Expression of multiple proteins within single primary cortical neurons using a replication deficient HSV vector.

The study of protein-protein interactions in the nervous system has become dependent on the ability to express foreign proteins (or to overexpress endogenous proteins) within neurons. Often, multiple genes need to be overexpressed in the same cell. To investigate the simultaneous co-expression of more than one virally introduced gene in primary cortical neurons, we infected cultures with two different herpes simplex virus (HSV) vectors and analyzed the proportion of singly and doubly infected cells. The vast majority of neurons expressed both gene products, with a smaller number expressing one or the other protein alone. Increasing the quantity of virus caused an increase in the proportion of doubly labeled cells at the expense of singly labeled cells, which is consistent with a model in which infection with one viral vector is independent of infection with the other. We conclude that co-infection with HSV vectors is an efficient way to obtain expression of multiple gene products within individual primary culture neurons.

The neuronal growth-associated protein GAP-43 interacts with rabaptin-5 and participates in endocytosis.

Structural plasticity of nerve cells is a requirement for activity-dependent changes in the brain. The growth-associated protein GAP-43 is thought to be one determinant of such plasticity, although the molecular mechanism by which it mediates dynamic structural alterations at the synapse is not known. GAP-43 is bound by calmodulin when Ca2+ levels are low, and releases the calmodulin when Ca2+ levels rise, suggesting that calmodulin may act as a negative regulator of GAP-43 during periods of low activity in the neurons. To identify the function of GAP-43 during activity-dependent increases in Ca2+ levels, when it is not bound to calmodulin, we sought proteins with which GAP-43 interacts in the presence of Ca2+. We show here that rabaptin-5, an effector of the small GTPase Rab5 that mediates membrane fusion in endocytosis, is one such protein. We demonstrate that GAP-43 regulates endocytosis and synaptic vesicle recycling. Modulation of endocytosis by GAP-43, in association with rabaptin-5, may constitute a common molecular mechanism by which GAP-43 regulates membrane dynamics during its known roles in activity-dependent neurotransmitter release and neurite outgrowth.

Olfactory bulb glycogen metabolism: noradrenergic modulation in the young rat.

The olfactory bulb exhibits high glycogen phosphorylase activity, the rate-limiting enzyme in the mobilization of glycogen. The bulb also receives dense noradrenergic innervation and noradrenaline is known to stimulate glycogen breakdown. We determined the levels of glycogen in the bulb over the course of development and then determined the ability of noradrenaline to mobilize bulb glycogen. At birth, olfactory bulbs have very high levels of glycogen, with levels declining as the pups develop. Picomolar levels of noradrenaline mobilize glycogen in the bulb,. Initially, beta-adrenergic receptors mediate teh glycogenolysis and subsequently, the alpha-noradrenergic receptors in the bulb stimulate the breakdown of glycogen. Carnosine is involved in the repletion of bulb glycogen levels. The stimulation of glycogen breakdown by noradrenaline may play a role in allowing the increased activity that accompanies early olfactory stimulation.

Extracellular dopamine increases in the neonatal olfactory bulb during odor preference training.

Young rats learn to approach an odor that has been paired with tactile stimulation. This attraction is accompanied by changes in the metabolism and anatomy within the olfactory bulb glomerular layer. In this study, we examined the changes that occur in the olfactory bulb during early olfactory learning, rather than after such pairings have occurred. Specifically, we determined whether the pairing of an odor with tactile stimulation would produce a modified response by olfactory bulb glomerular-layer neurons. To monitor one large subgroup of these neurons during early learning, we used in vivo microdialysis to assess the activity of dopaminergic neurons in the olfactory bulb of postnatal day (PND) 3 rats during simultaneous presentation of odor and tactile stimulation, tactile stimulation alone, odor alone, or clean air alone. Clean air evokes no change in extracellular dopamine (DA), while both odor alone and stroking alone induce prolonged increases in DA peaking at about 200% of baseline. The combination of odor and tactile stimulation, which allows an olfactory preference to be formed, induces a prolonged increase in DA which peaks at about 400% of baseline. The level of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) increases only in pups receiving both odor and tactile stimulation and peaks at about 200% of baseline. With the exception of the pups exposed to clean air, all groups show an increase in homovanillic acid (HVA) of between 150-200% following stimulation. The large and prolonged increase in DA may be linked to the longer term anatomical and physiological changes in the glomerular layer of the bulb that form as a consequence of early olfactory preference training.

