Michael J. Neal

Effects of Ischaemia on Neurotransmitter Release from the Isolated Retina

Abstract: The effects of “ischaemia” (glucose‐free Krebs‐bicarbonate medium gassed with N2/CO2 on the release of glutamate and other major neurotransmitters in the retina were examined using the isolated rat and rabbit retina. Amino acid transmitters, acetylcholine, and dopamine were measured by HPLC. The release of glutamate, aspartate, GABA, and glycine from ischaemic retinas was more than doubled after 30 min, and after 90 min of ischaemia the release of amino acids was ∼ 15–20‐fold that of control values. Ischaemia also produced large increases in the release of dopamine from both the rat and especially the rabbit retina. In contrast, the release of acetylcholine from the rat retina was significantly decreased by ischaemia, although the release of choline was increased. Because the ischaemia‐induced release of glutamate, aspartate, and GABA from the rat retina was completely Ca independent, and exposure of the retina to high K (50 mM) did not stimulate amino acid release, it is concluded that the mechanisms underlying the ischaemia‐induced release do not involve an initial release of K or an influx of calcium.

Rescue of photoreceptor function by AAV-mediated gene transfer in a mouse model of inherited retinal degeneration

Knowledge of the mutations leading to inherited retinal degenerations provides a foundation for the development of somatic gene therapy in which potentially corrective genes are transferred to the target photoreceptor cells. Towards this end, we have evaluated the efficacy of a recombinant adeno-associated virus (AAV) vector to deliver and express the correct form of the cGMP phosphodiesterase-β (PDE-β) gene in the retinas of rd mice, which suffer rapid retinal degeneration due to recessive mutation in the endogenous gene. A truncated murine opsin promoter was used to drive expression of the PDE-β cDNA. Following intraocular injection of AAV.PDE-β, increased retinal expression of immunoreactive PDE protein was observed, including within photoreceptor cell bodies. Compared with age-matched controls, treated eyes showed increased numbers of photoreceptors and a two-fold increase in sensitivity to light as measured by in vitro electroretinography. These findings provide evidence that rescue of functional photoreceptor neurons can be achieved by somatic gene therapy.

Evidence for a Complex Influence of Nicotinic Acetylcholine Receptors on Hippocampal Serotonin Release

Abstract: The effects of nicotine on 5‐hydroxytryptamine (5‐HT)release from serotonergic nerve endings in rat dorsal hippocampal slices werestudied. Nicotine (50‐500 μM) caused a concentration‐dependentincrease in 5‐HT release. This effect was antagonised by mecamylamine (0.5μM), indicating an action at nicotinic receptors. Nicotine‐evoked5‐HT release was not affected by tetrodotoxin (3 μM), cadmiumchloride (0.1 mM), or the absence of Ca2+ orNa+ in the superfusion medium. Unexpectedly, higher concentrationsof mecamylamine alone (1‐50 μM) increased 5‐HT release. Thissuggested the presence of inhibitory input to 5‐HT neurones and that theseinhibitory neurones possess tonically active nicotinic receptors. The effectof mecamylamine (50 μM) on 5‐HT release was reduced by themuscarinic M1 receptor agonist, McN‐A‐343 (100 μM), butpirenzepine (0.005‐1 μM), which blocks M1 receptors,alone increased 5‐HT release. Hippocampal serotonergic neurones are known topossess both excitatory nicotinic receptors and inhibitory M1receptors. Although there may be several explanations for our results, onepossible explanation is that nicotine stimulates 5‐HT release by activatingnicotinic heteroreceptors on 5‐HT terminals. Mecamylamine (0.5 μM)antagonises this effect, but higher concentrations increase 5‐HT releaseindirectly by blocking the action of endogenous acetylcholine on nicotinicreceptors situated on cholinergic neurones that provide muscarinic inhibitoryinput to 5‐HT neurones.

Modulation by endogenous ATP of the light‐evoked release of ACh from retinal cholinergic neurones

The retina is an area of the central nervous system that possesses intrinsic cholinergic neurones which release acetylcholine (ACh) in response to stimulation with flickering light. Using an eye‐cup preparation in anaesthetized rabbits we found that when the retina was exposed to the P2‐purinoceptor antagonist, PPADS, the light‐evoked release of ACh was strikingly increased (by over 40%). In contrast, ATP reduced the light‐evoked release of ACh by 20%. The inhibitory effect of ATP was not due to its catabolism to adenosine because it was not affected by the Aradenosine receptor antagonist, DPCPX, in combination with adenosine deaminase. The actions of both ATP and PPADS were completely blocked by strychnine. We conclude that during physiological stimulation of the retina with light, ATP is co‐released with ACh and partially inhibits ACh release by activating (with ACh) an inhibitory glycinergic feed‐back loop.

Immunocytochemical evidence that vigabatrin in rats causes GABA accumulation in glial cells of the retina

Vigabatrin (gamma-vinyl-GABA, GVG) is an irreversible inhibitor of GABA-aminotransferase (GABA-T) that is under clinical trial as an antiepileptic drug. Rats were injected (i.p.) with GVG and killed 18 h later. GVG administration reduced retinal GABA-T activity to undetectable levels and increased the GABA content 5-fold. Immunocytochemistry using a GABA antiserum clearly revealed the presence of GABA-IR in the glial Muller cells of retinas from GVG-treated rats but not from controls. This experiment indicates that the administration of drugs which inhibit GABA-T may cause the accumulation of GABA in retinal cells that do not normally possess enough endogenous GABA to be detected by immunocytochemistry.

