels only with the feet dependant. This prospective, double-blind study provides numerical data to support the observation that a simple Doppler probe can detect patent distal vessels which may not be demonstrated by routine arteriography. This allows selection of patients for further more detailed arteriograms or for operative exploration with a view to distal bypass grafting.
Assuntos
Tornozelo/irrigação sanguínea , Arteriosclerose/diagnóstico , Pé/irrigação sanguínea , Ultrassonografia , Idoso , Angiografia , Artérias/patologia , Arteriosclerose/diagnóstico por imagem , Arteriosclerose/patologia , Ensaios Clínicos como Assunto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Extracellular adenosine 5'-triphosphate (eATP) acts as a neurotransmitter within the retina and brain, activating a range of ionotropic P2X and metabotropic P2Y receptors. In this study, the specific localization of the P2X4 receptor (P2X4-R) subunit was evaluated in the retina using fluorescence immunohistochemistry and pre-embedding immuno-electron microscopy. Punctate P2X4-R labeling was largely localized to the inner and outer plexiform layers of mouse, rat and cat retinae. In the mouse outer retina, double-labeling of P2X4-R with the horizontal cell marker, calbindin, revealed P2X4-R immunoreactivity (P2X4-R-IR) on horizontal cell somata and processes. In the inner retina, P2X4-R expression was found closely associated with rod and cone bipolar cell terminals, and the punctate labeling was observed on calretinin-positive amacrine cells. Using immuno-electron microscopy, P2X4-Rs were observed on processes post-synaptic to photoreceptor and bipolar cell terminals, likely representing horizontal, amacrine and ganglion cells, respectively. Furthermore, P2X4-R expression was also observed on Müller cells, astrocytes and microglia. These data suggest a role for P2X4-Rs in the lateral inhibitory pathways of the retina, modulating neuronal function of photoreceptors and bipolar cells. The expression on macro- and microglial cells implicates a role for P2X4-Rs in glial signaling, tissue homeostasis and immunosurveillance within the mammalian retina.