Early hypothyroidism in rats causes increased adult testis and reproductive organ size but does not change testosterone levels.

The role of thyroid hormones in the testis is unclear, although recent evidence indicates they may be important for testicular development. Here we describe a novel method for increasing adult testicular size in the rat by induction of transient hypothyroidism during neonatal life. Rats were treated with a reversible goitrogen, 6-propyl-2-thiouracil from birth to day 25 when treatment was stopped, allowing return to a euthyroid state. At days 90, 135, 160, and 180, wt and DNA content of the testis, epididymis, ventral prostate, seminal vesicle, and those of some nonreproductive organs were determined, as well as serum levels of testosterone (T) and thyroid hormones. Despite decreased body wts in 90-day and older 6-propyl-2-thiouracil-treated rats, testis wt was increased by 40% and 60% at 90 and 135 days, respectively; maximal increase (80%) occurred at 160 days. These wt increases were accompanied by proportional changes in DNA content. Significant enlargements were also seen in other reproductive organs, but they occurred after a time lag and were smaller in magnitude. Interestingly, serum T levels showed no increase at any age. Weight and DNA content of nonreproductive organs, like body wts, were less than controls at all ages but thyroid hormone levels were normal. Thus, transient hypothyroidism in neonatal rats is associated with lasting enlargements in the ultimate size of testis and other reproductive organs in the adult. These changes are not related to excess T levels. The results indicate early critical influences of thyroid hormones on growth and development of the reproductive system and suggest an experimental model for inducing lasting enlargements in testis and reproductive organs. The model may also be useful for studying regulation of reproductive growth and final size.

Increased sperm production in adult rats after transient neonatal hypothyroidism.

In the preceding paper it was shown that transient neonatal hypothyroidism induced by treatment of rats from birth to day 25 with the goitrogen 6-propyl-2-thiouracil (PTU) is associated with increases in testis wt and DNA content of up to 80% during adulthood. The testis changes were accompanied by similar, though less marked, increases in the wt and DNA content of epididymis and accessory organs. The purpose of this study was to assess sperm production in these enlarged testes and measure changes in sperm reserves in the epididymis. Testes and epididymides were obtained from control rats or rats given PTU from birth to day 25 (designated "treated") at 90, 135, 160, and 180 days of age. Daily sperm production (DSP), efficiency of sperm production (DSP/g testis), and epididymal sperm reserves were measured in all animals. Compared to controls, DSP of the treated rats was increased by 83%, 86%, 136%, and 132% at 90, 135, 160, and 180 days, respectively. Thus, in the treated rats, DSP, like testis wt, plateaued at day 160. In addition, efficiency of sperm production was increased by 15%-30% at all ages in treated animals. Epididymal sperm reserves were also increased in treated rats at all ages, but the correlation between DSP and epididymal sperm reserves was weak. Sperm motility and concentration in caudal epididymal fluid of adult males treated from birth to day 25 with PTU were normal. These males were fertile and sired litters in which pup wt and pup number were normal. These results indicate that neonatal hypothyroidism in rats is associated not only with increased testis size but also with increased efficiency of sperm production, resulting in increases in DSP of up to 140% in these animals during adulthood. Maximal sperm production is reached at 160 days of age in treated rats (compared to 100 days in controls), coinciding with the attainment of final testicular size. This system represents the first experimental model in which such large increases in sperm production can be produced. The neonatal PTU treatment does not appear to impair fertility or alter sperm characteristics when these animals become adults and may be a useful system with which to study factors which normally regulate sperm production.

Arrhenius plots of membrane-bound enzymes of mitochondria and microsomes in the brain cortex of developing and old rats.

