Supplementary material to:

"Specialised sympathetic neuroeffector associations in rat iris arterioles"
(Sandow, S.L., Whitehouse, D. and Hill, C.E.).
Submitted to the Journal of Anatomy, 7.3.97.

(Viewing notes - clicking on the thumbnail images will give a large version of the image. The large images should ideally be viewed on 24 bit color displays.)

Series 1

Images 1 to 7 are reconstructions of a single varicosity in close apposition with a vascular smooth muscle cell (VSM) and a melanocyte from 33 serial sections over approximately 3.3 um of tissue (Images 1 to 3 correspond to Figs 5a to 5c, in the manuscript).

Image 1	This is a lateral view of the varicosity (grey) with Schwann cell (yellow) overlying the varicosity and VSM cell (red), with associated caveolae (yellow) below.
Image 2	The same lateral view as image 1 above, but with the axolemma removed, thus showing synaptic vesicle distribution. Three vesicle types can be seen; large granular vesicles (green), small granular vesicles (blue) and small agranular vesicles (pink). Vesicle accumulations at the neuroeffector regions with the smooth muscle cell (1), and the melanocyte (2), can be seen.
Image 3	Shows an underlying view of the varicosity with the region of VSM cell-varicosity close apposition highlighted (light green). The VSM cell, caveolae, and Schwann cell have been removed so that the region of close association can be clearly seen.
Image 4	Represents the same view as image 3 above, but with the caveolae present and the axolemma removed so that the association between caveolae and vesicle distribution can be seen. No association between caveolae and the region of close association can be observed.
Image 5	Represents the same view as in image 4, but from "above".
Image 6	Represents the same view as in image 5, but with the synaptic vesicles removed and the axolemma present as a wire frame mesh. No specific association of the varicosity with the caveolae can be observed.
Image 7	Represents the view from "above" with the Schwann cell overlying the axolemma and VSM cell.

Series 2

Images 8 to 14 are reconstructions of a bundle of three fibres surrounded by a single Schwann cell (yellow), from 52 serial sections over approximately 5.2 um of tissue (Images 8 to 10 correspond to Figs 5d to 5f, in the manuscript).

Image 8	This image shows the same features as seen in image 1 above. That is, a lateral view of the varicosity (grey) with Schwann cell (yellow) overlying the varicosity and VSM cell (red), with associated caveolae (yellow) "underneath".
Image 9	This image shows the same lateral view as image 8 above, but with the axolemma removed,thus showing synaptic vesicle distribution. As in image 2 above, three vesicle types can be seen; large granular vesicles (green), small granular vesicles (blue) and small agranular vesicles (pink). Vesicle accumulations at the neuroeffector regions with the smooth muscle cell (1), and the melanocyte (2), can be seen.
Image 10	Represents an underlying view of the varicosity with the region of VSM cell-varicosity close apposition highlighted (light green). As in image 3 above, the VSM cell, caveolae, and Schwann cell have been removed so that the region of close association can be clearly seen.
Image 11	Represents the same view as image 10 above, but with the caveolae present and the axolemma removed so that the association between caveolae and area of VSM close association can be seen. Due to the density of the caveolae, the region of close association between the VSM cell and varicosity is almost obscured.
Image 12	Represents the same view as in image 11, but from "above", showing synaptic vesicle, caveolae and VSM cell-varicosity close apposition. Again, the area of close apposition is a little obscured due to the density of caveolae and synaptic vesicles.
Image 13	Represents the same view as in image 12, but with the axolemma present as a wire frame mesh. No specific association of the varicosity with the caveolae can be observed.
Image 14	Represents the same view as in image 13 (ie. from "above"), but with the axolemma removed and the melanocyte-varicosity region of close apposition present (bright red). From table 5 (see manuscript) this region can be seen to be consistently larger than the region of close apposition to the VSM cell.

Series 3

Images 15 to 19 are reconstructions of a single varicosity in close association with a vascular smooth muscle cell and a melanocyte. The images represent information from 19 sections over approximately 1.9 um of tissue.

