The spatial organization of the peripheral olfactory system of the hamster. Part I: Receptor neuron projections to the main olfactory bulb.

The spatial organization of projections from olfactory receptor neurons to the main olfactory bulb (MOB) was studied in hamsters by using fluorescent stilbene isothiocyanates as retrograde tracers. Injections confined to small sectors of the MOB produce labeling of receptor neurons that is more restricted circumferentially (i.e., with respect to the medial-lateral and dorsal-ventral axes) than longitudinally (i.e., with respect to the rostral-caudal axis) along the mucosal sheet. This restricted labeling is also discontinuous, giving an initial impression that the peripheral input is only crudely organized with respect to the medial-lateral and dorsal-ventral axes of the nasal cavity. However, from analyses of serial sections, it is apparent that each set of mucosal segments shares convergent projections to a circumferential quadrant of the MOB with other segments that are positioned around a common domain of the nasal cavity airspace. The primary afferent projections to the MOB, thus, are organized rhinotopically (i.e., with respect to the three-dimensional position of receptor neurons in olfactory space) rather than mucosotopically.

The spatial organization of the peripheral olfactory system of the hamster. Part II: Receptor surfaces and odorant passageways within the nasal cavity.

The spatial organization of olfactory receptor surfaces and odorant passageways within the nasal cavity was studied in hamsters through descriptive and morphometric analyses of a complete stereotaxically defined series of coronal, sagittal, and horizontal sections through the snout. These analyses reveal that the caudal two-thirds of each cavity is divided into two longitudinally oriented medial and lateral channels. The olfactory mucosa that lines these two channels projects selectively onto the medial and lateral halves of the main olfactory bulb (MOB), respectively. Moreover, the ethmoturbinates of the caudal recesses create highly convoluted channels, lined by ventrally projecting mucosa, that lie ventral, lateral, and dorsal to a relatively smooth central channel lined by dorsally projecting mucosa. The rhinotopic map makes equivalent representations of medial and lateral olfactory space to the MOB but gives the smooth space lined by dorsally projecting mucosa a disproportionately larger representation on the MOB than the convoluted space lined by the more expansive ventrally projecting mucosa. Recent descriptions of the spatial distribution of probes for odorant receptor proteins conform closely to this organization, giving credence to the idea that rhinotopy is a basis for representing to the MOB the specific molecular features of odorant molecules.

Replication and differentiation of olfactory receptor neurons following axotomy in the adult hamster: a morphometric analysis of postnatal neurogenesis.

Regeneration of olfactory receptor neurons following unilateral olfactory nerve section was studied in Syrian golden hamsters by morphometric procedures. Characteristic structural and histochemical features of olfactory receptor neurons were compared on the sectioned and intact sides of the nasal septum at 6, 12, 18, 33, and 130 days following axotomy. The parameters measured were epithelial thickness and the numbers of nuclei, hematoxylin-staining olfactory vesicles, olfactory marker protein (OMP)-containing neuronal perikarya, and OMP-containing olfactory vesicles. The olfactory receptor neuron population was severely depleted 6 days after axotomy. In the succeeding 12-day period there was an initially rapid, then slower return of receptor neuron numbers. Though the regenerating olfactory epithelium appeared normal by gross inspection 33 days after the lesion, morphometric analysis revealed a substantial increase in the number of olfactory receptor neurons between 33 and 130 days postlesion. At our longest survival interval, all quantitative parameters had returned to 91-99% of control values. The numbers of OMP-containing perikarya and olfactory vesicles on the sectioned side were unchanged between the sixth and 12th postlesion days, thus suggesting that neural turnover was depressed for several days following surgery. Marker protein developed in newly formed receptor neurons between 6 and 12 days after the formation of the olfactory vesicle. Consequently, the ratio between OMP-containing olfactory vesicles and hematoxylin-staining vesicles (O/H) was used as an index of neural differentiation. The O/H ratio on the sectioned side was minimal 12 days following axotomy when stem-cell division was well under way but OMP antigenicity had not yet been expressed in newly formed neurons. At 33 days postlesion, O/H ratios were slightly but significantly greater than the control value, reflecting a degree of synchrony in the regenerating receptor neuron population. The average O/H ratio on the unlesioned side was 0.667. This value, together with our other observations, was used to derive an estimate of the life span of hamster olfactory receptor neurons, approximately 25-35 days.

