Cytochrome oxidase staining marks dendritic zones of the rat olfactory bulb external plexiform layer

Abstract

Cytochrome oxidase staining of the rat olfactory bulb external plexiform layer (EPL) produces a darkly stained intermediate zone bordered by lightly stained superficial and deep zones. Similar zonal staining was seen in cats, rabbits, and hamsters. These zones vary in relative thickness around the circumference of the olfactory bulb; the deep zone is proportionally thicker in the most dorsal and ventral parts of the bulb. Tufted cell somata are unevenly distributed within the EPL; the outer part of the EPL has more somata. The distribution of the cytochrome oxidase reaction product shows that the darkly stained intermediate zone is not produced by staining of tufted cell somata. Zones of cytochrome oxidase staining correspond to the sublaminar distribution of mitral and tufted cell basal dendrites. This was demonstrated by labeling mitral and tufted cells with small extracellular horseradish peroxidase injections and processing alternate sections for horseradish peroxidase and for cytochrome oxidase. Because there was cross-reaction of the cytochrome oxidase procedure with horseradish peroxidase, it was possible to trace many neurons through both series of sections. Middle tufted cells of the superficial EPL have basal dendrites confined to the superficial zone of light cytochrome oxidase staining. Internal tufted cells and middle tufted cells of the intermediate zone send their basal dendrites into the intermediate zone. One group of mitral cells (type I) has basal dendrites confined to the deep zone of lighter cytochrome oxidase staining. A second group of mitral cells (type II) and tufted cells of the intermediate EPL has basal dendrites primarily confined to the intermediate zone of dark cytochrome oxidase staining. The correlation of the enzyme staining with the dendritic laminar patterns supports the existence of three relatively distinct sublaminae in the EPL and supports the designation of two types of mitral cells. The staining pattern also provides an independent method for evaluating the sublaminae of the EPL without the necessity of labeling individual groups of cells. Finally, the staining pattern suggests that the intermediate zone of the EPL may be subjected to more tonic synaptic input, causing it to have an increased level of metabolic activity.

Two distinct olfactory bulb sublaminar networks involved in gamma and beta oscillation generation: a CSD study in the anesthetized rat

“…The same authors demonstrated that granule cell innervation of the EPL was also inhomogeneous, with more superficial cells of the GRL innervating the upper half of EPL, and with deeper cells of the GRL innervating the deeper EPL (Orona et al, 1983). Since then, other studies have shown that anatomic or metabolic markers can display differences between the lower and upper EPL in mice or rats, including cytochrome oxidase (Mouradian and Scott, 1988) and α3-GABA subunits (Panzanelli et al, 2005; Sassoè-Pognetto et al, 2009), or between the lower and upper GRL, including perisomatic targeting granule cells (Naritsuka et al, 2009) and the functional properties and spatial distribution of newborn cells (Carleton et al, 2003; Lemasson et al, 2005). Overall, these anatomical studies provide support for a sublaminar organization of the EPL and GRL, similar to the functional observations in the present CSD study (see Figure 9 for a parallel between the anatomical substrates and the CSD profiles).…”

Neuronal organization of olfactory bulb circuits

“…Hence, the EPL could be subdivided into at least two sublayers, the superficial/outer and the deep/inner layers, although a clear marker for the sublayers is not known. Interestingly, an early report identified a third intermediate sublayer, which is labeled by cytochrome oxidase (Mouradian and Scott, 1988). …”

“…The majority of these cells extend long secondary dendrites predominantly in the deep EPL and are termed type-I mitral cells (Mori et al, 1983; Orona et al, 1984). However, some mitral cells extend secondary dendrites predominantly in the intermediate EPL and are termed type-II mitral cells (Orona et al, 1984; Mouradian and Scott, 1988). In addition, subsets of mitral cells extend secondary dendrites in the superficial EPL (Imamura and Greer, unpublished data) and may not represent either type-I or type-II mitral cells.…”

Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat

“…Past studies on CytOx histochemical staining pattern in the olfactory system have been performed primarily in the olfactory bulb and cortex (Deiss et al, 2001;Holtzman et al, 1993;Meisami & Sendera, 1993;Mouradian & Scott, 1988;Onoda & Imamura, 1984a, b;Pedersen, et al, 1987;Woo et al, 1987).…”

“…It is noteworthy that whereas in the CNS, the staining pattern for CytOx is mainly associated with synapserich bands, such as the glomeruli of the OB (Meisami & Sendera, 1993;Onoda & Imamura, 1984b) or the sublayers of the external plexiform layer of the OB (Mouradian & Scott, 1988), the dense staining pattern of CytOx in the OE reflects mainly the non-synaptic dendritic microzones of the ORNs and the apical regions of supporting cell cytoplasm (see also Rash et al, 2005), both of which have abundant mitochondria. That the apices of OE supporting cells are highly metabolically active and contain many mitochondria is supported by ultrastructural evidence (e.g., Moran et al, 1992;Dahl & Ding, 2003).…”