Glycine receptor subunit composition alters the action of GABA antagonists

GABA receptor antagonists produce an unexpectedly significant inhibition of native glycine receptors in retina and in alpha1 or alpha2 homomeric glycine receptors (GlyRs) expressed in HEK 293 cells. In this study we evaluate this phenomenon in heteromeric glycine receptors, formed by mixing alpha1, alpha2, and beta subunits. Picrotoxinin, picrotin, SR95531, and bicuculline are all more effective antagonists at GlyRs containing alpha2 subunits than alpha1 subunits. Inclusion of beta subunits reduces the inhibitory potency of picrotoxinin and picrotin but increases the potency of SR95531 and bicuculline. As a result of these two factors, bicuculline is particularly poor at discriminating GABA and glycine receptors. Picrotin, which has been reported to be inactive at GABA receptors, blocks glycine currents in retina and in HEK293 cells, suggesting its utility as a selective glycine antagonist. However, picrotin is a more potent inhibitor of GABA than glycine in retinal neurons. We also tested if GABA and glycine receptor subunits can combine to form functional receptors. If GABAAR gamma2S subunits are co-expressed with GlyR alpha subunits, the mixed receptor is glycine-sensitive and GABA-insensitive. But the mixed receptor exhibits a non-competitive picrotoxinin inhibition that is not observed in the homomeric GlyRs. This suggests that glycine and GABA subunits can co-assemble to form functional glycine receptors.     

Localization of the alpha(1F) calcium channel subunit in the rat retina

PURPOSE: The molecular identity of the calcium channels that mediate glutamate release from photoreceptors is unknown. Mutations in the recently identified, retina-specific alpha(1F) calcium channel subunit cause incomplete X-linked congenital stationary night blindness (CSNB2), the phenotype of which is consistent with a defect in neurotransmission within the retina. The purpose of this study was to determine the cellular distribution of the alpha(1F) subunit in the retina. METHODS: Antibodies were raised against a unique peptide from the human alpha(1F) sequence. Rat retina sections were labeled with affinity-purified alpha(1F) antibodies and the immunofluorescence analyzed by confocal microscopy. The alpha(1F) staining was compared with that obtained with a pan-alpha(1) antibody, used to reveal the distribution of known voltage-gated calcium channels in the retina. Some sections were double labeled for alpha(1F) and the photoreceptor synaptic ribbon marker, bassoon. RESULTS: Staining of retina sections with anti-alpha(1F) resulted in strong punctate labeling in the outer plexiform layer (OPL) and weak punctate labeling in the inner plexiform layer (IPL), consistent with a synaptic localization. Staining was also observed in the outer nuclear layer. Within the OPL, alpha(1F) immunoreactivity was clustered in discrete, horseshoe-shaped patches, the shape and dimensions of which are characteristic of rod active zones. Similar structures were labeled with the pan-alpha(1) antibody. Localization of alpha(1F) immunoreactivity to rod active zones was confirmed by double labeling for bassoon, a component of photoreceptor synaptic ribbons. CONCLUSIONS: The distribution of alpha(1F) immunoreactivity in the OPL suggests that calcium influx through alpha(1F) or alpha(1F)-like channels mediates glutamate release from rod photoreceptors.     

GABA(A) and GABA(C) receptor antagonists increase retinal cyclic GMP levels through nitric oxide synthase