Biochemical quantitation and histochemical localization of glucose-6-phosphate dehydrogenase activity in the olfactory system of adult and aged rats.

The activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the hexose monophosphate shunt, was examined in olfactory epithelium, respiratory epithelium, olfactory bulb, and occipital cortex in Fisher 344 rats aged 4 and 24 months. Marked differences in this enzyme were found in olfactory compared to nonolfactory tissues. Olfactory epithelium and olfactory bulb have much greater glucose-6-phosphate dehydrogenase activity than respiratory epithelium and occipital cortex at both ages. Glucose-6-phosphate dehydrogenase remains fairly constant between adulthood and senescence in respiratory epithelium and occipital cortex. However, glucose-6-phosphate dehydrogenase activity decreases during the same time in both of the olfactory tissues examined. Previous studies of changes in this enzyme with aging have shown increases in enzyme activity in some brain regions, but never the decreases that we describe in olfactory tissues. Glucose-6-phosphate dehydrogenase histochemistry revealed intense staining of both the apical layer of olfactory epithelium and of Bowman's glands along with their ducts. Histochemistry of the olfactory bulb showed strongest staining in the nerve and glomerular layers of the bulb. The functional implications of these findings are discussed.

Glutathione levels in olfactory and non-olfactory neural structures of rats.

Olfactory receptor neurons are a CNS entry point for a wide variety of airborne substances. Therefore, it is probable that detoxification mechanisms are present in these neurons to neutralize such agents. Glutathione (GSH) is an essential component of several detoxification schemes, and in this study we examined the distribution and levels of GSH in the olfactory epithelium, olfactory bulb, cortex, hippocampus and cerebellum in neonatal, weanling, adult and aged rats. We report that GSH is primarily localized to the olfactory receptor neurons and their oxons within the olfactory epithelium. It is also localized within the glomerular neuropil and granule cells of the olfactory bulb. Levels of GSH in the olfactory epithelium and hippocampus do not change as a function of age, although GSH levels decrease in several brain regions, including the olfactory bulb, cerebellum and cortex.

Glucose-6-phosphate dehydrogenase activity in the olfactory system of the young rat: an enzyme histochemical study using computerized image analysis.

An understanding of olfactory system glucose metabolism is necessary for the interpretation of radiolabeled 2-deoxyglucose studies of odor processing since the relationship between glucose uptake and neural activity is based on assumptions regarding cellular glucose utilization. As part of an ongoing study examining divergent pathways of glucose metabolism in the olfactory system, the relative activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the hexose monophosphate shunt, was examined among cells of the rat olfactory bulb and anterior olfactory nucleus, by using enzyme histochemistry on fresh frozen tissue. Optical density measurement of formazan reaction product in stained tissue were quantified by computerized image analysis. To aid in the identification of histochemically stained neurons, alternate sections were Nissl-stained. The highest olfactory bulb dehydrogenase levels were found in the olfactory nerve and glomerular layers. Individual mitral and tufted cells also showed high dehydrogenase activity. In most stained neurons, formazan reaction product filled the cytoplasm and sometimes extended into the proximal part of dendrites and axons. The external plexiform and granule cell layers had low enzyme activity. High activity also was seen in pyramidal cells of pars dorsalis and pars lateralis of the anterior olfactory nucleus, one of the first, and most rostral of the olfactory bulb projection sites. High glucose-6-phosphate dehydrogenase activity in the olfactory system indicates that a significant amount of glucose can be channeled through the hexose monophosphate shunt in these neurons, with a concomitant production of NADPH. This may reflect high activity of cellular detoxification enzymes that rely on NADPH for reducing power. Such detoxification processes may be engaged in response to the potential entry and transsynaptic movement of airborne chemicals into the brain via the olfactory system.

