Dominic Man-Kit Lam

Localization of GABA immunoreactivity in the auditory brainstem of guinea pigs

Using an antibody against GABA, neurons within the guinea pig hindbrain, midbrain and forebrain auditory nuclei were identified which demonstrate GABA-like immunoreactivity. GABA-positive cells were localized in the cochlear nucleus, superior olivary complex, lateral lemniscus, inferior colliculus, and medial geniculate body. GABA-positive terminals could be seen surrounding globular and spherical cells in ventral cochlear nucleus and principal cells in medial nucleus of the trapezoid body. In addition, numerous positive, punctate terminals appeared throughout the hindbrain auditory nuclei and, although fewer in number, in midbrain and forebrain auditory nuclei.

The emergence, localization, and maturation of neurotransmitter systems during development of the retina in Xenopus laevis. III. Dopamine

The uptake, synthesis, and release of dopamine was studied in retinas of Xenopus laevi. In the tadpole and adult retina, 3H‐dopamine is accumulated by cells located in the inner nuclear layer. Retinas preloaded with 3H‐dopamine release this compound in response to high K+ concentrations in the medium. This release is probably Ca++‐dependent as it is inhibited by Co++ in the medium. Adult retinas are also capable of synthesizing 3H‐dopamine from 3H‐tyrosine. The appearance and maturation of these dopaminergic properties were followed during retinal development. Our data indicate that synthesis of dopamine can first be detected as early as stage 35/36 whereas uptake of dopamine first occurs at stage 43. K+‐stimulated release of preloaded 3H‐dopamine from putative dopaminergic neurons is, however, not evident until stage 46. These results show that similar to the development of GABA‐ergic and glycinergic properties, the uptake, synthesis, and release mechanisms for dopamine emerge at different stages during retinal differentiation in Xenopus Laevis.

GABAergic ganglion cells in the rabbit retina

The ganglion cells are the output neurons of the retina. There is, however, relatively little known about the neurotransmitters used by these cells. In the present study, ganglion cells identified with a ganglion cell-specific monoclonal antibody (AB5) are shown in separate double-label experiments to be gamma-aminobutyric acid (GABA)-like immunoreactive and to possess a high-affinity uptake mechanism for [3H]GABA accumulation. The localization of these markers of GABA activity to AB5-labelled ganglion cells provides the first definitive evidence for the presence of a classical transmitter in retinal ganglion cells and suggests that GABA may perform a role as a neurotransmitter in these cells.

THE UPTAKE AND RELEASE OF [3H]DOPAMINE IN THE GOLDFISH RETINA

Abstract— The uptake and release of [3H]dopamine was studied in the goldfish retina with the following results: (1) when goldfish retinas were incubated with 2 ± 10‐7m‐[3H]dopamine for less than 20min and processed for autoradiography. most of the label was associated with dopaminergic terminals that contact certain horizontal cells. Biochemical analysis showed that > 93% of this label was [3H]‐dopamine. (2) [3H]dopamine uptake saturated with increasing dopamine concentration and followed Michaelis‐Menten kinetics. This uptake could be explained by a single ‘high‐affinity’ mechanism with a Km of 2.61 ± 0.41 ± 10‐7m and a Vmax of 66 ± 12 ± 10‐12 mol/min/mg protein. (3) [3H]dopamine uptake was temperature‐dependent with a temperature coefficient of 1.7 and an energy of activation of 11.4 kcal/mol. (4) The initial rate of uptake was unaffected by the absence of Ca2+ or the presence of Co2+; however, more than 85, uptake was blocked in the absence of external Na+. (5) Neither 1 mm‐cyanide nor 5 mm‐iodoacetate blocked more than 30% of uptake individually; however, in combination > 70% of uptake was blocked. (6) Centrally acting drugs benztropine and diphenylpyraline inhibited at least 60–70% of [3H]dopamine uptake. (7) [3H]dopamine in the retina could be released by increasing the external K+ concentration. This release was Ca2+ ‐dependent and was blocked by 10mm‐Co2+ or 2Omm‐Mg2+. The amount of [3H]dopamine released was not affected by the presence of benztropine, diphenylpyraline or fluphenazine in the incubation medium. These studies add further support for dopamine as a neurotransmitter used by interplexiform cells of the goldfish retina.