To study changes in lipid-protein-interaction and fluidity in mitochondrial and microsomal membranes of brain during development and aging, the Arrhenius plots of marker enzymes for mitochondrial inner and outer membranes as well as those of microsomal membranes were compared at different ages. The enzymes were, beta-hydroxybutyrate dehydrogenase (BDH) for the inner mitochondrial membrane and rotenone-insensitive-NADH-cytochrome c reductase (Mit-NADH-CytR) for the outer membrane; also antimycin-insensitive-NADH-cytochrome c reductase (Micr-NADH-CytR) and NADPH-cytochrome c reductase for the microsomal membranes. The ages studied were, 1, 5, 30, 60 days postnatal and 2 years. In the microsomes, the plots of NADH-cytochrome c reductase were found to be biphasic at all ages except in the newborn where no break temperature was observed. The activation energy of this enzyme in the physiological range of temperature was found to be high in the newborn, declining with brain maturation. The plot of this enzyme in the old rat showed no difference when compared with the mature. The specific activity of the enzyme, however, was markedly reduced in the old brain microsomal fraction. In contrast, comparison of the plots of microsomal NADPH-cytochrome c reductase at the various ages revealed no break temperature and very similar energies of activation. For the inner mitochondrial membrane beta-hydroxybutyrate dehydrogenase, the Arrhenius plots were generally biphasic at all ages studied with a break temperature of about 20 degrees C. However, the newborn plot was only barely biphasic showing a high energy of activation in the physiological range of temperature. In contrast, for the outer membrane NADH-cytochrome c reductase, the newborn plots were definitely biphasic, exhibiting low activation energy above the breaks. There was also a significant decline in the break temperature with brain development. No significant differences in the plots of this enzyme were found between the old and mature brain cortex. It is concluded that the enzymes of inner and outer mitochondrial membranes as well as the oxidative enzymes of microsomal membranes may show differential patterns of change in lipid-protein-interaction during development and aging, the changes being more marked in development than in aging.

Early responses of skeletal muscle in recovery from hypothyroidism.

Postnatal growth of skeletal muscle (m. gastrocnemius) was compared in rats under euthyroid, hypothyroid and hypothyroid-rehabilitated conditions. In normal (euthyroid) animals, gastrocnemius muscle grows significantly in terms of weight (150 x) from birth to the young adult and, in terms of total contractile myofibril protein (15 x) and myosin ATPase activity (10 x) between days 25 and 90. Rats made hypothyroid (with 0.1% w/v propylthiouracil, PTU) from birth show reduced growth. At 25 days (weaning), compared with euthyroid, muscle weight is only 25% of normal, and a similar reduction is found in total DNA, RNA, protein, myofibril protein, and myosin ATPase activity. These deficits, already significant by day 10, are more marked by day 50 due to the near arrest of growth. Hypothyroid rats allowed to recover by PTU withdrawal after day 25 (rehabilitated) undergo marked compensatory muscle growth. By day 90, muscle weight and protein content increase 50 x, DNA 7 x and RNA 17 x. Over this period, total myofibrillar protein and myosin ATPase increase 20-40 x, but are still below those of 90-day controls, suggesting that the severe growth retardation had not yet been fully compensated. Early thyroid deficiency drastically reduces the normal age-related growth of skeletal muscle and severely retards the development of contractile elements, affecting muscle hypertrophy (protein content) more than cell proliferation (DNA content). Rehabilitation compensates to a major degree for this growth retardation. These results underline the key role of thyroid hormones in regulating development and maturation of skeletal muscle throughout the preweaning and postweaning phases of growth.

Paradoxical hypertrophy and plasticity of the testis in rats recovering from early thyroid deficiency: a growth study including effects of age and duration of hypothyroidism.

