Tropism of engineered and evolved recombinant AAV serotypes in the rd1 mouse and ex vivo primate retina.

There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery-intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration (rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.

Signalling by melanopsin (OPN4) expressing photosensitive retinal ganglion cells.

Over the past two decades there have been significant advances in our understanding of both the anatomy and function of the melanopsin system. It has become clear that rather than acting as a simple irradiance detector the melanopsin system is in fact far more complicated. The range of behavioural systems known to be influenced by melanopsin activity is increasing with time, and it is now clear that melanopsin contributes not only to multiple non-image forming systems but also has a role in visual pathways. How melanopsin is capable of driving so many different behaviours is unclear, but recent evidence suggests that the answer may lie in the diversity of melanopsin light responses and the functional specialisation of photosensitive retinal ganglion cell (pRGC) subtypes. In this review, we shall overview the current understanding of the melanopsin system, and evaluate the evidence for the hypothesis that individual pRGC subtypes not only perform specific roles, but are functionally specialised to do so. We conclude that while, currently, the available data somewhat support this hypothesis, we currently lack the necessary detail to fully understand how the functional diversity of pRGC subtypes correlates with different behavioural responses, and ultimately why such complexity is required within the melanopsin system. What we are lacking is a cohesive understanding of how light responses differ between the pRGC subtypes (based not only on anatomical classification but also based on their site of innervation); how these diverse light responses are generated, and most importantly how these responses relate to the physiological functions they underpin.

Melanopsin-dependent photoreception provides earliest light detection in the mammalian retina.

BACKGROUND: The visual system is now known to be composed of image-forming and non-image-forming pathways. Photoreception for the image-forming pathway begins at the rods and cones, whereas that for the non-image-forming pathway also involves intrinsically photosensitive retinal ganglion cells (ipRGCs), which express the photopigment melanopsin. In the mouse retina, the rod and cone photoreceptors become light responsive from postnatal day 10 (P10); however, the development of photosensitivity of the ipRGCs remains largely unexplored. RESULTS: Here, we provide direct physiological evidence that the ipRGCs are light responsive from birth (P0) and that this photosensitivity requires melanopsin expression. Interestingly, the number of ipRGCs at P0 is over five times that in the adult retina, reflecting an initial overproduction of melanopsin-expressing cells during development. Even at P0, the ipRGCs form functional connections with the suprachiasmatic nucleus, as assessed by light-induced Fos expression. CONCLUSIONS: The findings suggest that the non-image-forming pathway is functional long before the mainstream image-forming pathway during development.

Addition of human melanopsin renders mammalian cells photoresponsive.

A small number of mammalian retinal ganglion cells act as photoreceptors for regulating certain non-image forming photoresponses. These intrinsically photosensitive retinal ganglion cells express the putative photopigment melanopsin. Ablation of the melanopsin gene renders these cells insensitive to light; however, the precise role of melanopsin in supporting cellular photosensitivity is unconfirmed. Here we show that heterologous expression of human melanopsin in a mouse paraneuronal cell line (Neuro-2a) is sufficient to render these cells photoreceptive. Under such conditions, melanopsin acts as a sensory photopigment, coupled to a native ion channel via a G-protein signalling cascade, to drive physiological light detection. The melanopsin photoresponse relies on the presence of cis-isoforms of retinaldehyde and is selectively sensitive to short-wavelength light. We also present evidence to show that melanopsin functions as a bistable pigment in this system, having an intrinsic photoisomerase regeneration function that is chromatically shifted to longer wavelengths.

Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice.

In the mammalian retina, besides the conventional rod-cone system, a melanopsin-associated photoreceptive system exists that conveys photic information for accessory visual functions such as pupillary light reflex and circadian photo-entrainment. On ablation of the melanopsin gene, retinal ganglion cells that normally express melanopsin are no longer intrinsically photosensitive. Furthermore, pupil reflex, light-induced phase delays of the circadian clock and period lengthening of the circadian rhythm in constant light are all partially impaired. Here, we investigated whether additional photoreceptive systems participate in these responses. Using mice lacking rods and cones, we measured the action spectrum for phase-shifting the circadian rhythm of locomotor behaviour. This spectrum matches that for the pupillary light reflex in mice of the same genotype, and that for the intrinsic photosensitivity of the melanopsin-expressing retinal ganglion cells. We have also generated mice lacking melanopsin coupled with disabled rod and cone phototransduction mechanisms. These animals have an intact retina but fail to show any significant pupil reflex, to entrain to light/dark cycles, and to show any masking response to light. Thus, the rod-cone and melanopsin systems together seem to provide all of the photic input for these accessory visual functions.

