Su-Ja Oh

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina

Connexin 36 (Cx36) is a channel‐forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double‐labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.

Upregulation of ciliary neurotrophic factor in reactive Müller cells in the rat retina following optic nerve transection.

We have investigated the expression and cellular localization of ciliary neurotrophic factor (CNTF) in the rat retina following optic nerve transection. In the normal retina, CNTF immunoreactivity was restricted to profiles in the ganglion cell layer. Following optic nerve transection, immunoreactivity appeared in Müller cell somata and processes and its intensity increased between three and seven days post-lesion. Quantitative evaluation by immunoblotting confirmed that CNTF expression increased continuously up to 7 days after optic nerve transection (to 430% of control levels), but decreased again to 250% of controls at 4 weeks post-lesion. Our findings suggest that CNTF supplied by Müller cells may play a protective role for axotomized ganglion cells in the rat retina.

Immunocytochemical localization of nitric oxide synthase in the mammalian retina

The localization of nitric oxide synthase (NOS) was investigated by immunocytochemistry and immunoblotting using an antiserum against neuronal NOS in the rat, mouse, guinea pig, rabbit and cat retinae. Western blot analysis of retinal tissue extracts showed that the NOS-immunoreactive band of 155 kDa was present in all species. In the rat, mouse, guinea pig and rabbit retinae, two types of amacrine cells and a class of displaced amacrine cells were consistently NOS-labeled. In the cat retina, unlike other mammals, one type of amacrine cells and two types of displaced amacrine cells showed NOS immunoreactivity. NOS immunoreactivity was further found in some bipolar cells of the rat and guinea pig, some interplexiform cells of the mouse, some photoreceptor cells of the rabbit and some Müller cells of the cat.

Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina

Aquaporin 1 (AQP1; also known as CHIP, a channel‐forming integral membrane protein of 28 kDa) is the first protein to be shown to function as a water channel and has been recently shown to be present in the rat retina. We previously showed (Kim et al. [ 1998 ] Neurosci Lett 244:52–54) that AQP1‐like immunoreactivity is present in a certain population of amacrine cells in the rat retina. This study was conducted to characterize these cells in more detail. With immunocytochemistry using specific antisera against AQP1, whole‐mount preparations and 50‐μm‐thick vibratome sections were examined by light and electron microscopy. These cells were a class of amacrine cells, which had symmetric bistratified dendritic trees ramified in stratum 2 and in the border of strata 3 and 4 of the inner plexiform layer (IPL). Their dendritic field diameters ranged from 90 to 230 μm. Double labeling with antisera against AQP1 and γ‐aminobutyric acid or glycine demonstrated that these AQP1‐like‐immunoreactive amacrine cells were immunoreactive for glycine. Their most frequent synaptic input was from other amacrine cell processes in both sublaminae a and b of the IPL, followed by a few cone bipolar cells. Their primary targets were other amacrine cells and ganglion cells in both sublaminae a and b of the IPL. In addition, synaptic output onto bipolar cells was rarely observed in sublamina b of the IPL. Thus, the AQP1 antibody labels a class of glycinergic amacrine cells with small to medium‐sized dendritic fields in the rat retina. J. Comp. Neurol. 452:178–191, 2002.

Nitric oxide is involved in sustained and delayed cell death of rat retina following transient ischemia

We have investigated the role of nitric oxide (NO) in the rat retina following ischemic injury induced by transient increase of intraocular pressure. The thickness of both the inner plexiform layer and inner nuclear layer decreased during early postischemic stages (up to 1 week). In late postischemic stages (2-4 weeks), the thickness of the outer nuclear layer (ONL) decreased markedly. Thus, mechanisms other than excitotoxic ones may contribute to postischemic retinal cell death. Treatment of rats with N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, significantly reduced ischemic damage. Our findings suggest that NO is involved in the mechanism of ischemic injury, and plays a key role in the delayed and sustained cell death in the ONL following transient retinal ischemia.

Up-regulated CNTF plays a protective role for retrograde degeneration in the axotomized rat retina.

Using reverse transcription-polymerase chain reaction and in situ hybridization, we investigated the expression and cellular localization of ciliary neurotrophic factor receptor α (CNTFRα) in the rat retina following optic nerve transection (ONT). Following ONT, a signal for CNTFRα mRNA appeared in a layer-specific and time-dependent manner. In the ganglion cell layer, the signal showed a peak value 1 day after ONT, and then gradually decreased. In the inner nuclear layer the signal reached a peak value at 14 days of about 500% of control level, but then decreased at 4 weeks. Our findings suggest that CNTF might play a protective role for the retrograde degeneration of retinal cells induced by ganglion cell death in the rat retina following ONT.