NMDA receptor activation and early olfactory learning.

Norway rat pups have an enhanced olfactory bulb response to odors which they have learned to prefer early in life. When N-methyl-D-aspartate receptors are blocked pharmacologically before olfactory preference training, both the behavioral preference and the enhanced olfactory bulb response to the learned odor are suppressed. These results implicate the activation of these receptors in the kind of neural and behavioral plasticity that normally occurs during development.

Localized changes in olfactory bulb morphology associated with early olfactory learning.

Young rats exposed to an odor while receiving reinforcing stimulation come to approach that odor upon subsequent presentation. In addition, such pups have increased 14C-2-deoxyglucose (2DG) uptake within focal areas of the glomerular layer in response to that odor, compared to control animals experiencing the odor for the first time. In this study, the morphology of the glomerular areas underlying these 2DG foci was examined to determine whether early olfactory learning imposed local structural changes that could produce the enhanced 2DG uptake. Alternate sections either were processed with a silver and a Nissl stain to label both cell bodies and their processes or were histochemically treated for the mitochondrial enzymes cytochrome oxidase (CO) or succinic dehydrogenase (SDH) to define the glomerular core of the bulb; 2DG autoradiographs were aligned with adjacent stained sections, and regions underlying the high 2DG uptake foci were examined. In odor-familiar animals, large glomerular clusters that protruded into the external plexiform layer or the olfactory nerve layer were associated with the focal areas of increased 2DG uptake. Morphometric analysis of these regions revealed that the glomerular layer underlying the foci of high 2DG uptake was 30% wider in odor-familiar animals than comparable areas in odor-unfamiliar animals; the cross-sectional areas of individual glomeruli were 21% larger in odor-familiar animals. The foci of enhanced 2DG uptake therefore appear to be associated with groups of enlarged glomeruli. These data demonstrate that early olfactory learning influences the morphology of the olfactory bulb.

Glycogen phosphorylase activity in the olfactory bulb of the young rat.

The activity of glycogen phosphorylase, the enzyme that controls glycogen breakdown, was histochemically mapped in the olfactory bulbs of 19-day-old rats. The effect of early odor experience on subsequent olfactory bulb phosphorylase activity was also examined. The highest level of phosphorylase staining in the bulb (and seemingly the highest in the brain) was in the glomerular layer, followed by the external plexiform, internal plexiform, granule cell, and olfactory nerve layers. Virtually no activity was visible in the large output neurons of the bulb, mitral, and tufted cells. Early peppermint odor experience, previously shown to increase metabolic activity in specific glomerular foci as measured by 2-deoxyglucose uptake, had no apparent effect on glomerular-layer phosphorylase activity. In some odor-familiar animals, however, patches of activity were seen in the internal plexiform layer in the area of the bulb where foci of high deoxyglucose uptake are seen in response to peppermint. The patches were directly in line with modified glomerular clusters often seen to underlie foci of enhanced deoxyglucose uptake. The existence of particularly heavy activity in the peripheral third of the glomerular layer, where glycogen-containing modified Schwann cells have been localized, raises the possibility that the glomerular-layer activity is at least partially glial in origin. Finally, because of its rich noradrenaline and serotonin innervation and high density of insulin receptors, the olfactory bulb is proposed as a model system to study the interaction of glycogen/glucose metabolism with neural activity in a relatively well-defined neuronal circuit.

Odor specificity of the enhanced neural response following early odor experience in rats.