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal transduction pathway plays a role in every retinal cell type. Previous studies have shown that excitatory glutamatergic synaptic pathways can increase cGMP-like immunoreactivity (cGMP-LI) in retina through stimulation of NO production, but little is known about the role of synaptic inhibition in the modulation of cGMP-LI. Gamma-amino-n-butyric acid (GABA) plays critical roles in modulating excitatory synaptic pathways in the retina. Therefore, we used GABA receptor antagonists to explore the role of GABAergic inhibitory synaptic pathways on the modulation of the NO/cGMP signal-transduction system. Cyclic GMP immunocytochemistry was used to investigate the effects of the GABA receptor antagonists bicuculline, picrotoxin, and (1,2,5,6-tetrahyropyridin-4-yl) methylphosphinic acid (TPMPA) on levels of cGMP-LI. Cyclic GMP-LI was strongly increased in response to the GABA(A) receptor antagonist bicuculline, while the GABA(C) receptor antagonist TPMPA had little effect on cGMP-LI. The GABA(A)/GABA(C) receptor antagonist, picrotoxin, caused a moderate increase in cGMP-LI, which was mimicked by the combination of bicuculline and TPMPA. The nitric oxide synthase inhibitor, S-methyl-L-thiocitrulline (SMTC), blocked the increased cGMP-LI in response to stimulation with either bicuculline or picrotoxin. Treatments with either of the glutamate receptor antagonists (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) partially blocked the increases in cGMP-LI seen in response to bicuculline, but a combination of MK-801 and CNQX completely eliminated these increases. These results suggest that inhibitory synaptic pathways involving both types of GABA receptors work through excitatory glutamatergic receptors to regulate the NO/cGMP signal-transduction pathway in retina.     

Retinal vascular leakage occurring in GABA Rho-1 subunit deficient mice

Recent studies demonstrate that GABAergic activity elicits relaxation of retinal arterioles leading to an increase in blood flow. It has also been found that GABAnergic activity in the retina of mice with diabetic retinopathy is suppressed. In this study, we provide further evidence that lack of GABAergic activity significantly alters vasculature development as well as the hypoxia-induced angiogenic response. Using GABA_C receptor ρ₁ subunit-knockout mice (rho-1^−/−), our results demonstrate that in hypoxia-induced retinas a severe vascular leakage occurred in 2 week-old rho-1^−/− mice compared with their wildtype counterparts. In addition, our results also showed that all of the rho-1^−/− mice developed significant retinal vascular leakages by 48 weeks-of-age. Microarray and real-time PCR experiments revealed a unique angiogenesis-related gene expression pattern. This suggests that retinal vascular disorders of rho-1^−/− mice results from significant up-regulation of angiogenic genes and concomitant down-regulation of anti-angiogenic genes. The study results are consistent with the pathological changes of the retinal vascular leakage seen in diabetic retinopathy. Our data indicate that the GABA_C ρ₁ subunit plays a role in maintaining both homeostasis and balance of retinal neurotransmitter function. Knockout of the retinal GABA_C ρ₁-subunit leads to changes in vascular permeability similar to the pathological changes induced by retinal hypoxic conditions.     

Distribution of the alpha1 subunit of the GABA(A) receptor on midget and parasol ganglion cells in the retina of the common marmoset Callithrix jacchus

The inhibitory neurotransmitter gamma aminobutyric acid (GABA) has been shown to influence the responses of ganglion cells in the mammalian retina. Consistently, GABA(A) receptor subunits have been localized to different ganglion cell types. In this study, the distribution of the alpha1 subunit of the GABA(A) receptor on the dendrites of midget and parasol ganglion cells was investigated quantitatively in the retina of a New World monkey, the marmoset. Ganglion cells were injected with Neurobiotin in a live in vitro retinal whole-mount preparation. Retinal pieces were then processed with an antibody against the alpha1 subunit of the GABA(A) receptor. Strong punctate immunoreactivity indicative of synaptic localization is present in the ON and OFF sublamina of the inner plexiform layer. Many of the immunoreactive puncta coincide with the dendrites of both midget and parasol ganglion cells. Immunoreactive puncta are present on distal and proximal dendrites of ON and OFF cells of both ganglion cell types. On average, parasol cells show a slight increase in the spatial density of immunoreactive puncta with distance from the soma, whereas the density of immunoreactive puncta on midget cells stays even. Parasol ganglion cells show a slightly higher average density of immunoreactive puncta (0.083 puncta/microm dendrite) than midget cells (0.054 puncta/microm dendrite).     