Visual cycle in the mammalian eye. Retinoid-binding proteins and the distribution of 11-cis retinoids

This work was designed to provide an insight into the mammalian visual cycle by investigating the possible function of retinoid-binding proteins in this system, and the distribution and type of 11-cis retinoids present in the interphotoreceptor matrix and the cytosols of the retinal pigment epithelium and retina. The total retinol and retinal in the soluble fractions from these three compartments was 8% (3.31 nmol/eye) of the retinyl palmitate and stearate stored in the pigment epithelium membrane fractions (39 nmol/eye). Only small amounts of retinoids were detected in the rod outer segment cytosol. The insoluble fractions also contained retinol, nearly all of which was found in the retina. The retinoids in the soluble fractions appeared to be bound to cellular retinol-binding protein (CRBP), cellular retinal-binding protein (CRA1BP) and interstitial retinol-binding protein (IRBP, a high-Mr glycoprotein). Using immunospecific precipitation, immunoblot and immunocytochemical techniques it was demonstrated that IRBP was localized in the interphotoreceptor matrix and was synthesized and secreted by the retina, a process that did not require the protein to be glycosylated. The amount of retinol bound to IRBP increased if the eyes were exposed to light, when it was estimated that the protein carried up to 30% of its full capacity for all-trans retinol. In addition to all-trans retinol, IRBP carried smaller amounts of 11-cis retinol. The proportion of 11-cis retinol was frequently higher in eyes that had been protected from illumination, suggesting that IRBP plays a role in rhodopsin regeneration during dark-adaptation. Additionally, endogenous 11-cis retinoids in the retina and RPE cytosols were bound to an Mr 33,000 protein tentatively identified as CRA1BP. The 11-cis retinoid in the retina cytosol was mainly in the form of retinol, while in the RPE cytosol it was mainly in the form of retinal. Substantial amounts of 11-cis retinol were also found in the insoluble (membrane) fraction from the retina. It is suggested that in the mammalian retina 11-cis retinol is generated from all-trans retinol (possibly in the Muller cells). Lack of an 11-cis retinol oxidoreductase in the retina prevents it from being utilized for rhodopsin regeneration until it has been transported to the pigment epithelium, where it is converted to 11-cis retinal and returned to the rod outer segments. It is also suggested that IRBP may be implicated in the transport of retinoids between the rod outer segments, the Muller cells and the pigment epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

The signature hypothesis: Co-localizations of neuroactive substances as anatomical probes for circuitry analyses

The recent discoveries that a neuron in the vertebrate retina may contain more than one neuroactive substance (transmitter or neuropeptide) raise the possibility that within each class of neurons, every morphologically and physiologically distinct cell type may be uniquely identified and categorized by the neuroactive substances that it contains. This article examines the evidence to-date for such a conjecture and discusses some of its potential applications and implications.

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

The localization of γ-aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [3H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [3H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Abstract Dopamine receptors in the goldfish retina have been examined by binding studies using 3 H-spiroperidol and 3 H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D 2 receptor antagonist, 3 H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D 1 type dopamine receptors and few, if any, D 2 type receptors.

Purification of L-glutamic acid decarboxylase from catfish brain.

—L‐Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS‐polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD‐anti‐GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti‐GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD‐anti‐GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.

The presence of three neuroactive peptides in putative glycinergic amacrine cells of an avian retina

Amacrine cells are retinal interneurons which serve to mediate transmission between bipolar and ganglion cells. To date, in addition to several classical neurotransmitters, a number of neuroactive peptides have been localized to these cells. We have previously demonstrated in the chicken that both peptide-transmitter and peptide-peptide colocalization exists in some amacrine cells. In this report, we show that 3 neuroactive peptides (enkephalin, neurotensin and somatostatin) are present in subpopulations of amacrine cells which also possess a high affinity uptake system for glycine. These observations suggest that the simultaneous visualization by autoradiography of [3H]glycine-uptake and immunocytochemistry of peptides may be useful for distinguishing between different types of putative glycinergic amacrine cells.