The effects of various durations of postnatal hypothyroidism followed by recovery were studied on testicular growth in Long-Evans and Sprague-Dawley rats from birth to 7 months. Hypothyroidism was induced by adding propylthiouracil (PTU) in drinking water (0.1%, w/v). Recovery was induced by withdrawal of PTU. Testicular growth was reduced in rat pups by 20, 65 and 90% at days 10, 25 and 50. Upon withdrawal of PTU at weaning (25 days), testicular growth resumed and became compensatory; catch-up growth occurred by day 65. Paradoxically, testicular growth progressively increased, surpassing the control weights by 40, 50 and 100% at days 75, 90 and 210. Maximal testicular growth rate in the recovery group was 35% higher, occurred 2 weeks later and lasted 2 weeks longer than controls. Testes of rats subjected to prolonged postnatal hypothyroidism (60 or 120 days) also showed recovery and hypertrophy, amounting to nearly twice the normal maximal growth levels, after at least 6 months of recovery. Body weights of recovering rats remained always significantly below those of controls. When the suckling pups were exposed to short, week-long regimes of PTU treatment, only rats treated during the early postnatal weeks (days 1-8 or 9-16) had enlarged testes; PTU treatment during the late suckling period (days 17-24) or later had no effects. Total duration of hypothyroidism in the suckling period was positively correlated with testicular enlargement. The results indicate that hypothyroidism early in life is stimulatory to testicular growth, resulting in a paradoxical twofold increase in final testicular size which occurs even if hypothyroidism is prolonged. These effects occur similarly in different strains of rat with differing sized testes. It is suggested that there is a sensitive period for this effect, i.e. during the first 2 weeks after birth. The marked plasticity of testicular growth as shown by its recovery and hypertrophy, even after long periods of hypothyroid retardation, is unique in the body and may be a useful model for studying hormonal factors regulating testicular growth and for animal breeding and research into infertility.

Rehabilitation from neonatal hypothyroidism: spontaneous motor activity, exploratory behavior, avoidance learning and responses of pituitary--thyroid axis to stress in male rats.

Long-Evans male rats were made hypothyroid from birth by the addition of 6-N-propylthiouracil (PTU) to their drinking water (0.1%). A group of animals was rehabilitated beginning at postnatal day 25 by withdrawal of the PTU from the drinking water. Subsequently, the rats were tested for a variety of behavioral tasks. Serum concentrations of thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3) were determined by radioimmunoassay. At 50 days of age, PTU-treated rats had non-detectable levels of T4 but an eight-fold increase of TSH. In 50-day-old, neonatally hypothyroid but rehabilitated rats, serum TSH and T3 were normal, although T4 was still significantly lower. At 90 days of age, basal levels of TSH and thyroid hormones were normal in the rehabilitated rats, but thyroid hormone secretion in response to various types of neural stress was markedly altered. Comparison of passive avoidance learning revealed no significant alteration in the memory retention of either PTU-treated or rehabilitated animals. The 50-day-old, rehabilitated rats showed increased locomotor activity both in running-wheel and in hole-board tests; this hyperactivity, though markedly reduced, still persisted at day 90. In the early phase of rehabilitation (50 days of age), decreases in exploratory activity and lack of habituation occurred with the hole-board test; by the late phase of rehabilitation (90 days of age) these behavioral parameters had become normal. These results suggest generally longer periods of plasticity of the brain and better prospects for rehabilitation from neonatal cretinoid retardation than commonly believed. Specifically, the pituitary-thyroid system and neural mechanisms integrating adaptive behavior possess considerable capacity for spontaneous recovery from hypothyroidism; certain types of altered neuroendocrine and behavioral responses appear to be less amenable to rehabilitation or require longer periods for complete rehabilitation.

Human olfactory bulb: aging of glomeruli and mitral cells and a search for the accessory olfactory bulb.

The aims of this study on the human olfactory bulb were two. First morphometry of the bulbs revealed marked declines during aging in the numbers of mitral cells and glomeruli, the bulb's principal integrative and relay elements. Numbers of glomeruli and mitral cells in each bulb of the young adult human were found to be approximately 8,000 and 40,000, respectively; these numbers declined steadily with age at an approximate rate of 10% per decade, so that in the ninth and tenth decades less than 30% of these elements remain in place. Such a marked decline with aging is suggested to underlie in part the decline in olfactory abilities (odor detection and identification) of humans with aging. In a separate study a systematic search for presence of an accessory olfactory bulb in the adult and aging bulbs was undertaken. No positive evidence for such an organized formation was found in the various regions of the adult bulbs of different age groups. The implications of these negative findings for the recent theories on human vomeronasal function and pheromonal perception are discussed.

Vomeronasal organ in bats and primates: extremes of structural variability and its phylogenetic implications.