GABA and GABA(A) receptor antagonists alter developing cone photoreceptor development in neonatal rabbit retina

Gamma aminobutyric acid (GABA) has been established as an important developmental signal in a number of regions of the central nervous system (CNS), including retina. Our previous studies have shown that GABAergic horizontal cells act as the initial synaptic target for developing cone photoreceptors in neonatal rabbit retina. Since intraocular injections of the GABA(A) receptor antagonists, picrotoxin or bicuculline, disrupt cone synaptogenesis in vivo, GABA released from horizontal cells may provide a necessary signal for cone axon growth and/or synapse formation. In the current report, we have used cultured retinal explants to examine the effects of GABA(A) receptor antagonists on other aspects of developing cones. These include the distribution pattern of cone cell bodies across the outer surface of the retina and the expression of GABA(A) receptors within both cone cell bodies and axonal processes. Peanut agglutinin (PNA), a plant lectin that specifically labels cone plasma membrane and extracellular matrix, was used to monitor cone development, and a GABA(A) receptor antibody against the beta2/3 subunits of the protein was used to label GABA(A) receptors. Results showed that cones maintained in the explant culture express GABA(A) receptors in a temporal and spatial pattern similar to that observed in vivo, namely a low expression of receptors on cone cell bodies at postnatal day 1 (P1), peaking around P3 and diminishing by P7. Neonatal retinal explants exposed to the GABA(A) receptor antagonists, bicuculline (10 microM) or SR95531 (5 microM), for 24 h in culture showed disruption of the normal distribution of cone cell bodies. When GABA (100 microM) was added along with either antagonist, cone cell bodies appeared normal. Neither bicuculline nor SR95531 alone had any effect on the general morphology of other retinal layers, suggesting that these GABA(A) receptor antagonists at the concentrations used were not acting as nonspecific disruption agents. The effects of GABA antagonists were confined to the first week after birth with no disruption seen in P9 or adult explant cultures. These data provide a direct demonstration of the necessity for GABAergic input to cones during active synaptogenesis. As we have previously shown, GABA(A) receptor activation causes a substantial increase in intracellular calcium concentrations in cones and thereby could provide a mechanism by which GABA regulates cone maturation.     

Opposite effects of GABA(A) and GABA(C) receptor antagonists on the b-wave of ERG recorded from the isolated rat retina

The largest component in the fully dark-adapted ERG is a corneal-positive response, known as the b-wave, and believed to originate from depolarizing (ON-type) bipolar cells. The two types of GABA receptors, GABA(A) and GABA(C) have been reported to exist on bipolar cells in rat retina. The goal of these experiments was to find whether these GABA receptors participate in the generation of the b-wave of electroretinogram (ERG). ERGs were recorded from the isolated rat retinas. The P(2)(t) component, obtained by subtracting the ERGs measured before the application of 50 micrograms APB from those measured after the application of 50 micrograms APB, was used as an indicator of depolarizing bipolar cell activity. Photovoltages, the fast P(3)(t) component of ERG, were registered between the two microelectrodes across the rod outer segments. Bicuculline and 3-aminopropylphosphonic acid (3-APA) were used as selective antagonists of GABA(A) and GABA(C) receptors, respectively. It was found that the GABA(A) and GABA(C) receptors antagonists have opposite effects on the b-wave: bicuculline increased the b-wave amplitude, while 3-APA reduced the amplitude of the b-wave. Neither bicuculline nor 3-APA affect photoreceptors.     

