Functional adaptation in female rats: the role of estrogen signaling.

BACKGROUND: Sex steroids have direct effects on the skeleton. Estrogen acts on the skeleton via the classical genomic estrogen receptors alpha and beta (ERα and ERß), a membrane ER, and the non-genomic G-protein coupled estrogen receptor (GPER). GPER is distributed throughout the nervous system, but little is known about its effects on bone. In male rats, adaptation to loading is neuronally regulated, but this has not been studied in females. METHODOLOGY/PRINCIPAL FINDINGS: We used the rat ulna end-loading model to induce an adaptive modeling response in ovariectomized (OVX) female Sprague-Dawley rats. Rats were treated with a placebo, estrogen (17ß-estradiol), or G-1, a GPER-specific agonist. Fourteen days after OVX, rats underwent unilateral cyclic loading of the right ulna; half of the rats in each group had brachial plexus anesthesia (BPA) of the loaded limb before loading. Ten days after loading, serum estrogen concentrations, dorsal root ganglion (DRG) gene expression of ERα, ERß, GPER, CGRPα, TRPV1, TRPV4 and TRPA1, and load-induced skeletal responses were quantified. We hypothesized that estrogen and G-1 treatment would influence skeletal responses to cyclic loading through a neuronal mechanism. We found that estrogen suppresses periosteal bone formation in female rats. This physiological effect is not GPER-mediated. We also found that absolute mechanosensitivity in female rats was decreased, when compared with male rats. Blocking of adaptive bone formation by BPA in Placebo OVX females was reduced. CONCLUSIONS: Estrogen acts to decrease periosteal bone formation in female rats in vivo. This effect is not GPER-mediated. Gender differences in absolute bone mechanosensitivity exist in young Sprague-Dawley rats with reduced mechanosensitivity in females, although underlying bone formation rate associated with growth likely influences this observation. In contrast to female and male rats, central neuronal signals had a diminished effect on adaptive bone formation in estrogen-deficient female rats.

The effect of tongue exercise on serotonergic input to the hypoglossal nucleus in young and old rats.

PURPOSE: Breathing and swallowing problems affect elderly people and may be related to age-associated tongue dysfunction. Hypoglossal motoneurons that innervate the tongue receive a robust, excitatory serotonergic (5HT) input and may be affected by aging. We used a rat model of aging and progressive resistance tongue exercise to determine whether age-related alterations in 5HT inputs to the hypoglossal nucleus can be modified. We hypothesized that tongue forces would increase with exercise, 5HT input to the tongue would decrease with age, and tongue exercise would augment 5HT input to the hypoglossal nucleus. METHOD: Young (9-10 months), middle-aged (24-25 months), and old (32-33 months) male F344/BN rats received tongue exercise for 8 weeks. Immunoreactivity for 5HT was measured in digital images of sections through the hypoglossal nucleus using ImageJ software. RESULTS: Tongue exercise resulted in increased maximum tongue forces at all ages. There was a statistically significant increase in 5HT immunoreactivity in the hypoglossal nucleus in exercised, young rats but only in the caudal third of the nucleus and primarily in the ventral half. CONCLUSION: Specificity found in serotonergic input following exercise may reflect the topographic organization of motoneurons in the hypoglossal nucleus and the tongue muscles engaged in the exercise paradigm.

Mechanical loading of a long bone induces plasticity in sensory input to the central nervous system.

Although the skeleton is extensively innervated by sensory nerves, the importance of this innervation to skeletal physiology is unclear. Neuronal connectivity between limbs is little studied and likely underestimated. In this study, we examined the effect of bone loading on spinal plasticity in young male Sprague-Dawley rats, using end-loading of the ulna and transynaptic tracing with the Bartha pseudorabies virus (PRV). PRV was inoculated onto the periosteum of the right ulna after 10 days of adaptation to a single period of cyclic loading of the right ulna (1,500 cycles of load at 4 Hz, initial peak strain of -3,750 micro epsilon). We found that neuronal circuits connect the sensory innervation of right thoracic limb to all other limbs, as PRV was detectable in the dorsal root ganglia (DRG) of left and right brachial and lumbosacral intumescences. We also found that mechanical loading of the right ulna induced plasticity in the spinal cord, with significant augmentation of the connectivity between limbs, as measured by PRV translocation. Within the spinal cord, PRV was predominantly found adjacent to the central canal and in the dorsal horns, suggesting that plasticity in cross-talk between limbs is likely a consequence of dendritic growth, and enhanced connectivity of propriospinal interneurons. In conclusion, the data clearly demonstrate that the innervation of the skeleton exhibits plasticity in response to loading events, suggesting the existence of a dynamic control system that may be of regulatory importance during functional skeletal adaptation.

Serotonergic projections from the caudal raphe nuclei to the hypoglossal nucleus in male and female rats.

The respiratory control system is sexually dimorphic. In many brain regions, including respiratory motor nuclei, serotonin (5HT) levels are higher in females than in males. We hypothesized that there could be sex differences in 5HT input to the hypoglossal nucleus, a region of the brainstem involved in upper airway control. Adult Fischer 344 rats were anesthetized and a retrograde transsynaptic neuroanatomical tracer, Bartha pseudorabies virus (PRV), was injected into the tongue. Sections through the medulla were reacted immunocytochemically for the presence of (i) PRV, (ii) tryptophan hydroxylase (TPH; marker of 5HT neurons), (iii) PRV combined with TPH, and (iv) 5HT. Sex hormone levels were measured in female rats and correlated with TPH immunoreactivity, as hypoglossal 5HT levels vary with the estrous cycle. The number of PRV neurons was comparable in male and female rats. The number and distribution of TPH immunoreactive neurons in the caudal raphe nuclei were similar in male and female rats. The subset of 5HT neurons that innervate hypoglossal motoneurons was also similar in male and female rats. With the exception of the ventrolateral region of the hypoglossal nucleus, 5HT immunoreactivity was similar in male and female rats. These data suggest that sex differences in 5HT modulation of hypoglossal motoneurons in male and female rats are not the result of sex differences in TPH or 5HT, but may result from differences in neurotransmitter release and reuptake, location of 5HT synaptic terminals on hypoglossal motoneurons, pre- and postsynaptic 5HT receptor expression, or the distribution of sex hormone receptors on hypoglossal or caudal raphe neurons.

Sex steroid hormones and the neural control of breathing.

We review evidence that sex steroid hormones including estrogen, progesterone and testosterone are involved in the central neural control of breathing. Sex hormones may exert their effects on respiratory motoneurons via neuromodulators, in particular, the serotonergic system. Recent studies have shown that levels of serotonin (5HT) in the hypoglossal and phrenic nuclei are greater in female than in male rats. Serotonin-dependent plasticity in hypoglossal and phrenic motor output also differs in male and female rats. Changing levels of gonadal hormones throughout the estrus cycle coincide with changing levels of 5HT in respiratory motor nuclei, and gonadectomy in male rats results in a decrease in 5HT-dependent plasticity in respiratory motor output. We speculate that sex steroid hormones are critically involved in adaptations in the neural control of breathing throughout life, and that decreasing levels of these hormones with increasing age may have a negative influence on the respiratory control system in response to challenge.