The primary visual pathway in humans is regulated according to long-term light exposure through the action of a nonclassical photopigment.

BACKGROUND: The mammalian eye shows marked adaptations to time of day. Some of these modifications are not acute responses to short-term light exposure but rely upon assessments of the photic environment made over several hours. In the past, all attempts at a mechanistic understanding have assumed that these adaptations originate with light detection by one or other of the classical photoreceptor cells (rods or cones). However, previous work has demonstrated that the mammalian eye contains non-rod, non-cone photoreceptors. This study aimed to determine whether such photoreceptors contribute to retinal adaptation. RESULTS: In the human retina, second-order processing of signals originating in cones takes significantly longer at night than during the day. Long-term light exposure at night is capable of reversing this effect. Here, we employed the cone ERG as a tool to examine the properties of the irradiance measurement pathway driving this reversal. Our findings indicate that this pathway (1) integrates irradiance measures over time periods ranging from at least 15 to 120 min; (2) responds to relatively bright light, having a dynamic range almost entirely outside the sensitivity of rods; (3) acts on the cone pathway primarily through a local retinal mechanism; and (4) detects light via an opsin:vitamin A photopigment (lambda(max) approximately 483 nm). CONCLUSIONS: A photopigment with a spectral sensitivity profile quite different from those of the classical rod and cone opsins but matching the standard profile of an opsin:vitamin A-based pigment drives adaptations of the human primary cone visual pathway according to time of day.

Diurnal daylight phase affects the temporal properties of both the b-wave and d-wave of the human electroretinogram.

Aspects of the anatomy and physiology of the cone pathway are known to vary according to the phase of the natural light cycle. Using a prolonged flash stimulus ( approximately 200 ms), we have examined the human electroretinogram (ERG) over a 24 h period. We report that whilst the a-wave of the photopic ERG does not alter, there are profound effects upon the implicit times of both the b-wave and d-wave components. Both components are significantly slower in the night-time period and systematically become faster (15-22% reduction in implicit time), reaching a peak at around midday. The daily variation in the temporal properties of the ERG is abolished by constant light, but is retained during constant darkness. The data suggest that the changes in the temporal properties of the cone pathway affect both cone-ON and cone-OFF pathways. This suggests that the diurnal effect is presynaptic to the second order neurones, and most likely resides in the cone synapse.

Functional dopamine deficits in the senile rat retina.

The activity of the endogenous retinal dopamine (DA) pathway has been examined in the pigmented rat using retinas obtained from normal adult (approximately 3 months) and senile adults (approximately 24 months) using an in vitro electrophysiological approach. By comparing the pharmacological sensitivity of the horizontal cells (HCs) to exogenous DA, a D1 receptor antagonist (SCH 23390) and a DA-transport inhibitor (nomifensine), it is suggested that there is a functional deficit in the endogenous DA activity in the senile retina. Cells recorded from retinae obtained from senile animals are more sensitive to exogenous DA, whilst the senile retina is insensitive to SCH 23390. In addition, nomifensine was effective in potentiating subthreshold DA applications, but only in the normal adult retina. The data may suggest that endogenous DA release upon the HCs and selective re-uptake are suppressed in these retinae. These functional deficits also appear to be associated with changes in the receptive fields of the HCs, suggesting there is a corresponding deficit in spatial processing at the outer plexiform layer (OPL) of the senile rat.

Long-term light history modulates the light response kinetics of luminosity (L)-type horizontal cells in the roach retina.

We have examined the effects of prolonged periods of darkness on the responses of luminosity-type horizontal cells (L-HCs) in the freshwater cyprinid, Rutilus rutilus. Two groups of retinae were compared, those recorded after 10 min dark adaptation (SA) and those recorded after 3 h dark adaptation (LA). The results suggest that long-term light history does not modify the general responsiveness of the L-HCs in this species. However, there are apparent changes in the receptive field of the cells and modifications to the kinetics of the light-evoked response. The kinetics changes involve both a delay in the onset of light response and a selective effect on the hyperpolarizing light-ON response. Thus the mean time constant (tau) for the SA cells was 32.4+/-2.39 ms (n=62), whilst that for the LA cells was 53.4+/-3.03 ms (n=61). These effects occur in the absence of changes in the relative spectral sensitivity or threshold sensitivity of the HCs. The results suggest that in some vertebrate retinae, prolonged darkness (light-history) may regulate long-term plasticity in the kinetics of the cone-HC pathway.

Diurnal variation in the b-wave implicit time of the human electroretinogram.