The mere appearance of a tubular, epithelially-covered, bilateral structure, no matter how minuscule, on the anteroventral nasal septum of tetrapods, is generally called the vomeronasal organ (of Jacobson). However, considering the functionality of this chemosensory structure, the presence of a non-cilated (microvillar) neuroepithelium (and not just any odd type of epithelium) encased in a variously shaped vomeronasal cartilage, along with vomeronasal nerve bundles and above all an accessory olfactory bulb connected to the limbic vomeronasal amygdala, are the absolute essential neurostructural characteristics and anatomic requirement for a functional VNO and the accessory olfactory system in any tetrapod. The distribution of the vomeronasal organ is reported here in two mammalian orders: Chiroptera and Primates. An impressive data pool on the vomeronasal organ of bats is now available, pointing to the fact that at this time bats may be the only group in which this organ system is extremely variable, ranging from total absence (even in the embryo) to spectacular development with numerous intervening stages in different chiropteran species. Of the eighteen bat families, only one family of New World leaf-nosed bats, family Phyllostomidae, exhibits functional vomeronasal organs. The vespertilionid bat Miniopterus, and the mormoopid bat Pteronotus, present exceptions to this rule. Among Primates, very few species have been rigorously studied. As a result, developmental variability of the vomeronasal organ is almost unknown; either the vomeronasal organ is well developed (such as in New World monkeys) or absent (as in Old World monkeys and great apes) in the adult. The concept whether adult humans or embryonic and fetal forms are endowed with this so-called sixth sense, is a controversial one and is under intense study in our laboratory and by others. The general phylogenetic implications based on our cladistic analysis of bats are that the vomeronasal organ complex has evolved several times. Among the prosimians and platyrrhine primates, the organ is well developed, although to a varying degree. Among catarrhine primates, its loss has occurred only once, as it is generally absent in the adult forms.

Structure and diversity in mammalian accessory olfactory bulb.

The accessory olfactory bulb (AOB) is the first neural integrative center for the olfactory-like vomeronasal sensory system. In this article, we first briefly present an overview of vomeronasal system organization and review the history of the discovery of mammalian AOB. Next, we briefly review the evolution of the vomeronasal system in vertebrates, in particular the reptiles. Following these introductory aspects, the structure of the rodent AOB, as typical of the well-developed mammalian AOB, is presented, detailing laminar organization and cell types as well as aspects of the homology with the main olfactory bulb. Then, the evolutionary origin and diversity of the AOB in mammalian orders and species is discussed, describing structural, phylogenetic, and species-specific variation in the AOB location, shape, and size and morphologic differentiation and development. The AOB is believed to be absent in fishes but present in terrestrial tetrapods including amphibians; among the reptiles AOB is absent in crocodiles, present in turtles, snakes, and some lizards where it may be as large or larger than the main bulb. The AOB is absent in bird and in the aquatic mammals (whales, porpoises, manatees). Among other mammals, AOB is present in the monotremes and marsupials, edentates, and in the majority of the placental mammals like carnivores, herbivores, as well as rodents and lagomorphs. Most bat species do not have an AOB and among those where one is found, it shows marked variation in size and morphologic development. Among insectivores and primates, AOB shows marked variation in occurrence, size, and morphologic development. It is small in shrews and moles, large in hedgehogs and prosimians; AOB continues to persist in New World monkeys but is not found in the adults of the higher primates such as the Old World monkeys, apes, and humans. In many species where AOB is absent in the adult, it often develops in the embryo and fetus but regresses in later stages of development. Finally, new areas in vomeronasal system research such as the diversity of receptor molecules and the regional variation in receptor neuron type as well as in the output neurons of the AOB and their projection pathways are briefly discussed. In view of the pronounced diversity of size, morphologic differentiation, and phylogenetic development, the need to explore new functions for the vomeronasal system in areas other than sexual and reproductive behaviors is emphasized.

Lasting effects of early olfactory deprivation on the growth, DNA, RNA and protein content, and Na-K-ATPase and AchE activity of the rat olfactory bulb.