Temporal matching of sensory-motor behavior and limbic theta rhythm deteriorates in aging rats.

The correlations between investigatory sniffing and rhythmic slow-wave activity (RSA) in the dorsal hippocampal formation were studied during free behavior in Fischer 344 rats aged 3, 18, 30, and 36 months. The amount and vigor of spontaneous exploratory behavior was reduced in older animals, and the frequency distributions of investigatory sniffing and hippocampal RSA both shifted with age toward the lower end of their normal ranges. In the youngest animals, the dominant frequency of sniffing matched that of hippocampal RSA (frequency entrainment) more often than would be predicted by chance; preferred phase differences between sniffing and hippocampal RSA were reliably observed in the 5-9 Hz range; and these preferred phase differences varied linearly as a function of frequency, implying an underlying latency relationship. These correlations changed progressively with age as follows: the incidence of frequency entrainments decreased; the frequency range within which preferred phase differences were observed became lower and narrower; and the incidence of preferred phase differences decreased. However, animals of all ages exhibited similar preferred phase differences for those frequencies at which significant preferences were expressed. These findings are discussed in relation to the hypothesis that alterations of forebrain theta rhythms may accompany aberrations of the medial septum-diagonal band-nucleus basalis complex and may be importantly involved in aging-related impairments of cognitive and learning abilities.

Aging-related morphological changes in the main olfactory bulb of the Fischer 344 rat.

Aging-related changes in several structural characteristics of the main olfactory bulb (MOB) were evaluated using Fischer 344 rats 3, 18, 30 or 36 months of age. Histological examination of the nasal mucosa revealed no evidence of concurrent rhinitis in any of the animals studied. The internal granular layer of the MOB exhibited continual growth, increasing in volume by a factor of 63% over the range of ages studied. The sizes of MOB mitral cell perikarya and nuclei, expressed either as observed cross-sectional areas or as estimated mean volumes, did not change significantly as a function of age. The numbers of mitral cells exhibiting 2 nucleoli in the plane of section decreased from about 22% in the youngest animals to about 4% in the 18- and 30-month old animals and to nil in the oldest animals. These results are discussed in relation to findings of other investigators using Sprague-Dawley or Wistar rats. It is concluded that major structural age changes in the rat MOB are strain dependent.

Power spectral analysis of the EEG following protein malnutrition.

In these studies, power spectral analysis techniques were utilized to quantify the EEG obtained from rats reared on either an 8% or 25% casein diet during various vigilance states at two stages of development: (1) adulthood-90 to 120 days old; and (2) immediately after weaning-22 to 23 days old. It was found that the cortical EEG contained relatively more power in the low frequencies (ie., 0.5 to 10 Hz) for the 22-23 day old animals than for the 90-120 day old rats, especially during the slow wave sleep states-SWS1 and SWS2. Theta activity (5-8 Hz) in the hippocampus was shown to have greater power for the 22-23 day old group than for the older animals during both REM sleep and waking. Analyses of power spectral data and other indices of the frequency distribution of the hippocampal EEG indicated that those animals subjected to protein malnutrition have significantly more power in the theta band during REM sleep than the normal adult group. Since it was also noted that the hippocampal EEG obtained from the 22-23 day old group contained relatively more power in the theta band than the 90-120 day old group, the dietary treatment effect might be intrepreted as an instance of retarded development associated with protein malnutrition. Thus, a significant effect of the dietary manipulation used in the study may be largely on the system responsible for regulating theta activity.

Influence of prenatal and continuous postnatal protein-deficient diets on the development of rats.