GABA(A) receptor binding and localization in the tiger salamander retina

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the retina and also appears to act as a trophic factor regulating photoreceptor development and regeneration. Although the tiger salamander is a major model system for the study of retinal circuitry and regeneration, our understanding of GABA receptors in this species is almost exclusively based on the results of physiological studies. Therefore, we have examined the pharmacological binding properties of GABA(A) receptors and their anatomical localization in the tiger salamander retina. Radioligand-binding studies showed that specific 3H-GABA binding to GABA(A) receptors was dominated by a single high-affinity binding site (Kd = 15.6+/-6.9 nM). Specific binding of 3H-GABA was almost completely eliminated by muscimol (Ki = 105+/-62 nM) and bicuculline (Ki = 14.3+/-2.2 microM); however, SR-95531 only displaced about 40% of specific 3H-GABA binding (Ki = 35.0+/-3.8 nM). These data indicate that there are at least two subtypes of GABA(A) receptors present in the salamander retina that can be distinguished by their antagonist binding properties: one sensitive to both bicuculline and SR-95531, and one sensitive to bicuculline but insensitive to SR-95531. Because localization of GABA receptors in the salamander retina by immunocytochemistry is problematic, GABA(A) receptors were localized by fluorescent ligand binding combined with immunocytochemical labeling for cell specific markers. Binding of fluorescently labeled muscimol to GABA(A) receptors was present in both plexiform layers and on photoreceptor cell bodies. GABA(A) receptors in the outer plexiform layer were localized to both photoreceptor terminals and horizontal cell processes.     

GABA(A) receptor immunoreactivity is transiently expressed in the developing outer retina

Extensive evidence has suggested a trophic role of gamma-aminobutyric acid (GABA) on developing cone photoreceptors in postnatal retina. In a previous study, we showed that GABA raises intracellular calcium levels in the developing cones via activation of GABA(A) receptors. Using confocal microscopy in conjunction with immunocytochemistry, we have now demonstrated that (1) GABA(A) receptor subunits are localized on cone cell bodies as well as on cone pedicles, indicating that GABA has a direct, rather than indirect, effect on cones and (2) the temporal expression of GABA(A) receptor subunits coincides with the developmental effects of GABA on cone synaptogenesis. An antibody against the beta 2/3 subunits of the GABA(A) receptor and a specific cone marker peanut-agglutinin lectin (PNA) were used to double-label wholemount neonatal retinal preparations. Results show that GABA(A) receptors are transiently expressed on cone photoreceptors in the early stages of postnatal retinal development. GABA (A)receptor immunoreactivity is clearly present on cone cell bodies and their processes and on other--as yet unidentified--elements (horizontal cells?) in the outer plexiform layer. Immunoreactivity decreases within cone photoreceptor somata after postnatal day 5, but persists in the processes of the outer plexiform layer until day 7. Our results provide support for the hypothesis that GABA acts as an important developmental regulator of cone photoreceptor maturation.     

Vanilloid receptor 1 (TRPV1/VR1) co-localizes with fatty acid amide hydrolase (FAAH) in retinal amacrine cells

Fatty acid amide hydrolase (FAAH) is the degradative enzyme for anandamide (AEA), an endogenous ligand for the vanilloid receptor (TRPV1) and cannabinoid receptor 1. As FAAH and TRPV1 are integral membrane proteins, FAAH activity could modulate the availability of AEA for TRPV1 activation. Previous studies in this laboratory reported an extensive endocannabinoid system in goldfish retina. Immunocytochemistry was used to determine the relative distributions of FAAH-immunoreactivity (IR) and TRPV1-IR in goldfish retina. Here, we show the first example in an intact neural system in which TRPV1-IR co-localizes in subpopulations of FAAH-immunoreactive neurons, in this case amacrine cells. These cells are rare and consist of three subtypes: 1. ovoid cell with granular-type dendrites restricted to sublamina a, 2. pyriform cell with smooth processes in sublamina b, and 3. fusiform cell with smooth processes that project to sublaminae a and b. The varied appearances of reaction product in the dendrites suggest different subcellular localization of TRPV1, and hence function of FAAH activity regarding TRPV1 stimulation among the cell types. Ovoid and pyriform amacrine cells, but not fusiform cells, labeled with GAD-IR and constituted subsets of GABAergic amacrine cells. TRPV1 amacrine cells, though rare, are represented in the ON, OFF and ON/OFF pathways of the retina. As TRPV1 stimulation increases intracellular calcium with numerous downstream effects, co-localization of TRPV1 and FAAH suggests an autoregulatory function for anandamide. Due to the rarity of these cells, the three vanilloid amacrine cell types may be involved in global effects rather than feature extraction, for example: sampling of ambient light or maintaining homeostasis.     