Photopic electroretinograms (ERGs) elicited by light flashes were recorded for three normal human subjects who were exposed, throughout, to natural ambient light cycles over 24-h day-night periods. ERGs were recorded either with the adaptation state of the eyes maintained at the level set by the natural ambient lighting, or after 10 min dark adaptation. The amplitudes and implicit times of both the a- and b-wave components of the ERG were examined and of these, only the b-wave implicit time exhibited significant diurnal variation, such that the nighttime values were 20-40% greater than those recorded during daytime. Such diurnal variations were observed under both recording conditions and cannot, therefore, be attributed to diurnal changes in the adaptation state of the cone photoreceptors. ERGs were recorded at midday and midnight during 24-h exposure to the natural light cycle, but during the recording period, the short-term adaptation state of the eye was controlled by exposure to rod saturating background field, so that visual sensitivity was the same at both recording times. The b-wave implicit times recorded at midnight were, nonetheless, greater than those recorded at midday. This difference is not, therefore, determined by the short-term state of retinal adaptation, but reflects long-term light history. Measurements performed under 24-h continuous light exposure showed no variation in the b-wave implicit time, whereas some measurements made during extended dark adaptation provided limited evidence for implicit time changes. By controlling the wavelengths to which the eye was exposed during the daylight phase of the diurnal cycle, it was shown that the shifts in b-wave implicit time associated with the change from dark to light are triggered by the rod system, although they are most clearly observed in the cone-dominant responses to long-wavelength light. The results demonstrate a diurnal variation in the temporal responses of the post-photoreceptoral cone pathways of the human retina, which is triggered by activation of the rod photoreceptors.

Neurobiology of retinal dopamine in relation to degenerative states of the tissue.

Neurobiology of retinal dopamine is reviewed and discussed in relation to degenerative states of the tissue. The Introduction deals with the basic physiological actions of dopamine on the different neurons in vertebrate retinae with an emphasis upon mammals. The intimate relationship between the dopamine and melatonin systems is also covered. Recent advances in the molecular biology of dopamine receptors is reviewed in some detail. As degenerative states of the retina, three examples are highlighted: Parkinson's disease; ageing; and retinal dystrophy (retinitis pigmentosa). As visual functions controlled, at least in part, by dopamine, absolute sensitivity, spatial contrast sensitivity, temporal (including flicker) sensitivity and colour vision are reviewed. Possible cellular and synaptic bases of the visual dysfunctions observed during retinal degenerations are discussed in relation to dopaminergic control. It is concluded that impairment of the dopamine system during retinal degenerations could give rise to many of the visual abnormalities observed. In particular, the involvement of dopamine in controlling the coupling of horizontal and amacrine cell lateral systems appears to be central to the visual defects seen.

Consequences of transient retinal hypoxia on rod input to horizontal cells in the rat retina.

The effects of hypoxia on horizontal cells were studied by intracellular recording in the superfused rat retina. Hypoxic challenge from the photoreceptor surface resulted in horizontal cell depolarization, associated with a reduction of the light-evoked hyperpolarization (S-potential). This depolarization, though not the reduction in S-potential, was reversed by non-NMDA receptor antagonists, but not by NMDA related antagonists. Horizontal cells were also depolarized during hypoxia when calcium-dependent synaptic transmitter release was blocked by Co2+. Diazoxide, an activator of ATP-sensitive K+ channels (K+ATP), had no effect on normal cells but blocked the depolarization and the reduction in S-potential. We conclude that retinal hypoxia results in increased activation of the non-NMDA receptors of horizontal cells, whilst the activation of K+ATP channels may be protective in retinal hypoxia.

Electrophysiological properties of neurones following mild and acute retinal ischaemia.

Early electrophysiological changes following acute retinal ischaemia were studied by recording single or multiunit retinal ganglion cells and the electroretinogram (ERG) in barbiturate anaesthetized cats. Retinal ischaemia was initiated photochemically by platelet aggregation in retinal vessels which had been irradiated with monochromatic green light following an intravenous injection of Rose Bengal dye. No physiologically active ganglion cells were found within, or close to, the irradiated sites with chorioretinal oedema. On the other hand, in the areas 5-20 degrees away from the irradiation sites, ganglion cells had abnormally raised spontaneous (background) firing which obscured visually driven firing. The retinal areas where no physiologically active ganglion cells were found showed histopathological changes which are similar to those described for glutamate-induced retinal damage. Retinal areas where depolarized retinal ganglion cells were located, however, showed only minor vacuolation of the ganglion cell fibre layer. Early global electrophysiological changes following photochemically induced retinal vascular lesion were consistent with those predicted from the findings in the single cell study. Vascular lesions produced with high irradiation energy (10-30 J), which promote extensive chorioretinal oedema, resulted in gradual loss of visually responsive ganglion cells. Lesions produced by low-energy irradiation (2 J), causing slight narrowing of the blood columns in the vessels, on the other hand, resulted in significant increases in the amplitude and the implicit time of the ERG b-wave and the background firing of multiunit retinal ganglion cells. Electrophysiological changes associated with mild retinal ischaemia are analogous to physiological effects associated with exogenous glutamate or blockade of glutamate uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-NMDA type excitatory amino acid receptors mediate rod input to horizontal cells in the isolated rat retina.

