Selective staining of photoreceptors

Trends in NeuroSciences February1982

Selective staining of photoreceptors A recent article b y de Monasterio, Schein and M c C r a n e 8 has s h o w n that a particular class o f photoreceptor takes up Procion yellow, so that the whole cell fluoresces. The result is interesting f r o m at least two points o f view: first, the location and spacing o f the receptors involved; second, the m e m b r a n e properties a n d cytology that are responsible f o r the p h e n o m e n o n .

The cells labelled by de Monasterio et al. are almost certainly the blue-sensitive cones. In the macaque retina they form a regular but sparse mosaic with highest density near the fovea. They tend to be absent in the central fovea. These properties agree with what is known about the bluesensitive cones in the primate from other techniques. The regularity of spacing of the cones, as shown by the Procion yellow, is quite striking. In the cat and rabbit very few photoreceptors take up the stain, which agrees with other evidence that there are very few blue absorbing cones in these species. These results build on previous work in which particular photoreceptors have been selectively labelled. Laties and Liebman6 showed that the outer segments of cones, but not rods, fluoresce with Procion yellow. The fact that cone outer segments were labelled and rods were not is believed to be due to the structure of the cone outer segment. The cone outer segment membrane is infolded, as shown by Cohen4, so that molecules can diffuse to the spaces among the lamellae without passing across the plasma membrane, whereas most of the rod discs are enclosed within the plasma membrane and separated from it. Labelling of a cone outer segment presumably relies on interaction of the tracer with some component of the plasma membrane which binds the marker molecule. Other substances, besides Procion yellow, such as horseradish peroxidase and microperoxidase =, label cone outer segments but not rod outer segments, probably for the same reason. This mode of action is supported by the observation that the bases of rod outer segments, which have newly forming discs open to extracellular space, also fluoresce following injection of a fluorescent dye5. In fact,

in rods so stained, a band of fluorescence moves up the rod, just as the discs in the rod are known to do when new discs are added at the base~, and the width of the band corresponds to the number of discs which are open. In de Monasterio et al's. result, the whole of the blue sensitive cone fluoresces, so an additional mechanism must be involved. They also found Procion yellow in the outer segments of other cones, as did Laties and Liebman: the blue-sensitive cones were the only ones in which the dye penetrated the cell membrane and filled the cell. Why does the dye get inside some cells but not others?

Research News Procion yellow does not normally cross the cell membrane, so presumably some damage of the membrane is involved. Laties et al. found some cell damage in their results 7, but in the de Monasterio paper higher doses of Procion yellow were used, and it seems that these are required to give the phenomenon regularity, de Monasterio et al. hypothesize that blue-sensitive cones are more susceptible to damage than red- or green-sensitive cones, and there is some clinical evidence to support this (K611ner's rule). Another specific marking procedure has become available which depends on the dif-

Fig. l. Cones of the macaque retinastained with Procion yellow appear as white elements in this micrograph of a radial section of central retina. Unstained rod and cone inner segments appear as darker structures. Notice that outer ~egmentsofall cones are ~tained. ,

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34 fering metabolic properties of rods and cones, rather than the organization of their membranes. BunP 2 has recently demonstrated that [3H]fucose is incorporated into a glycoprotein which is a component of cone outer segments but not rod outer segments. In the retina of cyprinid fish this substance is most prevalent in the outer segments of red- and blue-sensitive cones, and less prevalent in the outer segments of greensensitive cones. It is not yet known whether the glycoprotein is part of the cone visual pigment, but it is clearly separable from rod opsi#. Why glycoproteins in red and blue cone outer segments should incorporate substantial amounts of fucose and glycoproteins in green cones should incorporate less, is still an intriguing mystery. The two types of receptor that do not incorporate much fucose - rods and green cones - have

spectral sensitivities very close to each other: this may have functional significance, or it may simply be a coincidence. The bearing of this research on the photoreceptor mosaic is inmtediate and obvious. With these markers one can see how the receptors are arranged in relation to each other, and how regular is the array. The significance of the results in relation to the membrane structure and cytology of photoreceptors is less obvious, but probably more intriguing. If red-sensitive cones take up more fucose than green-sensitive cones, this must bear some relationship to differences in their function. Similarly, if bluesensitive cones can he damaged by Procion yellow, this must be telling us something about their membranes. This will clearly be a fruitful area of research for some time to come.