It is shown that when one of the olfactory bulbs (OB) of the rat is deprived of olfactory stimulation from birth, as compared to the normal OB on the opposite side, significant and permanent reductions in its growth and in several biochemical parameters take place as follows: weight gain, 25%; total DNA, 20--30%; total RNA, 30%; total protein, 30%; total Na-K-ATPase activity, 50%; total AChE activity, 20%. The concentrations of DNA, RNA and protein and the specific activity of AChE were not significantly affected but the specific activity of Na-K-ATPase was significantly reduced. In general, these interbulbar differences were seen in every experimental animal and were not due to hypertrophy or hyperplasia of the normal OB. These results suggest that olfactory stimulation during the early postnatal period has a significant influence on cell proliferation and cell growth as well as on the proliferation of neuronal membranes and synapses in the developing OB. It appears that the effects of olfactory deprivation are exerted during the first few weeks after birth when the bulb shows its most rapid period of growth and development, since (with the exception of DNA) the magnitude of the interbulbar difference did not increase appreciably by prolongation of olfactory deprivation beyond the early period. Thus in the rat the existence of a critical period encompassing the second and third postnatal weeks is suggested during which the developing OB appears to be especially vulnerable to the absence of olfactory stimulation.

Relative contribution of brain and peripheral connections to postnatal growth and cell accretion in the rat olfactory bulb.

Total weight and DNA (cell number) determination in whole olfactory bulbs (OB) revealed that unilateral transection of the olfactory peduncle in newborn rats causes 2 X more (40%) deficit in cell number in the operated OB than section of the olfactory nerve. The deficit due to combined operations ('isolation' of OB) were additive. Similar effects were observed on weight gain. It is inferred that both olfactory nerve and sources in the brain exert marked influences on postnatal cell accretion and growth in the OB and that the greater share of the brain is due to its direct contribution of new microneurons via the 'rostral migratory stream'.

Acetylcholinesterase activity in the developing olfactory bulb: a biochemical study on normal maturation and the influence of peripheral and central connections.

In the newborn rat olfactory bulb (OB), the specific activity of acetylcholinesterase (AChE) is about 20% of the adult value. During postnatal development, the specific activity remains unchanged until day 10; a growth spurt of 5X is observed between days 10 and 25, when adult values (90 nmoles ACh/min/mg protein; 6 nmol/min/mg wet wt.) are reached. However, total activity shows continuous increase slowly at first and rapidly between days 10 to 30, reaching a plateau by day 60. Between birth to day 60 total activity increases 65X. To determine the influence of peripheral and central connections on the development of AChE activity in the OB, rats were subjected to unilateral olfactory denervation and/or transection of olfactory peduncle, carried out either neonatally or at day 30; the bulbs were assayed a month later (days 30 and 60 respectively). It was found that in both neonatal and 30-day-old rats, denervation caused a 15% decrease in total activity, while transection led to more than 60% reduction. In the older rats, the reduction due to transection represented the degenerative loss of activity, but in the neonatally transected bulbs the growth of some cholinergic elements continued although very slowly. Between birth to day 30, total AChE activity increased only 12X in completely isolated OB, 25X in transected OB and 40X in denervated OB, compared to 45-50X in control OB. In the transected and isolated bulbs specific activity of AChE was also reduced significantly (25-50% depending on age and operation). These results suggest that while centrifugal fibers are the main source of cholinergic activity in the mature as well as the developing OB, the olfactory nerve and some intrabulbar sources such as cholinergic cells or cholinoceptive membranes also contribute to AChE activity in the OB. These intrinsic sources of AChE activity can persist and even show some growth in the developing olfactory bulb in the absence of the centrifugal fibers and/or the olfactory afferents.

A quantitative study of the effects of early unilateral olfactory deprivation on the number and distribution of mitral and tufted cells and of glomeruli in the rat olfactory bulb.