The influence of prenatal and continuous postnatal protein-restricted diet on the development of rats was investigated. Virgin female rats were maintained for 35 days on isocaloric diets containing 8 or 25% casein, and then mated. The animals were supplied their respective diets during gestation and nursing. It was observed that the offspring of mothers kept on the 8% casein diet had 50% less body weight, and 10-15% less brain weight than the corresponding controls. The peak of rapid brain growth for both groups of animals occurred at age 6 days. The rate of de novo biosynthesis of sterols, following the injection of (2-14C)-mevalonic acid into the midline thalamic area and the cerebellum, was also investigated. A significant shift of the age of peaking of 14C-incorporation into sterols in the cerebellum was noted. Our results indicate that although unlimited amounts of food were supplied to the mothers on the 8% casein diet, this could not offset the detrimental effect of the protein deficiency on the offspring.

Dorsal raphe, substantia nigra and locus coeruleus: interconnections with each other and the neostriatum.

Using a retrograde axonal transport method, direct projections to the neostriatum were demonstrated from the dorsal raphe nucleus, a large area of the ventral midbrain tegmentum (including the ventral tegmental area of Tsai, the substantia nigra pars compacta, reticulata and suboculomotoria), and the tegmentum ventral to the caudal red nucleus. A direct projection was also found from the mediodorsal part of the substantia nigra to the rostral part of the dorsal raphe nucleus. Projections from the entopeduncular nucleus (pallidum) and the lateral hypothalamic area to the lateral habenular nucleus, and from the latter to the dorsal raphe nucleus were also found. This habenular projection arises primarily from large neurons in the medial part of the lateral habenula and also from another group of small cells immediately adjacent to the medial habenular nucleus. A non-reciprocal connection of the dorsal raphe nucleus to the locus coernuleus was also found. On the basis of these results and the data available in the literature on the possible neurotransmitters used by these various structures, it is suggested that the dorsal raphe nucleus may play an important role in brain stem modulation of neostriatal function.

Locus coeruleus-to-dorsal raphe input examined by electrophysiological and morphological methods.

In order to examine the hypothesis that the locus coeruleus (LC) projects directly to the nucleus raphe dorsalis (DR), electrical stimulation was applied to the LC of rats while recording from single neurons in the region of the DR. Slow firing units of the DR were not influenced by the stimulation, although faster firing units in the nearby substantia grisea centralis (SGC) were. These latter cells become oscillatory in their firing rates during LC stimulation. In parallel studies a retrograde transport technique was imployed to obtain morphological evidence regarding projections to DR. Placements of horseradish peroxidase precisely in the DR resulted in very sparse labeling in the LC, although positive transport occurred to other areas. The results indicate that the LC does not project directly to slow firing DR neurons, but does influence faster firing celles in the region of the SGC, probably by complex routes. Suggestions are made for the integration of these findings with earlier fluorescence studies.

Feeding and drinking in rats maintained on a low protein diet.

Male and female rats born of protein malnourished mothers were fed a low-protein diet (8% casein) for 150 days after weaning and daily food and water intakes and body weights were monitored. Although daily intakes of diet throughout the study were significantly lower than those of rats maintained on a normal protein diet (25% casein) or stock diet, intakes/100 g body weight were significantly greater. Daily increments in body weight, as percent of previous day's weight, were consistently higher in rats fed the low-protein diet in comparison to rats fed the normal protein diet. Marked retardation of body growth was evident throughout the study although feeding efficiency ratios (daily body wt. increment per g daily food intake) were comparable among the various dietary groups. Compensation for reduction of the protein component of the diet by increased daily food intake/100 g body weight did not alleviate growth retardation.

Effects of maternal dietary protein restriction on growth of the brain and body in the rat.

We studied the growth of the brain and body in rats born of dams fed a low-protein (8% casein) diet ad lib beginning 5 weeks prior to mating and continuing throughout gestation and lactation. Control dams were fed an isocaloric 25% casein diet. Litters were culled at birth to 8 pups. At birth, brain weights and body weights of pups of protein restricted dams were similar to those of control pups. During the period of lactation, pups of restricted exhibited severely retarded body growth but only mildly retarded brain growth resulting in an elevated brain/body weight ratio. This relative macrocephaly was maximal at 10-20 days of age, declining completely by 35 days of age. The relative macrocephaly could not be accounted for by increased retention of water in the brain of the malnourished rats. Following weaning, pups were maintained ad lib on the diets fed their mothers. At adulthood brain/body weight ratios were normal in the protein restricted group.