Development of glutamate receptor subunit 2 immunoreactivity in postnatal rat retina

Previous studies have shown that the expression of glutamate receptor subunits is developmentally regulated and have been implicated in processes of cell differentiation during postnatal life. The tissue localization and developmental pattern of the glutamate receptor 2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptor were investigated by means of immunohistochemistry and immunoblotting. Labeling of amacrine and ganglion cells and the inner plexiform layer appeared early during development, while glutamate receptor 2 subunit expression in the outer plexiform layer started after the first postnatal week. The distribution of labeling within the inner plexiform layer changed from nonorganized to laminated appearance prior to eye-opening. There was an increasing number of positive amacrine and ganglion cell somata during the first 2 weeks, but their number decreased considerably as the retina matured and were seen at least up to 35 days of postnatal development. Little labeling was found in the ganglion cell layer and in the inner plexiform layer of late postnatal and adult retina. Labeling in the outer plexiform layer and of bipolar cell somata appeared to increase in the developing retina. Glur2 labeling of these cells and the outer plexiform layer became discernible during the second postnatal week, and this labeling was present in the adult as well. Immunoblotting showed that GluR2 protein levels were similar at postnatal days 7 and 10, but slightly decreased between the second and fourth postnatal weeks. Our data imply that the immunological expression of glutamate receptor 2 subunit in the inner plexiform layer decreases as a function of age, and is correlated with developmental event(s) in the postnatal retina.     

Modulation of horizontal cell function by GABA(A) and GABA(C) receptors in dark- and light-adapted tiger salamander retina

The physiological function of GABA transporters and GABA receptors in retinal horizontal cells (HCs) under dark-and light-adapted conditions were studied by whole-cell voltage clamp and intracellular recording techniques in retinal slices and whole-mounted isolated retinas of the larval tiger salamander. Puff application of GABA in picrotoxin elicited a NO-711 (a potent GABA transporter blocker)-sensitive inward current that did not exhibit a reversal potential in the physiological range, consistent with the idea that these HCs contain electrogenic GABA transporters. Application of GABA in NO-711 elicited a chloride current in HCs; about half of the current was suppressed by bicuculline or I4AA (a GABA(C) receptor antagonist), and the remaining half was suppressed by bicuculline + I4AA or picrotoxin. In whole-mount retinas, NO-711, bicuculline, I4AA, or picrotoxin hyperpolarized the HCs and enhanced the light responses under dark-adapted conditions, and blocked the time-dependent recovery of HC membrane potential and light responses during background illumination. Based on the parallel conductance model, GABA released in darkness mediates a chloride conductance about three times greater than the leak conductance or the glutamate-gated cation conductance. About half of this chloride conductance is mediated by GABA(A) receptors, and the other half is mediated by GABA(C) receptors. These results suggest that GABA released from HCs through the NO-711-sensitive GABA transporters activates GABA(A) and GABA(C) receptors, resulting in chloride conductance increase which leads to a HC depolarization and reduction of the light response. Additionally, GABA transporters also mediate GABA release in background light that is responsible for the recovery of HC membrane potential and light responses.     

Epidermal growth factor receptor transactivation by the cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) induces differential responses in corneal epithelial cells