Peptidergic transmission in ganglia Although immunohistochemical studies reveal more and more peptide-containing nerve fibres in the CNS, one would still be hard placed to state exactly how any single peptidergic system operates. This, o f course, reflects the formidable difficulty o f doing the appropriate experiment in the labyrinthine maze o f the CNS. To a large extent our understanding o f other forms o f central transmission, such as cholinergic transmission, is equally vague (with one or two notable exceptions), yet we feel more secure with cholinergic transmission because we have excellent analogies in the autonomic nervous system on which to base our concepts. Now, it seems, the autonomic nervous system is also beginning to illuminate our view o f peptidergic transmission. Two examples o f recent research illustrate this point: they concern transmission by an LHRH-Iike peptide in frog sympathetic ganglia and by substance P (SP) and enkephalin (ENK) in guinea-pig inferior mesenteric ganglia. When the preganglionic supply to bullfrog lumbar sympathetic ganglia is stimulated, a complex sequence of post-synaptic potentials is recorded from the ganglionic neutones, comprising (in chronological order) a fast excitatory post-synapdc potential (fast epsp), a hyperpolarizing slow inhibitory post.synaptic potential (slow ipsp), a slow epsp and a very delayed late slow epsp~ (see Fig. 1). The first three result from the action of acetylcholine released from the cholinergic fibres but the late slow epsp is resistant to acetylcholine antagonists. Over the last two years Jan, Jan, and KuffieP -~'m have demonstrated that the latter results from the release of a peptide resembling LHRH from a discrete, and probably separate, population of preganglionic fibres. Thus, the peptide is present in the fibres and their terminals; is lost after denervation; is released when the fibres are depolarized or stimulated electrically; both imitates and occludes the late slow epsp; and the effects of both LHRH and nerve stimulation are blocked by inhibitory analogues. Although the precise chemical structure of the peptide is in doubP, this catalogue of tests appears sufficiently comprehensive to satisfy the most stringent criteria for a transmitter

function. Further, because the neurones can be studied using voltage-clamp techniques, we are in the unusual position of being able to appreciate exactly how the transmitter works: it selectively inhibits a uniquely identifiable time- and voltage-dependent K-current called the M-currenO (though other currents may also be affected outside the normal range of membrane potentialsm). Since the M-current exerts a partial brake on normal cell discharge, peptide release acts as an enabling device to increase the excitability of the neurone over quite long time periods. One hesitates to use the overworked word neuromodulation in this context, but this action very much fits in with a concept of peptidergic transmission as being a half-way house between the fast, puuctate neurotransmission exemplified at the neuromuscular junction and the slow, diffuse effect of a hormone. While the frog ganglion experiumnts yield a clear conceptual image of how a peptidergic system works, the experiments on the guinea-pig inferior mesenteric ganglion (IMG) suggest an even more direct analogy with the mammalian CNS. The cells in the IMG also show dual excitatory response, one fast and cholinergic and one slow and

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peptidergic. In this cam, however, the slow response is probably mediated by the undecapeptide SP n't4 The evidence for this is very nearly as complete as that for the role of the LHRH-Iike peptide in the frog ganglion: only the effect of an SP-antagonist is missing, and no doubt this will be rapidly rectified now that such antagonists are available5 (if not already completed by the time this report has been written). The most interesting aspect of this system is that the SP-contalning fibres are sensory fibres (or branches thereof), whose cell bodies are in the dorsal root ganglion and whose axons course to, and through, the IMG via the lumbar sptanchnic sympathetic nerves4,14 This means that the SP-pathway can be selectively stimulated at the dorsal root whereas the cholinergic outflow to the splanchnic nerves comes (as usual) from the ventral roots. The demonstrable release of SP from the peripheral end of sensory fibres, or from sensory axon collaterals, sustains the view that SP is an important transmitter at the central ends of some sensory nerves since, as Dale ~ expressed it: 'Musste man darum nicht einen gleichen chemischen Mechanismus an diesen zwei Endigungen desselben Neurons, der peripheren und der zentrale, erwarten?'*. Perhaps even more provocative is the recent evidence for a second tier of peptidergic control in the 1MG at the presynaptic site, mediated by ENK. ENKcontaining fibres have previously been detected immunohistochemically in several sympathetic ganglia, including the IMG 17, though where they come from is unclear; and application of ENK was found to depress both the cholinergic last epsp and the SP-mediated slow epsp in the IMG 4.'~,12 'Must one therefbre not anticipate one common t 'hemical mechanism at these two ends - central and peripheral - o f the .~ame neurones'?'