Horseradish peroxidase tracing of the lateral habenular-midbrain raphe nuclei connections in the rat.

Connections of the habenular complex to the nuclei of the midline in the midbrain (interpeduncularis, medianus raphe, and dorsalis raphe) have been studied classically by anterograde degeneration in the monkey, the cat, and marsupials. Passing fibers from the medial septal nucleus and lateral preoptic area, however, have also been demonstrated which can complicate interpretation of these results. In this paper the habenular projections were studied in the rat by the retrograde axonal transport of horseradish peroxidase (HRP). After HRP injections in the medianus raphe nucleus labelled neurons appeared in the lateral habenular nucleus and parafascicular nucleus. Labelled neurons were also found in the lateral habenular nucleus after injections in either the dorsalis raphe nucleus or the caudal central gray substance. The habenular projections were always bilateral. There were no labelled neurons in the medial habenular nucleus after HRP injections in the medianus raphe especially on the dorsalis raphe neurons which have usually been thought of as functionally related to other brainstem structures. The present results suggest also that in the rat the lateral habenular nucleus might be the link between basal forbrain inputs and the limbic midbrain area. Thus, the raphe nuclei of the midbrain appear to be crucial regions for integrating two descending circuits: first, a limbic (through septum) circuit, and, second, a basal forebrain (through lateral habenular-preoptic area) circuit.

Effects of protein malnutrition during development on protein synthesis in brain and peripheral tissues.

Rats born of mothers fed a low protein diet (8% casein versus a normal 25% casein diet) starting 5 weeks prior to mating showed a 50-100% increase in protein synthesis in the brain and kidney on the day of birth. This effect was due to a 50-100% increase in the uptake of IP injected 14C-leucine in the malnourished rats. The proportion of total tissue radioactivity in the trichloroacetic acid-protein precipitates was the same in the 8% and 25% casein groups. For the most part, there were no significant diet related changes in uptake or incorporation of 14C-leucine in the brain, liver or kidney in the 8% and 25% casein groups on Days 5, 10-11 and 21. While the physiological basis of the diet related changes seen on the day of birth is unknown, the present data represent a previously undescribed effect of prenatal protein malnutrition.

The effect of chronic protein malnutrition on trans-callosal evoked responses in the rat.

Studies were carried out on the trans-callosal evoked response in rats born of dams fed either a low (8%) or normal (25%) protein diet beginning 5 weeks prior to mating and throughout gestation and lactation. After weaning, pups were fed the same diets as their mothers. Bipolar (surface vs depth) stainless steel stimulating and recording electrodes were positioned at corresponding loci in the right and left sensorimotor cortices. Trans-callosal evoked responses were measured under urethane anesthesia using twice-threshold bipolar pulses of .1 msec duration at ages 13, 21, and 60-66 days. Evoked response latency was significantly greater in malnourished animals at 13 days of age, whereas at adulthood no latency differences were seen. Poststimulation excitability (15-100 msec range) was not significantly affected by the dietary treatment. These results are interpreted as corroborating previous reports on rats undernourished during development using sensory evoked potentials. By avoiding the use of extrinsic sensory stimulation, the present study demonstrates a dietary effect upon ontogeny of cortical evoked potentials independent of any possible effect on sensory receptor mechanisms.

Effects of growth hormone on brain biogenic amine levels.

The effects of IP administered bovine growth hormone (GH) on regional brain serotonin, 5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine levels in rats were examined. GH decreased the levels of both monoamines and 5-HIAA in the diencephalon and brainstem while not affecting telencephalic concentrations. In hypophysectomized rats, however GH produced significant elevations of monoamine and 5-HIAA levels in all brain regions. In normal rats the decreases in norepinephrine content produced by GH were correlated with a reduction in the stimulatory action of d-amphetamine on general activity levels. These results demonstrate that GH can affect brain biogenic amines and that these effects have behavioral consequences.