This is a study of the chemoanatomical organization of the projection from the raphe nuclei to the main olfactory bulb in the rat. A heavy projection from the dorsal and median raphe nuclei to the main olfactory bulb was shown by both retrograde and anterograde tracing techniques. Following injections of 1% wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the main olfactory bulb, up to 1300 neurons were retrogradely labeled in the dorsal and median raphe nuclei, a much larger number than has been suggested by previous studies. By combining 5-HT immunofluorescence with True blue retrograde fluorescent labeling, it was determined that the vast majority of raphe neurons that project to the olfactory bulb contain serotonin. Injections of WGA-HRP into dorsal and/or median raphe produced dense anterograde labeling in the glomeruli, while fewer labeled fibers were observed in the external plexiform layer, internal plexiform layer, and granule cell layer. In contrast to the number of other centrifugal afferents to the bulb, a significant contingent of fibers from the raphe nuclei enters the main olfactory bulb (MOB) from outside in, i.e., via the olfactory nerve layer. Serotonergic fibers in MOB were visualized by immunocytochemistry, and the distribution of specific 5-HT fibers closely matched the distribution of anterograde terminal label resulting from injections of WGA-HRP in the raphe nuclei. The serotonergic fibers have a specific laminar distribution and morphology in MOB. Thus, the density of the serotonergic innervation to the glomerular layer is 2-3 times that of any other layer in MOB. In addition to their preferential innervation of the glomeruli, glomerular and infraglomerular serotonergic fibers are morphologically different. Serotonergic fibers located in the glomerular layer are generally thick (0.25-0.60 micron) compared to the thinner (0.25-0.35 micron) fibers that predominate in infraglomerular layers. Another difference is that the glomerular fibers often contain varicosities that are greater than 1 micron in diameter, while varicosities along infraglomerular fibers are usually barely larger than the axonal diameter. Finally, glomerular fibers are much more intensely stained than infraglomerular fibers. Electrolytic lesions of the dorsal and median raphe caused a total depletion of serotonin fiber staining in the bulb, demonstrating that the raphe nuclei are the sole source of the serotonergic input to the main olfactory bulb. Thus, it has been demonstrated that serotonergic neurons in dorsal and median raphe project very heavily to glomeruli and less heavily to other layers in the main olfactory bulb.(ABSTRACT TRUNCATED AT 400 WORDS)

The developmental expression of tyrosine hydroxylase (TOH) was studied in a large, specific population of dopaminergic (DA) neurons in the main olfactory bulb (MOB) of the rat. These DA neurons comprise an anatomically distinctive population that has been well characterized in the adult hamster (Davis and Macrides, 1983) and rat (Halasz et al., 1981; Baker et al., 1983, 1984). We addressed a basic question in developmental neurobiology: What factors regulate the expression of neuronal transmitter phenotype during development? Olfactory bulb DA neurons are born in the ventricular and subependymal zones and migrate through all intervening layers to the most superficial layer in the bulb (Altman, 1969; Bayer, 1983). The time of TOH expression in these neurons was determined using immunohistochemistry and light microscopic image-analysis techniques. The results indicate that TOH phenotype is not expressed when the cells are born in the subependymal zone nor during their migration to the periglomerular region but only after they reached their final destination, the glomerular layer. This suggests that epigenetic factors associated with the glomeruli initiate the expression of the key transmitter synthesizing enzyme in these neurons. Primary olfactory neurons in the nasal epithelium project exclusively to glomeruli of the MOB; removal of this input in adult rats (Kawano and Margolis, 1982; Baker et al., 1983, 1984), mice (Nadi et al., 1981; Baker et al., 1983), dogs (Nadi et al., 1981), and hamsters (Kream et al., 1984) appears to down-regulate the expression of the TOH in periglomerular cells. The present results suggested that the input from the primary olfactory nerve is also necessary for the initial expression of the TOH phenotype. In support of this notion, we found that lesions of the olfactory nerve during the first postnatal week caused a significant reduction in the number of TOH-positive juxtaglomerular neurons in the following weeks. Thus, the olfactory nerve appears to be necessary for both the initiation and maintenance of TOH expression in olfactory bulb neurons. These findings suggest that specific cell-cell interactions play a key role in CNS neuronal transmitter phenotype regulation.(ABSTRACT TRUNCATED AT 400 WORDS)