Anatomic effects of early unilateral olfactory deprivation on the developing olfactory bulb were investigated in the albino rat. Unilateral anosmia was experimentally induced by neonatal cauterization of the left or right nare; regenerating the tissue permanently blocked the nostril. The anosmic olfactory bulb (ipsilateral to the blocked nare) and its contralateral counterpart, serving as the normal control, were compared for the following quantitative anatomic parameters: total number and distribution of mitral and tufted cells and olfactory glomeruli; average diameters of mitral cells and glomeruli; relative dimensions of the bulb and its layers. The effects on mitral cells and glomeruli were studied at the ages of 25 and 60 days and at 2 years; other studies were carried out in the 25-day-old animals only. In the normal mature bulb, the number of mitral cells, tufted cells and glomeruli was found to be about 70,000, 160,000 and 3000, respectively. It was found that the absence of early olfactory stimulation was invariably correlated with a significant and permanent loss of mitral cells, amounting to more than a quarter of the total number. This loss apparently occurred rapidly during the first 3 weeks, as it was already evident by day 25 and did not increase appreciably with prolongation of deprivation. Tufted cells were apparently even more susceptible, because their number decreased by about 45%. As evident from this distribution profiles, the loss of mitral and tufted cells occurred uniformly throughout the bulb. It is shown that these differences were due neither to inherent interbulbar differences, nor to a hyperplasia in the normal bulb. Early anosmia had no significant effects on the number or average diameter of the glomeruli. Morphometric studies revealed that the dimensions and thickness of layers (internal and external plexiform and granular) of the anosmic bulb were significantly reduced. It is suggested that early olfactory stimulation is necessary for survival of the developing mitral and tufted cells; thus the first 2-3 postnatal weeks, covering the final developmental stages of these cells, would constitute a vulnerable period in the development of the rat olfactory system.

Deficient lipid-protein interaction in mitochondrial cytochrome oxidase from newborn and old rat brain.

In contrast to the mature brain, Arrhenius plots of mitochondrial cytochrome c oxidase from newborn and old rat brain show no breaks (transition temperature); the apparent activation energy of the enzyme, in the physiological range of temperature, is also significantly higher. The results indicate deficient lipid-protein interaction as well as differential fluidity status in the inner mitochondrial membranes of the newborn and aged brain. These changes may contribute to the known deficiency in oxygen consumption of the newborn and old brain.

Early olfactory deprivation and the mitral cells of the olfactory bulb: a Golgi study.

Golgi impregnation was used to study the morphology, morphometry and number of mitral cells in control and stimulus-deprived olfactory bulbs (OB) of 25-day-old albino rats. The rats were made unilaterally anosmic by closure of one nostril in the early neonatal period. The previously described reductions in the size of OB and thickness of OB layers were confirmed. Quantitative comparison of the control and anosmic OB revealed that in the odor deprived OB the number of Golgi impregnated mitral cells was 40% less and the impregnation of dendritic neuropil in the external plexiform layer was greatly reduced. Of the remaining mitral cells of the odor-deprived OB, a small portion exhibited an 'atrophic' appearance with markedly reduced impregnation of the intracellular material, while the majority showed relatively 'normal' morphology. Morphometric measurements and statistical comparison of the means revealed that in the remaining 'normal' mitral cells, there were significant reductions in the following parameters: (1) the size of the cell soma as measured by the vertical and transverse diameters (17 and 12%); (2) length of the apical dendrite (27%); (3) diameter of the glomerular tuft (22%); (4) total length of the basal dendrites (18%); (5) diameter of the dendritic field of the basal dendrites (22%). The pattern of impregnation did not differ between the control OB of the experimental rats and the normal OB of normal rats. The results indicate that an absence of olfactory stimulation during the postnatal period exerts deleterious transneuronal influences on the developing mitral neurons, leading to atrophy and loss for some and diminished somatic and dendritic growth for others.

Selective effects of thyroid hormonal deprivation on growth and development of olfactory receptor sheet during the early postnatal period: a morphometric and cell count study in the rat.