The actions of excitatory amino acid agonists and antagonists on rod driven horizontal cells were studied in the isolated retina of the rat. Horizontal cells were depolarised by L-glutamate, kainate and quisqualate but not by N-methyl-D-aspartate (NMDA). The broad band excitatory amino acid antagonist, kynurenate and the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), hyperpolarised horizontal cells, blocking the light responses and also the effects of agonists. In contrast, the competitive NMDA antagonists, 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (CPP) and 2-amino-5-phosphono-pentanoate (AP5) were without effect. Thus, rat horizontal cells possess excitatory amino acid receptors of the non-NMDA type and these mediate their rod driven inputs.

Actions of baclofen and phaclofen upon ON- and OFF-ganglion cells in the cat retina.

The effects of the GABAB (gamma-amino butyric acidB) receptor agonist, baclofen and its antagonist, phaclofen on physiologically identified retinal ganglion cells were studied in the optically intact eye of pentobarbitone-anaesthetized cats. These results were compared with the effects of the GABAA receptor agonist, muscimol and its antagonist, bicuculline. Baclofen inhibited the total visually driven firing of both ON- and OFF-cells more effectively upon OFF- than ON-cells; this action was weaker and slower than that of muscimol. Whilst bicuculline raised the firing level of only ON-cells in the area centralis together with all peripheral cells, phaclofen raised that of all OFF-cells. Paradoxically, in OFF-cells, baclofen enhanced the driven transient component and suppressed the sustained component, whilst phaclofen raised the sustained component. Thus, GABAB receptors activated by tonically released GABA may modulate the sustained and transient excitatory inputs to OFF-cells.

Desensitization and capsaicin-induced release of substance P-like immunoreactivity from guinea-pig ureter in vitro.

Substance P-like immunoreactivity was assayed in superfusates of guinea-pig ureters following stimulation of afferent fibres with capsaicin, potassium chloride and the calcium channel agonist Bay K 8644. Capsaicin-evoked release of substance P-like immunoreactivity was calcium-dependent but unaffected by cobalt. Under appropriate conditions release was dose-related (ED50 = 610 nM) and reproducible. Selective desensitization to capsaicin could be demonstrated following prolonged exposure to different doses of capsaicin. No desensitization to capsaicin was observed following afferent fibre stimulation with a combination of Bay K 8644 and K+, which released a similar amount of substance P-like immunoreactivity as a desensitizing capsaicin stimulus. These data suggest that depletion of releasable substance P-like reactivity is unlikely to account for selective desensitization of ureteric primary afferent fibres to capsaicin.

Non-cholinergic synaptic potentials mediated by lumbar colonic nerve in the guinea-pig inferior mesenteric ganglion in vitro.

Non-cholinergic slow synaptic potentials mediated by the lumbar colonic nerve have been investigated using an in vitro preparation of the guinea-pig inferior mesenteric ganglion attached to a distal colonic segment. Non-cholinergic potential responses to colonic nerve stimulation, colonic distension and chemical activation of sensory afferents were recorded intracellularly from neurons in the inferior mesenteric ganglion. Electrical stimulation of the lumbar colonic nerve produced either a slow excitatory postsynaptic potential, or a slow inhibitory postsynaptic potential followed by a slow excitatory postsynaptic potential. The extrapolated reversal potential of the slow excitatory postsynaptic potential was in the range of 0 to -20 mV and that of the slow inhibitory postsynaptic potential was -90 to 110 mV. The slow excitatory postsynaptic potential and the slow inhibitory postsynaptic potential were reversibly abolished by perfusion of the ganglion with tetrodotoxin (1 microM), or perfusion with low calcium (200 microM), high magnesium (12 mM) containing solution. Capsaicin (1 microM) evoked a reversible depolarization of inferior mesenteric ganglion cells after which desensitization occurred and the slow excitatory postsynaptic potential was abolished but the slow inhibitory postsynaptic potential was enhanced in amplitude and prolonged in duration. Bath application of substance P (2 microM) evoked a prolonged depolarization of inferior mesenteric ganglion neurons, during which the slow excitatory postsynaptic potential but not the slow inhibitory postsynaptic potential was abolished. Distensions of the colon to pressures in the range of 2-25 cm of water produced a stimulus graded non-cholinergic slow depolarization which was occasionally followed by a late slow hyperpolarization. Both types of response were abolished by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of afferent fibres of the guinea-pig ureter evokes potentials in inferior mesenteric ganglion neurones.

