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The development of new axonal tract tracing and cell labelling methods has revolutionised neurobiology in the last 30 years. The aim of this review is to consider some of the key methods of neuroanatomical tracing that are currently in use and have proved invaluable in charting the complex interconnections of the central nervous system. The review begins with a short overview of the most frequently used tracers, including enzymes, peptides, biocytin, latex beads, plant lectins and the everincreasing number of¯uorescent dyes. This is followed by a more detailed consideration of both well established and more recently introduced neuroanatomical tracing methods. Technical aspects of the application, uptake mechanisms, intracellular transport of tracers, and the problems of subsequent signal detection, are also discussed. The methods that are presented and discussed in detail include: (1) anterograde and retrograde neuroanatomical labelling with¯uorescent dyes in vivo, (2) labelling of post mortem tissue, (3) developmental studies, (4) transcellular tracing (phagocytosis-dependent staining of glial cells), (5) electrophysiological mapping combined with neuronal tract tracing, and (6) simultaneous detection of more than one axonal tracer. (7) Versatile protocols for three-colour labelling have been developed to study complex patterns of connections. It is envisaged that this review will be used to guide the readers in their selection of the most appropriate techniques to apply to their own particular area of interest. 7

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Experiments were designed to determine the contribution of endothelial cells to the heterogeneous behavior of the arterial and venous wall. Rings of canine femoral, pulmonary, saphenous, and splenic arteries and veins, with and without endothelium, were mounted for isometric tension recording in Krebs-Ringer bicarbonate solution. Endothelium-dependent inhibitory responses to acetylcholine, adenosine triphosphate, bovine thrombin, and arachidonic acid were prominent in the arteries. In the veins, only transient endothelium-dependent relaxations to these substances were observed. Removal of the endothelium decreased the augmentation of the response to norepinephrine caused by anoxia in both arteries and veins. In the veins, arachidonic acid and thrombin caused endothelium-dependent increases in tension during contractions evoked by norepinephrine. The endothelium-independent inhibitory effects of isoproterenol and adenosine and the excitatory effects of acetylcholine and ATP were more pronounced in the veins than in the arteries. These experiments demonstrate that in the arterial and venous wall the endothelial cells can contribute to both inhibitory and excitatory responses of the smooth muscle cells of the media. Inhibitory endothelial responses prevail in the arteries, and excitatory ones in the veins.

To monitor the cascade of events initiated by injury of adult neurons, and to explore whether and how neighboring microglial cells contribute to the degradation of lesioned neurons, axotomy-induced ganglion cell degeneration was investigated in adult rats. Suppression of macrophage and microglia activity during the weeks following transection of the optic nerve was performed with the immunoglobulin-derived tripeptide Thr-Lys-Pro, which is a macrophage inhibitory factor (MIF) and retards the activity of cells of monocytic origin. Single or repeated injection of MIF into the vitreous body during and after transection of the optic nerve resulted in significant retardation of axotomy-induced ganglion cell degradation in the retina as detected by specific labeling with the retrogradely transported fluorescent dye 4Di-10ASP. MIF specifically altered the morphology of labeled microglial cells from a ramified to an oval, less ramified shape, indicating that these cells were targets of its activity. Injection of the tetrapeptide macrophage stimulating factor, also known as tuftsin (Thr-Lys-Pro-Arg), revealed effects opposite to those described for the MIF: it increased the number of labeled microglial cells and enhanced the devastating effects of axotomy on ganglion cells. The viability of rescued ganglion cells in retinas treated with the various drugs was assessed both in vivo and in vitro. (1) Intravitreal injection of MIF to prevent degradation of neurons combined with transplantation of autologous peripheral nerve grafts, which facilitate regrowth of the transected neurites, revealed that significantly more ganglion cells contributed to axonal regeneration (17.1%) than in untreated controls (9.5%). (2) Explantation of retinas that were pretreated with MIF in situ revealed higher incidence of axonal outgrowth in organ cultures than untreated control explants or retinas treated with either the basic fibroblast growth factor or brain-derived neurotrophic factor. The present results demonstrate that axotomy initializes a cascade of microglia-mediated autodestructive retinal responses, which culminate in degradation of "sick," but obviously viable neurons. We postulate that the retinal microglial system has a key role in recognizing and eliminating severed neurons.