Light microscopic numerical and morphometric studies were conducted on the olfactory epithelium of postnatal normal and hypothyroid rats. The normal rat olfactory epithelium undergoes marked growth and development during the suckling period (days 1-25): thickness, 50%; area, x 8, total number of olfactory neurons, basal and supporting cells, x 10, x 11 and x 8, respectively. The effects of thyroid hormonal deprivation on these proliferative postnatal growth changes were studied by adding PTU (n-propylthiouracil, a reversible antithyroid goitrogen) to the litter's drinking water from birth to weaning (day 25). The general architecture of naso-olfactory cavities as well as the histology and thickness of the olfactory epithelium were unaffected in the hypothyroid pups. However, the surface area of the olfactory receptor sheet was reduced by 40%, the reduction occurring throughout the cavity, though not uniformly. The total number of olfactory neurons, supporting and basal cells were reduced by 33, 45 and 47%, respectively. These results indicate that the postnatal vertical accretion of olfactory neurons occurring across the epithelial thickness is unaffected in the hypothyroid pups, while the horizontal proliferation of neurons accompanying the expansion of the sheet's surface area is markedly reduced. The results suggest differential effects of thyroid hormones on these modes of proliferative growth and imply further that in addition to possible direct effects, the influence of thyroid hormones on developmental growth of the olfactory epithelial sheets may be secondary to effects on the underlying submucosal connective tissue.

Lack of thyroid hormones but not their excess affects the maturation of olfactory receptor neurons: a quantitative morphologic study in the postnatal rat.

To study quantitatively actions of thyroid hormones on maturation of olfactory receptor neurons (ORN), surface density and total number of receptor knobs (1 knob/ORN) were measured in 1 mu sections from septal olfactory epithelium of newborn, 12- and 25 day normal, hypo- and hyperthyroid rats. Hypothyroidism was induced by adding to drinking water n-propylthiouracil (0.1% w/v) from birth. Hyperthyroidism was induced by daily injection of pups with T4 (1-thyroxine, 0.3 microgram/g b.w., s.c.). Experimental pups showed all the signs of hypo- and hyperthyroidism. Between days 1-25, normal pups showed marked increase in surface area of septal olfactory epithelium (6x), total number (12x) and surface density (#/mm2, 2x) of mature ORNs. Thyroid deficient rats showed, by day 12, marked reductions in epithelial surface area and total number of mature ORNs; these and the surface density deficits became very pronounced by 25 day (30% area, 27% density, 47% # mature ORNs). Hyperthyroid rats, however, did not show an increase in any of these parameters over controls. Although total number of ORNs (mature and immature), as measured by number of nuclei, was also reduced in hypothyroid pups, surface density was not altered, indicating that maturation of ORNs, but not their local accretion is altered in thyroid deficiency. The results indicate that thyroid hormones are essential for normal proliferative expansion of olfactory epithelium and for maturation of ORNs postnatally. These actions of thyroid hormones are not increased or accelerated by excess T4 suggesting saturation of the hormone receptor system at the normal plasma level.

Essential role of thyroid hormones in maturation of olfactory receptor neurons: an immunocytochemical study of number and cytoarchitecture of OMP-positive cells in developing rats.

Neurogenesis and proliferation of olfactory receptor neurons (ORNs) in the olfactory epithelium (OE) are reduced in postnatal hypothyroid rats and upregulated following restoration of thyroid function, leading to compensatory growth and restitution of these deficits [Paternostro M.A. and Meisami E. (1993). Dev. Brain Res. 76, 151-161; Paternostro M.A. and Meisami E. (1994). Dev. Brain Res. 83, 151-162]. To investigate thyroid hormonal role on maturation of ORNs, serial sections of the septal OE from normal newborn, 25- and 90-day-old rats were immunostained for olfactory marker protein (OMP), a marker for mature ORNs, and compared with the same from age-matched hypothyroid rats and those allowed to recover from thyroid deficiency at the time of weaning (day 25). The parameters studied were the localization and distribution of the OMP(+) cells within the OE and their density and total number. Hypothyroidism was induced by adding the reversible goitrogen propylthiouracil (PTU) to the rats' drinking water (1 g/l) from birth to days 25 or 90. Recovery from hypothyroidism was induced by withdrawal of PTU at day 25. The OMP(+) cells occupied a distinct, broad band in the normal rat OE, while in hypothyroid animal, this band was narrow and restricted to OE's apical zones. Recovery resulted in broadening of the OMP(+) cell band and normalized distribution of OMP(+) cells as evident in the 90-day-old recovery animals. In normal control rats, density of OMP(+) cells increased by 2.5- and 1.3-fold during the suckling and post-weaning period (days 25-90), while total numbers of these cells increased by 12- and 3-fold, respectively, during the same age periods. Hypothyroidism decreased the growth in density by 25 and 30%, while total number of OMP(+) neurons were reduced by 40 and 70% in the 25- and 90-day-old animals, respectively. Withdrawal of PTU resulted in marked restoration of these deficits so that, at 90 days, the total number of OMP(+) cells were only 20% less than 90-day-old controls. These results indicate that thyroid hormones are essential for maturation of single ORNs and accretion of new mature ORNs in the OE of suckling and post-weaning rat. Also, the process of maturation and the final number of mature ORNs show remarkable recovery from hypothyroid-induced growth retardation.