Differential expression and cellular localization of doublecortin in the developing rat retina

Doublecortin is 40 kDa microtubule‐associated phosphoprotein required for neuronal migration and differentiation in various regions of the developing central nervous system. We have investigated the expression and cellular localization of doublecortin in the developing rat retina using immunocytochemistry and Western blot analysis. The expression of doublecortin was high from embryonic day 18 (E18) until E20 and was low during the postnatal period. The doublecortin immunoreactivity first appeared in a few radially orientated cells in the mantle zone of the primitive retina at E15. From E16 onward, the immunoreactivity appeared in two different regions: the inner part of the retina and middle of the neuroblastic layer. In the inner part, the somata of cells in the ganglion cell layer, in the distal row of the neuroblastic layer and profiles in the inner plexiform layer showed doublecortin immunoreactivity up to postnatal day 1 (P1). Afterwards, the doublecortin immunoreactivity persisted in the inner plexiform layer until P15, although the intensity decreased gradually with the maturation of the retina. In the middle of the neuroblastic layer, doublecortin immunoreactivity appeared in the radially orientated cells. These cells transformed into horizontal cells. The doublecortin immunoreactivity persisted in these cells up to P21. Given these results, doublecortin may play an important role in the migration and differentiation of specific neuronal populations in developmental stages of the rat retina.

Neuronal nitric oxide synthase immunoreactive neurons in the mammalian retina.

The development of immunocytochemistry has led to a better understanding of synaptic transmission carried out by neuroactive substances in the mammalian brain, including the retina. In the mammalian retina, nitric oxide (NO) is widely accepted as a neuromodulator. Histochemistry based on NADPH‐d and immunocytochemistry based on nitric oxide synthase (NOS) have been used to identify the presence of nitric oxide in the mammalian retina. Certain types of amacrine cells and a class of displaced amacrine cells have been labeled consistently in all mammalian retinae studied to date. Other cell types showing NADPH‐d reactivity or NOS immunoreactivity varied between species. NADPH‐d reactive or NOS immunoreactive amacrine cells may serve as a source of NO for amacrine, bipolar, and ganglion cells in the inner retina, whereas interplexiform cells, bipolar cells, and horizontal cells may serve as a source of NO for the outer retina of mammals. Microsc. Res. Tech. 50:112–123, 2000.

Light and electron microscopical analysis of nitric oxide synthase-like immunoreactive neurons in the rat retina.

We have investigated the morphology of the NOS-like immunoreactive neurons and their synaptic connectivity in the rat retina by immunocytochemistry using antisera against nitric oxide synthase (NOS). In the present study, several types of amacrine cells were labeled with anti-NOS antisera. Type 1 cells had large somata located in the inner nuclear layer (INL) with long and sparsely branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). Somata of type 2 cells with smaller diameters were also located in the INL. Their fine processes branched mostly in stratum 3 of the IPL. A third population showing NOS-like immunoreactivity was a class of displaced amacrine cells in the ganglion cell layer (GCL). Their soma size was similar to that of the type 1 cells; however, their processes stratified mainly in strata 4 and 5 of the IPL. Labeled neurons were evenly distributed throughout the retina, and the mean densities were 57.0 +/- 9.7 cells/mm2 for the type 1 cells, 239.3 +/- 43.4 cells/mm2 for the type 2 cells and 121.2 +/- 27.5 cells/mm2 cells for the displaced amacrine cells. The synaptic connectivity of NOS-like immunoreactive amacrine cells was identified in the IPL by electron microscopy. NOS-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in all strata of the IPL. The most frequent postsynaptic targets of NOS-immunoreactive amacrine cells were other amacrine cell processes. Ganglion cell dendrites were also postsynaptic to NOS-like immunoreactive neurons in both sublaminae of the IPL. Synaptic outputs onto bipolar cells were observed in sublamina b of the IPL. In addition, a few synaptic contacts between labeled cell processes were observed. Our results suggest that NOS immunoreactive cells may be modulated by other amacrine cells and ON cone bipolar cells, and act preferentially on other amacrine cells.

Neuronal nitric oxide synthase is expressed in the axotomized ganglion cells of the rat retina

This study investigated the expression and cellular localization of neuronal nitric oxide synthase in the rat retina following optic nerve transection (ONT). In the normal rat retina, nNOS immunoreactivity was localized to amacrine cells and displaced amacrine cells. A few bipolar cells were also labeled. In the axotomized retina, ganglion cells showed nNOS immunoreactivity from 3 days after ONT, and these cells increased in number, peaking 5 days after ONT. Quantitative evaluation using immunoblotting confirmed that nNOS expression showed a peak value (255% of control levels) 5 days after ONT and decreased to 137% of controls by 28 days. These findings suggest that axotomized ganglion cells degenerate via NO-mediated excitotoxicity.