The present studies have defined the developmental time course and distribution patterns of serotonergic fibers in the main olfactory bulb (MOB) using immunocytochemistry, anterograde tracing and image analysis. The results indicate that the deployment of serotonergic fibers to the main olfactory bulb is essentially a postnatal event in the rat. During the first 4 d after birth, 5-HT fibers infiltrate and begin to arborize in the MOB. The density of fibers in each layer is sparse during this period, but increases rapidly. By postnatal day 8 all layers are much more heavily innervated by 5-HT fibers. The surge of fiber growth into all layers is rapid; the fibers arborize earlier at caudal than at rostral levels. This may be related to the increased metabolic activity that is reported to occur selectively in the caudal parts of the immature olfactory bulb. After the second postnatal week, 5-HT fiber density increases much more gradually in all layers except the glomerular layer; in the glomerular layer, 5-HT fiber density continues to increase rapidly. It is also during this time that the olfactory bulb begins to grow substantially in volume. Bulb volume increases from the second week into adulthood (greater than 60 d); during the same period, the density of 5-HT remains relatively constant in the infraglomerular layers. Thus, from the second week onward, the growth of 5-HT fibers appears to be closely linked to the increasing volumes of these layers. The density of 5-HT fibers in the glomerular layer, however, continues to increase from the second postnatal week. Thus, the density of fibers in the glomerular layer increases more than the increase in glomerular size, indicating that the glomerular 5-HT fibers are proliferating more than could be accounted for by simple glomerular expansion. In the adult, 5-HT fibers are 2-3 times denser in the glomerular than the infraglomerular layers (McLean and Shipley, 1987). This preferential innervation of glomeruli may be the result of a protracted period of arborization by glomerular versus infraglomerular fibers. This could be due to the prolonged focal release of a trophic factor by glomerular-associated neurons or to the earlier production of an inhibitory factor by infraglomerular neurons.

The midbrain periaqueductal gray (PAG) has been shown to be a site where various manipulations induce pain suppression. Recent physiological evidence (Behbehani and Pert, 1984; Behbehani et al., 1987) suggests that neurotensin has pronounced physiological actions in PAG and effects pain suppression. We have performed immunohistochemical studies in order to determine the magnitude and distribution of neurotensin-like immunoreactive (NT-IR) cell bodies and fibers in PAG. NT-IR cell bodies were common throughout PAG, although there were more in the caudal than the rostral half. NT-IR neurons were much more numerous in the ventral than the dorsal half of PAG, and some appeared to be located within the dorsal raphe nucleus. The pattern of NT-IR fibers was analyzed with the aid of image enhancement/analysis and densitometry. The fibers were found to be heterogeneously distributed, being most heavily concentrated in the region adjacent to the cerebral aqueduct in the caudal two-thirds of PAG. The distribution of NT fibers closely matches sites where exogenously applied NT elicits long-lasting excitation of PAG neurons (Behbehani et al., 1987). Based on the known physiological and behavioral actions of NT in PAG, the present anatomical results suggest that NT acts on elements located predominantly in the medial and ventrolateral parts of PAG. Neurons activated by NT may project directly to the nucleus raphe magnus and adjacent ventral medulla (Behbehani and Pert, 1984) to activate neurons that project to the spinal cord and modulate nociceptive circuits.

The effect of neurotensin (NT) on periaqueductal gray (PAG) neurons was measured with extra- and intracellular recording methods in an in vitro preparation. Neurons excited by NT were heterogeneously distributed, being preferentially located in a region of PAG rich in NT-like fiber immunoreactivity. The majority of the responsive neurons were located in regions surrounding the aqueduct and the medial part of PAG. In 90% of the neurons, application of NT produced excitation that lasted for more than 2 min, while in the remaining cells, the excitatory effect lasted for less than 1 min. An inhibitory action of NT was rarely observed. Intracellular recordings showed that NT produced a depolarization leading to an increase in the spontaneous activity and multiple spiking with only a slight decrease in membrane resistance. The excitatory effect of NT was observed in neurons that were maintained in a solution containing cobalt. These results support the hypothesis that NT action on PAG neurons is due to the depolarization of the membrane and that this effect is mediated through a postsynaptic mechanism.