Digital in-line holography with numerical reconstruction has been developed into a new tool, specifically for biological applications, that routinely achieves both lateral and depth resolution, at least at the micron level, in three-dimensional imaging. The experimental and numerical procedures have been incorporated into a program package with a very fast reconstruction algorithm that is now capable of real-time reconstruction. This capability is demonstrated for diverse objects, such as suspension of microspheres and biological samples (diatom, the head of Drosophila melanogaster), and the advantages are discussed by comparing holographic reconstructions with images taken by using conventional compound light microscopy.

* Ci rcadi an Size Changes m Monopol ar Cel l Axons * Ci rcadi an Size Changes i n Monopol ar Cel l Axons

Two sets of wide-field neurons extend neurites into the fly's optic lamina, where monopolar cells receive photoreceptor input. They exhibit immunoreactivity to antibodies raised against either 5-hydroxytryptamine or the crustacean peptide PDH, respectively. Both are proposed whole-field neuromodulators of vision, apparently regulating a circadian rhythm of monopolar cell size. Seeking functional correlates, we have re-examined the electroretinogram for circadian rhythmicity, and for responses to locally injected 5-hydroxytryptamine and peptide. Long-term electroretinogram recordings from Calliphora entrained to a light/dark cycle and then transferred to constant darkness, uncovered a gradual, modest increase during the subjective night in the electroretinogram's ON- and OFF-transients, from the lamina's monopolar cells. Five to twenty nl of 5-hydroxytryptamine (10(-3) mol.1(-1)) injected into the head haemolymph strongly enhanced the electroretinogram transients, an action reversed by 5-hydroxytryptamine antagonists. Injected into the eye, 5-hydroxytryptamine (10(-4) mol.1(-1)) had the opposite effect; the rapid onset there suggests direct action, whilst the opposing effect from haemolymph injection suggests a different receptor site. Pigment-dispersing hormone (2.2 x 10(-5) mol.1(-1)) injected into the haemolymph increased the electroretinogram transients along a biphasic course, with a slow partial recovery; injected into the eye, it lacked effect.

Many invertebrates have supplementary extraocular photoreceptors that often are implicated in circadian rhythms. An extraretinal group of candidate photoreceptors in the fruit fly, Drosophila melanogaster, has been revealed previously at the posterior margin of the compound eye by using a photoreceptor‐specific monoclonal antibody (Hofbauer and Buchner [1989] Naturwissen 76:335–336), but it never has been characterized. Here, we report the fine structure of this cell cluster reported by Hofbauer and Buchner, which is called “eyelet,” as well as the further candidacy of their visual pigment and neurotransmitter. Eyelet forms a specialized, pigmented organ with cells that have numerous microvilli arranged into coherent rhabdomeres. The presence of rhabdomeric microvilli is a defining feature of a photoreceptor, reported here for the first time in eyelet. The rhabdomeres exhibit Rh6 opsin‐like immunoreactivity, which provides evidence that the photoreceptors are functional: they fail to immunostain with antibodies against NINAE (Rh1), Rh4, or Rh5. The photoreceptors have been shown previously to exhibit histamine‐like immunoreactivity, but they also stain with a monoclonal antiserum raised against Drosophila choline acetyltransferase (ChAT), suggesting that the photoreceptors not only may contain histamine but also can synthesize acetylcholine. A ChAT‐immunoreactive axon bundle originating from eyelet terminates in the cortex of the anterior medulla. This bundle also is seen with reduced silver stains. Electron microscopic examination revealed four axon profiles of similar size in this bundle, indicating that eyelet contains at least four photoreceptors. The pathway of eyelet's axon bundle coincides with the precocious pathway of Bolwig's nerve that arises from the larval organ of sight. The origin and possible function of eyelet are discussed. J. Comp. Neurol. 412:193–202, 1999. © 1999 Wiley‐Liss, Inc.

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