1. Intracellular recordings were made from neurones of the guinea-pig inferior mesenteric ganglion (IMG) maintained in vitro with both ureters and major nerve trunks attached. Afferent fibres in the ureteric nerve were activated by electrical, chemical and mechanical stimuli. 2. Repetitive stimulation of a ureteric nerve branch evoked a non-cholinergic, synaptic slow excitatory potential (slow EPSP) in 48% of neurons. The amplitude of the slow EPSP was dependent on membrane potential and was decreased by membrane depolarization and increased by hyperpolarization. 3. The slow EPSP was attenuated or abolished by capsaicin (1 microM), which itself depolarized IMG neurones. Substance P (2 microM) or neurokinin A (2 microM) also depolarized IMG neurones and in the presence of these tachykinins the slow EPSP was attenuated or abolished. 4. Distension of the ureter evoked a non-cholinergic slow depolarization in 45% of IMG neurones which was abolished by tetrodotoxin (1 microM) and by capsaicin (1 microM). 5. Chemical stimulation of ureteric afferent nerve terminals by intralumenal perfusions of the ureter with capsaicin (1 microM) produced a slow depolarization in the IMG which was prevented by blocking nerve conduction with TTX. 6. These data demonstrate that electrical stimulation of ureteric afferent fibres produces a non-cholinergic slow EPSP in the IMG. Primary afferent (capsaicin-sensitive) C fibres are also activated by distension of the ureter and evoke a slow depolarization in the IMG. The synaptic mediator of these events is likely to be tachykinin(s) released from capsaicin-sensitive C fibres. These fibres may be mechanosensory and/or nociceptive.

Actions of neuropeptide K and calcitonin gene-related peptide on inferior mesenteric ganglion cells--tachykinin interactions with non-cholinergic potentials evoked by ureteric nerve stimulation.

Neuropeptide K (NPK) induced a slow depolarization in principal ganglion cells of the guinea pig inferior mesenteric ganglion (IMG) in vitro. This effect was due to a postsynaptic action and prevented by pre-exposure of the IMG to neurokinin A (NKA) or substance P (SP). The non-cholinergic slow postsynaptic excitatory potential (s-EPSP) evoked by ureteric nerve stimulation was depressed during NPK, SP or NKA application. Calcitonin gene-related peptide (CGRP) applied in concentrations up to 10 microM had no effect on the membrane potential in 90% of IMG cells nor did it influence the s-EPSP. We suggest that NPK may depolarize IMG neurones via similar mechanisms/in a similar fashion, to other tachykinins and that the s-EPSP, induced by stimulation of the afferent ureteric nerve fibres, is mediated by a tachykinin whereas there is little indication/evidence for an involvement of CGRP.

Neuropharmacological actions of kynurenic and quinolinic acids on horizontal cells of the isolated fish retina.

We have studied the effects of naturally occurring metabolites of tryptophan, kynurenic and quinolinic acids, on the electrophysiological responses of retinal horizontal cells in the fish (Rutilus rutilus, the roach). Quinolinic acid usually hyperpolarizes the cells and reduces their light evoked responses (S-potentials) but on occasion, it causes a slight depolarization of the membrane potential. These actions are similar to those found with N-methyl-D-aspartate (NMDA) and our results are consistent with the proposal that it acts at NMDA binding sites. Kynurenic acid (greater than or equal to mM) invariably hyperpolarizes horizontal cells to their potassium Nernst equilibrium potential and, more significantly, blocks the depolarizing actions exerted on them by excitatory amino acids, such as kainic and quisqualic acids. We show that this action persists in the presence of the synaptic blocker, cobalt chloride, and thus is not mediated by chemical synaptic activity. Kynurenic acid does not reverse depolarization of horizontal cells by dopamine or gamma-aminobutyric acid, thus its inhibitory effects are selective to the actions of excitatory amino acids. Neither xanthurenic acid, a close structural analogue of kynurenic acid, nor quinolinic acid are effective in blocking depolarizations by excitatory amino acids.