The enhanced neural response in the olfactory bulbs of rat pups following early olfactory experience is specific to the familiar odor. Pups were exposed daily to either peppermint or cyclohexanone odor for the first 18 postnatal days. On day 19, peppermint-familiar pups exposed to peppermint had significantly higher [14C]2-deoxyglucose (2-DG) uptake in a focal glomerular area compared with the response to peppermint by cyclohexanone-familiar pups. We also found that cyclohexanone-experienced pups had a subsequent enhanced response to cyclohexanone odor in glomerular areas medial and caudal to those responding to peppermint. None of the 2-DG uptake differences were attributable to respiration differences between the groups during any part of the odor test.

Enhanced neural response by adult rats to odors experienced early in life.

The enhanced olfactory bulb neural response to familiar odors by young rats persists into adulthood. Ninety-day-old rats who had received neonatal odor exposure had an enhanced uptake of [14C]2-deoxyglucose (2-DG) when exposed to the familiar odor. The odor-familiar rats did not have an increased respiration rate during the 2-DG test. A long-lasting change in neuronal response is consistent with the observation of behavioral effects of early odor experience persisting into adulthood.

Olfactory bulb responses after odor aversion learning by young rats.

The increased olfactory bulb response by young rats to familiar odors was not observed in response to odors which have attained their familiarity in aversive situations. Odor experience associated with toxicosis induced a behavioral aversion to the odor which was not accompanied by the enhanced uptake of [14C]2-deoxyglucose (2-DG) that accompanies attractive familiar odors. A single odor exposure on day 17 was sufficient to induce a small increase in 2-DG uptake in specific glomerular areas. We hypothesize that a different neural substrate underlies familiarity associated with an aversive odor than that associated with an attractive odor.

Stressors produce a concurrent decrease and increase in reflex amplitude in rats treated with naloxone.

The effect of inescapable foot-shock on the tail-flick response and on the startle response to brief shocks and brief tones was studied in rats. In the first experiment, 25 minutes of inescapable foot shock (stressor) produced a significant increase in tail-flick latency which was antagonized by the opioid antagonist naloxone (2.0 mg/kg). In the second experiment, the startle response to an electric shock to the tail was significantly diminished by the stressor, and this effect was not significantly reduced by naloxone. However, the size of the startle response to a brief tone was significantly increased in rats treated with naloxone. Thus, rats injected with naloxone had a decreased startle to shock but an increased startle to tone following inescapable foot shock. Finally, tones which preceded shocks by one second produced a facilitation of the startle response to the shocks in tests that followed exposure to the stressor. This facilitation was not affected significantly by naloxone. These results indicate that the changes in the startle response following the stressor were not mediated exclusively by endogenous opioids.

Enhanced neural response to familiar olfactory cues.

Norway rat pups have an enhanced olfactory bulb response to a familiar odor. A specific complex of glomeruli showed increased carbon-14-labeled 2-deoxy-D-glucose uptake in response to peppermint odor in 19-day-old pups exposed to peppermint on days 1 to 18 after birth, relative to control pups that had been exposed to clean air. The increased activity was not due to increased respiration of the familiar odor.

Diurnal cycle of mother-young contact in Norway rats.

Mother rats maintained on a LD 12:12 photoperiod (lights on 0800 hrs) had longer contact bouts with their offspring during the day than during the night and maternal brain temperature peaked during the night. When the daily temperature cycle was suppressed by removal of adrenal and ovarian hormones, the daily maternal contact cycle was also suppressed. These data are consistent with a thermal model for the limitation of mother-young contact bout duration.

Development of olfactory bulb organization in precocial and altricial rodents.

The structural organization of the olfactory bulbs of spiny mice, Norway rats and Mongolian gerbils was followed over the course of their development. The pups of all 3 species normally begin to approach the odor of their dams at a time when their olfactory bulbs are at a similar stage of development. The data suggest that there may be a common aspect of olfactory bulb development that underlies the onset of olfactory guided approach behavior in rodents.