Image 15	This is a lateral view of the varicosity (grey) with Schwann cell (yellow) overlying the varicosity and VSM cell (red), with associated caveolae (yellow) below.
Image 16	The same lateral view as image 15 above, but with the axolemma removed, thus showing synaptic vesicle distribution. Three vesicle types can be seen; large granular vesicles (green), small granular vesicles (blue) and small agranular vesicles (pink). Vesicle accumulations at the neuroeffector regions with the smooth muscle cell (1), and the melanocyte (2), can be seen. A greater accumulation of vesicles can be seen at the melanocyte region of close association.
Image 17	Shows an underlying view of the varicosity with the region of VSM cell-varicosity close apposition highlighted (light green). The VSM cell, caveolae, and Schwann cell have been removed so that the region of close association can be clearly seen.
Image 18	Shows an underlying view of the varicosity with the region of VSM cell-varicosity close apposition highlighted (light green). The VSM cell, axolemma and Schwann cell have been removed so that the region of close association can be clearly seen. No clear association between the area of varicosty-VSM cell close association and caveolae can be seen.
Image 19	Shows a view from "above" of the varicosity with the axolemma, Schwann cell and VSM cell removed. The regions of varicosity-VSM cell (light green) and varicosity-melanocyte close association (bright red) can be seen. The melanocyte region of close apposition occurs on two separate regions of the varicosity, away from the VSM cell region.

Series 4

Images 20 to 23 are reconstructions of a bundle of three fibres with varicose regions of close apposition with melanocyte only. The images are from 53 serial sections over approximately 5.3 um of tissue. One of the fibres forms a terminal varicosity, whilst the other two become varicose at some points along there length. These images highlight one of the problems in viewing three-dimensional reconstructions - due to the large amount of information present, features often become obscured; even when viewing images from a variety of angles.

Image 20	Shows a slightly oblique view of the fibres (grey), and nearby VSM cells (red) with associated caveolae below (yellow).
Image 21	Represents the same view as in image 20 above, but with the axolemma removed. A number of synaptic vesicle localisations can be seen (1 - arrow). These correspond to regions of close association with melanocyte.
Image 22	Represent the view from "above" with the Schwann cell and VSM cell removed. The region of melanocyte close association (bright red) can be seen, although it is partially obscured by the fibres. In these regions much of the melanocyte association occurs between the VSM cells surface (where caveolae are present) and the varicosities.
Image 23	Represents the same view as in image 22 above, but with the Schwann cell and VSM cell present. A large region of the fibres has no covering of Schwann cell and is not in close association with melanocyte or VSM cell.

Series 5

Images 24 to 29 are reconstructions of a single varicosity with regions of close apposition to two VSM cells and melanocyte. The images are from a reconstruction from 58 serial sections over approximately 5.8 um of tissue.

Image 24	Represents a slightly oblique view of a single fibre (grey), overlying two VSM cells (red) with associated caveolae "underneath" (yellow).
Image 25	Represent the same view as image 24 above, but with the axolemma removed. Typical varicosity associated accumulations of synaptic vesicles can be seen at three points. Only the varicosity in the mid-region of the image was reconstructed in full. The region of close association of the VSM cell and varicosity can be seen (1).
Image 26	Shows an underlying view of the varicosity with the region of VSM cell-varicosity close apposition highlighted (light green). The VSM cell, caveolae, and Schwann cell have been removed so that the region of close association can be seen.
Image 27	Represents the same view as image 26 above, but with the caveolae present and the axolemma removed so that the association between caveolae and the region of close association can be seen. No association between caveolae and this region was observed.
Image 28	Represents the same view as in the previous image looking through the varicosity, but from "above" with the melanocyte-varicosity region of close apposition highlighted (bright red). As with images 10 and 14 above the varicosity-melanocyte region of close association is considerably larger than that of the varicosity-VSM cell (cf/. images 26 and 28). Note that the melanocyte contact region virtually completely obscures the accumulation of synaptic vesicles present in the varicosity.
Image 29	Represents the same view as in image 28 above, but without the varicosity-melanocyte region of close association and with the Schwann and VSM cell present. Note that a large region of the fibre has no covering of Schwann cell and is not in close association with melanocyte.

Series 6

Images 30 and 31 are reconstructions of a single varicosty with a region of close association with melanocyte only. These images are from a reconstruction of 14 serial sections over approximately 1.4 um of tissue. A continuous layer of melanocyte was present between the VSM cells and the varicosity (not shown).

Image 30	This is a lateral view of the varicosity (grey) with Schwann cell (yellow) overlying the varicosity and VSM cell (red), with associated caveolae (yellow) below.
Image 31	The same lateral view as image 30 above, but with the axolemma removed, thus showing synaptic vesicle distribution. Three vesicle types can be seen; large granular vesicles (green), small granular vesicles (blue) and small agranular vesicles (pink).