A proposed relationship between increases in the number of olfactory receptor neurons, convergence ratio and sensitivity in the developing rat.

In the rat neonate, as in other altricial young, olfaction, in contrast to vision and hearing, is functional at birth, being critically important in feeding, growth and other aspects of infant-mother interaction. Yet, olfactory sensitivity, i.e., the ability to detect odors at low levels, is poor in the newborn, improving dramatically in the first few postnatal weeks. To find a neural explanation for this phenomenon at the level of peripheral olfactory system, we present quantitative light microscopic data which reveal that during the suckling period of postnatal development the surface area of the olfactory receptor sheet and the total number of olfactory receptor neurons increase by about 8- and 12-fold respectively, being about 15 mm2 and 1.0 million on each nasal half of the newborn. Since the number of mitral cells, the principal relay neurons of the olfactory bulb, is already established at birth, at about 40,000 per olfactory bulb, it may be estimated that the convergence ratio of the olfactory neurons to mitral cells increases by more than 10-fold in the suckling period. We propose that the increased number of primary sensory afferent units and the higher convergence upon the central relay cells enhances the physiological capacities of the olfactory afferent pathway, increasing the opportunity for spatial summation and facilitation. The latter changes may lead to reduced olfactory thresholds and improved sensitivity with development. The relative contribution of these peripheral changes in enhancement of olfactory sensitivity during growth is discussed in the light of our knowledge on the developing olfactory system.

Developmental plasticity of the rat olfactory receptor sheet as shown by complete recovery of surface area and cell number from extensive early hypothyroid growth retardation.

To assess the effects of early thyroid deficiency, and recovery from this condition on growth and development of olfactory epithelium (OE), male Sprague-Dawley rat pups were rendered hypothyroid by addition of propylthiouracil (PTU) to their drinking water from birth. At weaning some rats continued to receive PTU while others ere allowed to recover by withdrawal of PTU. Body weights and plasma thyroxine levels were determined in all groups. At the ages of 25, 50 and 90 days, the OE of these hypothyroid and 'recovery' rats were compared with age-matched controls for surface area, epithelial thickness, density and total number of olfactory receptor neurons, basal cells and supporting cells, using morphometric and cell counting methods. Normal rats showed marked and highly significant increases in the OE surface area and olfactory neuron number (2.6- and 2.3-folds) during the post-weaning period. In the hypothyroid rats, body growth and thyroxine levels were severely suppressed. The OE in the 25-day-old hypothyroid rats showed more than 40% reduction in surface area and cell number, compared to controls, but mean epithelial thickness and surface density of cells were unchanged. In the post-weaning hypothyroid rats, the expansion of surface area was severely retarded, and increase in cell number ceased entirely. In rats allowed to recover by PTU withdrawal, by 90 days of age, body weight and size had markedly increased but had not caught up completely; however, thyroxine levels were restored to normal and the surface area and cell number in the OE had increased in a compensatory manner, completely restoring the deficiencies in OE growth, including surface area, numbers of receptor neurons, basal cells and supporting cells. The results indicate marked growth plasticity of OE in the post-weaning rats. This pronounced ability to recover from early growth retardation contrasts with that seen in central neural structures, and indicates the great potential of OE for use as a model neural system for the study of recovery from early damage and growth retardation.