Cheryl K. Mitchell

GABA and GABA-A receptors are maximally expressed in association with cone synaptogenesis in neonatal rabbit retina

Previous studies have shown the cone photoreceptors form reciprocal synapses with horizontal cells during the first week after birth in rabbits. These synapses constitute pioneering elements of the developing outer plexiform layer. We now report that antibodies against the alpha-1 and against the beta-2/3 subunits of the GABA-A receptor label a highly restricted sublamina in the developing outer plexiform layer known to contain nascent cone photoreceptor terminals. Staining is relatively weak at birth, increases to maximal levels between postnatal days 5 and 7, and is significantly reduced in the adult. These results support recent calcium imaging studies which have shown that the activation of GABA-A receptors causes an increase in intracellular free calcium in cones, an effect which is observed only at 3-9 days after birth. The transient expression of GABA-A receptors in this region coincides with the period of peak expression of GABA immunoreactivity in horizontal cells. A direct functional link between GABAergic transmission and cone synaptogenesis is suggested by previous reports that GABA-A receptor antagonists cause disruption of cone synaptogenesis. Together these findings support the notion that GABA functions as a developmental neurotransmitter which is produced by horizontal cells and interacts with developing cone axons in order to facilitate synaptic linkage between these two cells types.

Darkness stimulates rapid synthesis and release of melatonin in rat retina

The presence of melatonin in retina has been widely reported for over two decades although studies of its functional importance within the retina have only recently been emphasized. We have analyzed the biochemical characteristics of melatonin synthesis and release, focusing on rapid changes in response to light/dark conditions. Our major findings are consistent with the following conclusions: (1) melatonin synthesis is stimulated within minutes after exposure to darkness, and may reflect an increase in N-acetyl transferase activity; (2) melatonin is not stored, but rather it diffuses freely throughout the retina immediately after it is synthesized; and (3) the dark-induced increase in retinal melatonin release is a synthesis-coupled response and does not involve separate secretion mechanisms. The characteristics of melatonin synthesis and release described herein would be consistent with the proposed role of melatonin as a local paracrine effector of dark-adaptive responses in retina.

Development of the glutamate system in rabbit retina

We have investigated two characteristics of the glutamate system in the developing rabbit retina. 1) Glutamate immunoreactivity was observed at birth within developing processes of four cell types; two of which, photoreceptors and ganglion cells, are known to be glutamatergic in the adult. Two other cell types, type A horizontal cells and amacrine cells, are immunoreactive to both glutamate and GABA at birth, suggesting that endogenous pools of glutamate in GABAergic neurons serve as precursor for GABA synthesis. Thus it appears that endogenous glutamate pools are present within neurons prior to synaptogenesis as part of the early expression of either the glutamate or GABA transmitter phenotype. 2) Analysis of3H-glutamate metabolism during retinal development showed that rapid conversion of glutamate to glutamine does not occur until the second postnatal week, coincident with the expression of Muller (glial) cell activity. In the absence of glial metabolism in the neonate, extracellular concentrations of glutamate remain relatively high and are likely to have major effects on neuronal maturation.

N-methyl-D-aspartate-mediated glutamate toxicity in the developing rabbit retina

Extracellular levels of endogenous glutamate are relatively high in the developing rabbit retina but nonetheless appear to promote cell survival and developmental processes at concentrations considered toxic in the adult. We wished to examine the development of retinal susceptibility to glutamate toxicity as well as the protective effects of two N‐methyl‐D‐aspartate (NMDA) antagonists, 2‐amino‐5‐phosphono‐5‐valeric acid (APV) and dextromethorphan (Dex), and the nitric oxide synthase (NOS) inhibitor, NG‐methyl‐L‐arginine (metARG). One day in vitro retinal explants of adult and neonatal rabbits were incubated with various agonists and antagonists, and stained with trypan blue to visualize necrotic cells. The density of the necrotic cells was analyzed using the Zeiss Videoplan 2. Immature neurons were approximately 10‐fold less sensitive to NMDA toxicity compared to the adult. Although both NMDA antagonists and met ARG provided marked protection for adult retinal neurons against glutamate toxicity, the modest susceptibility of the immature neuron was blocked only by Dex and not APV or metARG. At least two factors may contribute to the ability or the neonatal retina to survive in the presence of high levels of endogenous extracellular glutamate. First, the 10‐fold developmental increase in NMDA toxicity occurs simultaneously with a 12–15‐fold downregulation of extracellular glutamate, probably through the actions of maturing Muller cells. Second, the NMDA/NO excitotoxic pathway may not be active at birth since an NOS inhibitor had little effect at this stage and our previous morphological data demonstrate that NOS‐containing cells are not present in their mature configuration until the second postnatal week. J. Neurosci. Res. 47:416–426, 1997.

Melatonin inhibits ACh release from rabbit retina.