Corneal epithelial injury induces release of endogenous metabolites that are cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1) agonists. We determined the functional contributions by CB1 and TRPV1 activation to eliciting responses underlying wound healing in human corneal epithelial cells (HCEC). Both the selective CB1 and TRPV1 agonists (i.e., WIN55,212-2 [WIN] and capsaicin [CAP], respectively) induced EGFR phosphorylation whereas either inhibition of its tyrosine kinase activity with AG1478 or functional blockage eliminated this response. Furthermore, EGFR transactivation was abolished by inhibitors of proteolytic release of heparin bound EGF (HB-EGF). CB1-induced Ca²⁺ transients were reduced during exposure to either the CB1 antagonist, AM251 or AG1478. Both CAP and WIN induced transient increases in Erk1/2, p38, JNK1/2 MAPK and Akt/PI-3K phosphorylation status resulting in cell proliferation and migration increases which mirrored those elicited by EGF. Neither EGF nor WIN induced any increases in IL-6 and IL-8 release. On the other hand, CAP-induced 3- and 6-fold increases, which were fully attenuated during exposure to CPZ, but AG1478 only suppressed them by 21%. The mixed CB1 and TRPV1 antagonist, AM251, enhanced the CAP-induced rise in IL-8 release to a higher level than that elicited by CAP alone. In conclusion, CB1 and TRPV1 activation induces increases in HCEC proliferation and migration through EGFR transactivation leading to global MAPK and Akt/PI-3K pathway stimulation. On the other hand, the TRPV1-mediated increases in IL-6 and IL-8 release are elicited through both EGFR dependent and EGFR-independent signaling pathways.     

Adenosine A2A receptor mediated protective effect of 2-(6-cyano-1-hexyn-1-yl)adenosine on retinal ischaemia/reperfusion damage in rats

AIMS: To determine the effect of 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado), an adenosine A2A receptor agonist, on retinal ischaemia/reperfusion damage in rats. METHODS: Retinal ischaemia/reperfusion damage was induced by elevating the intraocular pressure of one eye to 130 mm Hg for 60 minutes and returning it to normal. 7 days later, retinal ischaemia/reperfusion damage was histologically quantified by measuring the thickness of retinal layers. Intraocular pressure was measured by pressure transducer. RESULTS: Retinal ischaemia/reperfusion caused cell loss in the ganglion cell layer and thinning of the inner plexiform and nuclear layer. Both ocular topical and intravenous administration of 2-CN-Ado caused a reduction of retinal ischaemia/reperfusion damage. A selective A2A receptor antagonist, 1,3,7-trimethyl-8-(3-chlorostyryl) xanthine (CSC), but not a selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), or a selective A2B receptor antagonist, alloxazine, reduced the protective effect of 2-CN-Ado. While ocular topical administration of 2-CN-Ado caused a sustained reduction of intraocular pressure, intravenous administration of 2-CN-Ado showed a transient ocular hypotensive effect. CONCLUSIONS: These results suggest that 2-CN-Ado attenuates retinal ischaemia/reperfusion damage, and at least some of this protective effect of 2-CN-Ado might be mediated via activation of the adenosine A2A receptor.     

Expression of the gamma-aminobutyric acid (GABA) plasma membrane transporter-1 in monkey and human retina

PURPOSE: To determine the expression pattern of the predominant gamma-aminobutyric acid (GABA) plasma membrane transporter GAT-1 in Old World monkey (Macaca mulatta) and human retina. METHODS: GAT-1 was localized in retinal sections by using immunohistochemical techniques with fluorescence and confocal microscopy. Double-labeling studies were performed with the GAT-1 antibody using antibodies to GABA, vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH), and the bipolar cell marker Mab115A10. RESULTS: The pattern of GAT-1 immunostaining was similar in human and monkey retinas. Numerous small immunoreactive somata were in the inner nuclear layer (INL) and were present rarely in the inner plexiform layer (IPL) of all retinal regions. Medium GAT-1 somata were in the ganglion cell layer in the parafoveal and peripheral retinal regions. GAT-1 fibers were densely distributed throughout the IPL. Varicose processes, originating from both the IPL and somata in the INL, arborized in the outer plexiform layer (OPL), forming a sparse network in all retinal regions, except the fovea. Sparsely occurring GAT-1 processes were in the nerve fiber layer in parafoveal regions and near the optic nerve head but not in the optic nerve. In the INL, 99% of the GAT-1 somata contained GABA, and 66% of the GABA immunoreactive somata expressed GAT-1. GAT-1 immunoreactivity was in all VIP-containing cells, but it was absent in TH-immunoreactive amacrine cells and in Mab115A10 immunoreactive bipolar cells. CONCLUSIONS: GAT-1 in primate retinas is expressed by amacrine and displaced amacrine cells. The predominant expression of GAT-1 in the inner retina is consistent with the idea that GABA transporters influence neurotransmission and thus participate in visual information processing in the retina.     