Characterization of Cell Death Pathways in Murine Retinal Neurodegeneration Implicates Cytochrome c Release, Caspase Activation, and Bid Cleavage

Apoptosis is considered to be the final common pathway of photoreceptor cell death in different inherited retinal diseases. However, apoptosis encompasses diverse pathways of molecular interactions culminating in cellular demise. To begin dissecting these interactions, we have investigated key participants in the rd (retinal degeneration) model of retinal neurodegeneration. By Western blot analysis and immunocytochemistry, we found that cytochrome c release occurs in rd retinas concurrently with the activation of the proapoptotic protein Bid. Active forms of caspase-8 and the mitogen-activated protein kinase p38, both of which are capable of cleaving Bid, were detected in rd retinas at the peak time of photoreceptor death. In addition, the activated form of the cell death effector caspase-3 was detectable particularly at the photoreceptors in parallel with this peak degenerative phase. These data suggest that activation of both major apoptotic pathways occurs during photoreceptor degeneration in the rd mouse model of inherited blindness.

Modulated expression of secreted frizzled-related proteins in human retinal degeneration.

Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive loss of photoreceptors, apparently by apoptosis, and our recent report of increased secreted Frizzled-related protein-2 (SFRP2) in RP retinas suggests altered Wnt signalling may be a component of the degenerative process. The present study shows that levels of SFRP1, SFRP3 and SFRP5 mRNAs are also abnormal in RP, giving rise to idiosyncratic expression patterns. In highly degenerative retinas, the SFRP proteins localize mainly to the inner limiting membrane, but in a well-preserved retina SFRP1 and SFRP5 are notably localized to the surviving photoreceptors. Together with increased c-jun mRNA expression in all cases examined, these results support the notion that disruptions of Wnt network signalling are involved in retinal neurodegeneration.

Modulation of extracellular GABA levels in the retina by activation of glial P2X‐purinoceptors

In the rat retina, γ‐aminobutyric acid (GABA) released as a transmitter is inactivated by uptake mainly into glial cells (Müller cells). Activation of P2‐purinoceptors in Müller cells increases [Ca2+]i and the present study was undertaken to see whether this action affected the glial release of [3H]‐GABA from the superfused rat isolated retina. Adenosine 5′‐triphosphate (ATP) and the P2X‐purinoceptor agonists, α,β‐methylene‐ATP (α,β‐meATP) and β,γ‐methyleneATP (β,γ‐meATP) significantly increased the KCl‐evoked release of [3H]‐GABA from the retina. Adenosine and the P2Y‐purinoceptor agonist, 2‐chloroATP, had no effect on the KCl‐evoked release of [3H]‐GABA from the retina. However, 2‐methylthioATP (2‐Me‐S‐ATP) significantly enhanced the evoked release of [3H]‐GABA. The effect of ATP on the glial release of [3H]‐GABA was abolished by the P2‐antagonist, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS). When the superfused retina was exposed to the GABA uptake inhibitor, SKF89976A, the enhancing effect of α,β‐meATP on the KCl‐evoked release of GABA was abolished. The KCl‐evoked release of [3H]‐GABA from the frog retina and rat cerebrocortical slices, which take up GABA mainly into neurones, was not affected by ATP or α,β‐meATP. We concluded that the glial Müller cells in the rat retina possess P2‐receptors, activation of which increases the ‘release’ of preloaded [3H]‐GABA apparently by reducing uptake. On balance, the results suggest the involvement of P2X‐purinoceptors, although we cannot exclude the possibility that P2Y‐purinoceptors may be involved. Our results suggest that ATP, as well as being a conventional transmitter in the retina, may be involved in neuronal‐glial signalling and modulate the extracellular concentration of GABA.

Nitric oxide inhibits depolarization-induced release of endogenous dopamine in the rabbit retina.

The effect of nitric oxide donor compounds (sodium nitroprusside, hydroxylamine and S-nitroso-N-acetyl-D,L-penicillamine) on depolarization-induced release of endogenous dopamine in the light-adapted, isolated retina of the rabbit was studied by HPLC. All three compounds had the same effect, reducing the amount of dopamine released by up to 90%. The effect was concentration dependent, saturating at 300 microM; it was blocked by the nitric oxide scavenger, mannitol (50 mM), which by itself had no effect on the basal release of dopamine. GABAA receptors were not involved. Possible cellular mechanisms underlying the findings are discussed. It is suggested that the inhibitory interaction between dopamine and nitric oxide could represent a higher order function in the light adaptation process in the retina.

Inhibition by nociceptin of the light-evoked release of ACh from retinal cholinergic neurones

The retina possesses cholinergic amacrine cells which release acetylcholine (ACh) in response to flickering light. Using an eye‐cup preparation in anaesthetized rabbits we found that when the retina was exposed to nociceptin, the light‐evoked release of ACh was reduced in a concentration‐dependent manner (IC50 = 100 nm), the maximum effect being 60% inhibition. Opioid receptors were not involved in the inhibitory effect of nociceptin because its action was not blocked by naloxone (1 μm) and furthermore μ‐opioids enhanced the light‐evoked release of ACh. Using rabbit retina homogenates we found that the retina possessed a substantial number of high‐affinity binding sites for [3H]‐nociceptin indicating the presence of ORLI‐receptors. Since [des‐Phe1]‐nociceptin, which has no affinity for the ORLI‐receptor, had no effect on the light‐evoked release of ACh it is unlikely that the action of nociceptin was simply non‐specific. We conclude that the inhibitory effect of nociceptin on retinal ACh release involves activation of the ORLI receptors.