Previous studies have suggested that melatonin, released from photoreceptors, may modulate retinal dark-adaptive responses by inhibition of dopamine release from retinal interneurons. We have broadened these studies to examine the effect of melatonin on release of another retinal neurotransmitter, acetylcholine (ACh). The ACh system in rabbit retina has been localized to starburst amacrine cells, which release ACh in response to a variety of experimental stimuli, including direct potassium depolarization, flashing light, and glutamatergic as well as GABAergic inputs. The effect of melatonin on release of endogenously synthesized [3H]-ACh was measured in perfusates from retinas or retinal synaptosomes preloaded with [3H]-choline chloride. Melatonin significantly inhibited ACh release stimulated by potassium in intact retina but not in synaptosomes. Stimulation of intact retina by flashing light or by the glutamate receptor agonist, kainic acid, was also inhibited by melatonin. In contrast, there was no significant effect of melatonin on picrotoxin-induced release. These findings suggest that melatonin does have an inhibitory effect on ACh release, either by direct interaction with the cholinergic amacrine cell, or indirectly via GABAergic but not glutamatergic neurons.

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.

2-Amino-4-phosphonobutyric acid and N-methyl-d-aspartate differentiate between [3H]glutamate and [3H]aspartate binding sites in bovine retina

Three pharmacologically and kinetically distinct binding sites for acidic amino acids were observed in synaptic membranes from bovine retina. One site preferentially binds aspartate with an apparent Kd of 6.3 nM and Bmax of 0.033 pmol/mg protein. [3H] Glutamate binds preferentially to two sites which have KdS of approximately 10 and 800 nM; and BmaxS of 0.148 and 0.417 pmol/mg protein, respectively. Only one of these sites, the 800 nM glutamate site, displayed a pharmacological specificity which was in extensive agreement with that reported for the putative glutamate receptor previously localized to a specific set of retinal neurons, the on-center, depolarizing bipolar cells.

Calbindin immunoreactivity of horizontal cells in the developing rabbit retina

Horizontal cells are retinal interneurons that establish inhibitory feedback loops within the outer plexiform layer of the primary visual pathway. Most mammalian retinas contain two types of horizontal cells. A-type horizontal cells have neuritic branches that contact cone photoreceptors exclusively, while the B-type horizontal cells have dendritic branches that contact cones, in addition to axons that form synapses with rod photoreceptors. Immunoreactivity for calbindin, a calcium binding protein involved in calcium transport, was used as a marker for horizontal cells during post-natal development of the rabbit retina. On post-natal days 1, 3 and 5, calbindin immunoreactivity is limited to a single population of A-type horizontal cells. They appear as a monolayer of cells with broad tapering processes, establishing the proximal border of the nascent outer plexiform layer and forming a target for ingrowing cone photoreceptor terminals. The size and density of the cell bodies and the length of neuritic processes are essentially unchanged during this period, which corresponds to the time of peak expression of GABAergic markers in horizontal cells. Coincident with a decrease in GABAergic markers and the completion of cone-to-horizontal cell synaptogenesis by day 7, changes within the horizontal cell mosaic are detected morphometrically. A delayed phase of overall cell growth results in a 70% increase in average somal diameter (representing a 3.7-fold increase in spherical volume), a six-fold increase in mean neurite length and a decrease in cell density to one-third of that found in the newborn. We conclude that the process of terminal differentiation of horizontal cells is not complete until some time after the second post-natal week. Furthermore, the expression of GABAergic markers is associated primarily with early maturational events, whereas expression of calbindin is sustained throughout post-natal development, suggesting a prominent role for calcium dependent mechanisms at all development stages.

GABA and GABAA 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.

Two-color fluorescent analysis of connexin 36 turnover: relationship to functional plasticity

ABSTRACT Gap junctions formed of connexin 36 (Cx36, also known as Gjd2) show tremendous functional plasticity on several time scales. Changes in connexin phosphorylation modify coupling in minutes through an order of magnitude, but recent studies also imply involvement of connexin turnover in regulating cell–cell communication. We utilized Cx36 with an internal HaloTag to study Cx36 turnover and trafficking in cultured cells. Irreversible, covalent pulse-chase labeling with fluorescent HaloTag ligands allowed clear discrimination of newly formed and pre-existing Cx36. Cx36 in junctional plaques turned over with a half-life of 3.1 h, and the turnover rate was unchanged by manipulations of protein kinase A (PKA) activity. In contrast, changes in PKA activity altered coupling within 20 min. New Cx36 in cargo vesicles was added directly to existing gap junctions and newly made Cx36 was not confined to points of addition, but diffused throughout existing gap junctions. Existing connexins also diffused into photobleached areas with a half-time of less than 2 s. In conclusion, studies of Cx36-HaloTag revealed novel features of connexin trafficking and demonstrated that phosphorylation-based changes in coupling occur on a different time scale than turnover. Summary: Fluorescent labeling of Cx36-HaloTag revealed that turnover occurs on a different time scale than phosphorylation-driven plasticity. Cx36-HaloTag is also partially mobile within gap junctions.