AMPA glutamate receptor subunit 2 in normal and visually deprived macaque visual cortex

Glutamate and its various receptors are known to play an important role in excitatory synaptic transmission throughout the CNS, including the primary visual cortex. Among subunits of the AMPA receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), subunit 2 (GluR2) is of special significance because it controls their Ca2+ permeability. In the past, this subunit has been studied mostly in conjunction with other AMPA subunits. The present study sought to determine if GluR2 alone has a distinct laminar distribution in the normal macaque visual cortex, and if its pattern correlated with that of cytochrome oxidase (CO) under normal and monocularly deprived conditions. In the normal adult cortex, GluR2 immunoreactivity (ir) had a patchy distribution in layers II/III, in register with CO-rich puffs. GluR2-ir highlighted the upper border of layer II, the lower border of layer IV (previously termed IVC(beta dark)) and, most prominently, layer VI. Labeled neurons were primarily of the pyramidal type present in the upper border and lower half of layer VI, layers II/III, and scattered in layers V and upper IVB. Labeled nonpyramidal cells were large in layer IVB and small in IVC(beta dark). Notably, the bulk of CO-rich layers IVC and IVA had very low levels of GluR2-ir. At fetal day 13, however, GluR2 labeling showed a honeycomb-like pattern in layer IVA not found in the adult. A fragment of GluR2 cDNA was generated from a human cDNA library, and in situ hybridization revealed an expression pattern similar to that of GluR2 proteins. After 1-4 weeks of monocular impulse blockade with tetrodotoxin (TTX), alternating rows of strong and weak GluR2-ir in layers VI and II/III appeared in register with CO-labeled dark and light ocular dominance columns in layer IVC and puffs in II/III, respectively. Our results indicate that various cortical layers are differentially influenced by glutamate. The bulk of the major geniculate-recipient layers IVC and IVA have low levels of GluR2, presumably favoring synaptic transmission via Ca(2+)-permeable glutamate receptors. GluR2 plays a more important role in supragranular and infragranular layers, where the initial geniculate signals are further modified and are transmitted to other cortical and subcortical centers. The maintenance of GluR2 in these output layers is governed by visual input and neuronal activity, as monocular impulse blockade induced a down-regulation of this subunit in deprived ocular dominance columns.     

Discoidin domain receptor (DDR) 1 and 2: collagen-activated tyrosine kinase receptors in the cornea

Discoidin domain receptor (DDR) 1 and 2 have recently been found to serve as receptors for several collagen types. These receptors have been found to modulate cell proliferation and metalloprotease expression in response to collagen stimulation. The purpose of this study was to examine expression of DDR1 and DDR2 in the cornea and to determine the effect of several collagen types on proliferation and response to pro-apoptotic cytokines by corneal fibroblasts. DDR1 and DDR2 mRNAs were detected by RT-PCR. Proteins were detected by immunocytochemistry and immunoprecipitation with Western blotting. Cell proliferation in response to acetic acid-solubilized collagen type I, II, IV, IX or X was determined by cell counting. The effect of these collagen types on Fas-stimulating antibody-induced cell death was determined by trypan blue assay. DDR1 and DDR2 mRNAs were detected in each major human cell type of the cornea. Both were also detected in ex vivo human corneal epithelium. DDR1 and DDR2 proteins were detected in all three major cell types in culture and in human corneal tissue. Collagen types I, II, IV, IX and X stimulated proliferation, but had no effect on Fas-mediated apoptosis, of corneal fibroblasts. DDR1 and DDR2 tyrosine kinase receptors are expressed in the cornea. Collagen-stimulated mitosis of corneal fibroblasts in culture is likely mediated by the DDR receptors. Collagen had no effect on Fas-mediated apoptosis of corneal fibroblasts.     

