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## Staining patterns   Cytochrome oxidase stains all cells but shows distinct patterns in brain areas depending on the distribution of mitochondria and the cytochrome oxidase density within the mitochondria (pp 17). (Gonzalez-Lima 1998).  Typically cell bodies and axon trunks have lower levels of cytochrome oxidase density compared to dendrites and axon terminals (pp 17). (Gonzalez-Lima 1998).  In the telost brain several features of the staining are apparent which are consistent with findings in mammal and lizard brains. 1) Gray matter has higher staining density than white matter, and fiber tracks are clearly visible (e.g. Figure 5 5A  black arrow). 2) The intensity of staining varies between different structures, particularly throughout the forebrain. structures.  For example, subdivisions of DM, DL, DA and DC, DP, and Vc Vs  are all clearly differentiated by the cytochrome oxidase stain (Figure 5). 5A). The teleost optic tectum is stained in great detail by COX histochemistry and can be seen as a layered cortical structure (Figure 5B black arrow), a staining pattern that is strikingly similar to the lizard optic tectum (Gonzalez-Lima 1998) 61.  3) COX reactivity is not necessarily correlated with the density of cell bodies. This can be clearly seen in the olfactory bulb (Figure 6). 5C).  In teleosts the olfactory receptor neurons terminate in the glomerular layer of the olfactory bulb and form comparmental structures called glomeruli with the apical dendrites of the mitrial cells. First order integration of olfactory signals is thought to occur within these structures (Laberge and &  Hara 2001). The glomeuli can be clearly seen as densly stained compartments in the cytochrome oxidase stain, but are not stained at all by Nissl, Nissl (Figure 5C black arrows),  because in teleosts this layer is devoid of cell bodies. In contrast the mitral cell layer has a high density of cell bodies, as seen in the Nissl stain, but stains only lightly with COX histochemistry because the COX is concentrated in the dendrites rather than the cell soma. soma (Figure 5C red arrows).  4) The teleost hypothamlamus is largely uniform in staining throughout. This has been observed previously in rodents. In gerbils only the SCN, PV, and VM nuclei were visually distinguishable, while other nuclei appeared uniform (Jones et al al.  1997). In the rat COX atlas (at least to my eye) only the PV and LH are clearly distinguishable while other prominent hypothamalmaic nuclei such as the POA, VMH and SCH appear uniform (I have photocopied the atlas in [appendix XX] for your convience). This can also be seen in the mouse brain which was included in this study as a positive control. For example, some brain areas like the hippocampus and blAMY are clearly differentiated by the COX stain while the hypothalamus is uniform in staining, based on the rat COX atlas and landmarks I believe this section is at the level of the VMH VMH, although I cannot be sure without the adjacent Nissl for reference  (Figure 5D). My hypothesis about this interesting and deeply conserved pattern is that cytochrome oxidase in the hypothalamus is distributed throughout a diffuse network of neuropil and is not concentrated in cell-dense brain nuclei. Where you do tend to see clearly defined nuclei such as the rodent PV and LH nucleus, the staining is usually lighter than the surronding areas, which is consistent with the hypothesis that COX is concentrated in neuropil rather than cell-dense regions. That being said many studies have found interesting differences between experimental groups within the hypothalamus and regions that appear uniform to the eye can sometimes be distiguished using quantitative densitomitry.