NMDA受体亚单位NR2A、NR2B在正常大鼠视皮层中的表达变化

目的 观察大鼠视皮层中N-甲基-D-天门冬氨酸受体(NMDAR)功能亚单位NR2A与NR2B的表达随发育的变化.方法 运用免疫组织化学和图像分析技术分析NR2A、NR2B在Wistar大鼠生后第0、14、21、28、35、42、60、90d视皮层的表达情况.结果 NR2A、NR2B在出生时的视皮层中即已存在,之后NR2A的表达缓慢上升,生后35d达峰值,后略有下降,至成年维持于较高水平;NR2B生后14d达到峰值,继而逐渐下降,至第60d后趋于平稳.结论 视皮层中NR2A、NR2B表达量的变化与视觉发育可塑性关键期的起始与终止在时间上具有一定程度的同步性,提示生后NR2A、NR2B表达的发育性时间差异可能是视觉发育可塑性调节的一个重要分子基础.     

Comparison of the biological principles underlying the action of monoclonal antibody (mAb) and decoy receptor anti-VEGF agents--on the example of ranibizumab (anti-VEGF-A mAb) and aflibercept (decoy VEGFR1-2 receptor)

Current state-of-the-art anti-angiogenic therapies target the VEGF pathway, which is the main essential signaling pathway for angiogenesis, including pathological angiogenesis in cancer and eye disease. Ranibizumab (Lucentis) and VEGF-Trap (aflibercept) represent two different approaches to inhibiting angiogenesis by targeting VEGF family signaling. The former is a relatively short monoclonal antibody fragment, which binds VEGF-A on the basis of antigen recognition by the variable region of an antibody, while aflibercept is not an monoclonal antibody, but a decoy receptor, binding VEGF-A on the basis of the molecular interaction between the ligand (VEGF) and its cognate cellular receptor (VEGFR-1 and VEGFR-2). VEGF-Trap has therefore a broader specificity, recognizing and binding VEGF-B and PIGF in addition to VEGF-A, following the specificity of VEGFR-1 and VEGFR-2. This broader specificity is considered as beneficial in cancer treatment and could be also beneficial in treatment of nAMD, this claim should, however, be backed by clinical studies. The presence of an Fc fragment in VEGF-Trap is also an important difference; even though this fragment does not participate in the recognition of the target molecule, it can influence the biological properties of the fusion protein. The relative merits of both approaches will become clear only after long-term laboratory and clinical testing, as their biological activity is also likely to differ. Given the clear differences in the mechanism of target molecule recognition, biochemical and biophysical properties (including molecular weight) and specificity, they cannot be considered as equivalent, unless extensive long-term clinical studies prove otherwise.     

N-甲基-D-天门冬氨酸受体亚单位NR2B在正常大鼠视皮层中的表达变化

目的观察从出生至50大的大鼠视皮层中N-甲基-D-天门冬氨酸受体（N—methyl—D—aspartate receptor，NMDAR）功能亚单位NR2B的表达变化，为研究视觉刺激对视皮层NR2B的表达调控机制作准备。设计实验性研究。研究对象30只新生正常健康纯种Wistar大鼠。方法30只大鼠随机分为10组，每组3只，正常饲养，分别于出生后第0、5、10、15、20、25、30、35、40、50天处死，大鼠脑视皮层行30um厚冠状冰冻切片，应用免疫组织化学链霉亲和素-生物素-过氧化物酶复合物（streptomycin—avidin—biotin—peroxidase complex，SABC）法显色，显微镜观察NR2B的表达情况并进行图像分析。主要指标平均光密度（averageo ptical density，AOD）与综合光密度（integrated optical density，IOD）。结果NR2B在出生时的视皮层中即已存在，之后呈逐渐上升趋势，至出生后第15天达到峰值，继而逐渐下降，至第50天趋于平稳。结论在大鼠出生后0-15天，NR2B水平呈逐渐上升状态，提示NR2B对出生后大鼠的视觉发育有着重要的生理作用。自第15天大鼠睁眼起，NR2B水平逐渐下降，表明NR2B的弱化表达与视觉刺激密切相关。