Modifications in gonadotropin control and reproductive behavior in the female rat by hypothalamic and preoptic lesions

Effects of small hypothalamic and preoptic area lesions on vaginal cyclicity, ovulation and ovarian weights of female rats were examined. Animals were then gonadectomized and tested for lordosis behavior following injections of estrogen alone and estrogen plus progesterone. Male sex behavior was also measured during daily treatment with testosterone. Relative to sham operated rats, lesions in the dorsal preoptic area produced a significant increase in lordosis behavior, virtual elimination of male sex behavior, and only marginal effects on ovarian function. Animals with lesions in the ventral preoptic area showed constant vaginal cornification, lack of ovulation and significantly smaller ovaries than the other groups. These rats also tended to show more female sex behavior and less male sex behavior than sham operated rats. Animals with lesions in the anterior hypothalamus and dorsomedial hypothalamus showed normal ovarian function and levels of female and male sex behavior comparable to the ventral preoptic lesioned rats. Animals with lesions in the ventromedial hypothalamus tended to show lower levels of lordosis behavior than sham animals but displayed a dramatic and significant increase in male copulatory behavior relative to the other groups. These data indicate a clear dissociation between the neural control of cyclic gonadotropin activity and sex specific reproductive behavior.     

Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: implications for the molecular basis of a sexually differentiated behavior

The ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM) contains many estrogen-concentrating neurons which mediate estrogen facilitation of reproductive behavior. Previous studies have shown that estrogen treatment increases proenkephalin (PE) gene expression in neurons of the VL-VM in ovariectomized female rats, and that enkephalin peptides may stimulate lordosis behavior. To determine whether there is a sex difference in steroid hormone regulation of PE gene expression we have examined the effects of estrogen and testosterone on PE mRNA levels in male rats. Slot blot hybridization analysis of RNA isolated from the ventromedial hypothalamus indicated that estrogen treatment increased PE mRNA levels in the VL-VM of ovariectomized female rats (2.2-fold), but had no measurable effect on PE mRNA levels in gonadectomized males. Testosterone treatment of gonadectomized males also had no effect on PE gene expression. To determine whether the sex difference in estrogen-inducibility of PE gene expression is due to the developmental effects of gonadal steroids, we have investigated the effect of estrogen on PE mRNA levels in the VL-VM of neonatally androgenized female rats. Unlike the genetic male, the androgenized females responded to estrogen treatment with a female-typical increase in PE mRNA levels (1.7-fold). Further, although the androgenized rats clearly exhibited signs of defeminization, they did exhibit estrogen-facilitated lordosis behavior when tested with manual stimulation. The PE mRNA induction in estrogen-treated androgenized rats correlated well with the lordosis scores obtained by manual stimulation testing. These results indicate that estrogen regulation of PE gene expression in the VL-VM is sexually differentiated and support the hypothesis that the enkephalinergic neurons of the VL-VM are involved in the regulation of female reproductive behavior.     

Effects of autoimmune NGF deprivation in the adult rabbit and offspring

An experimental autoimmune approach to the production of nerve growth factor deprivation, which we have previously described in the rat and guinea pig, has been applied to the rabbit. This species was chosen for study because of several potential advantages. The rabbit produces large litters and has a relatively short gestation period. More importantly, rabbits generate high titers of antibody against mouse NGF and large amounts of maternal antibody are passively transferred to the developing rabbit fetus compared to most other species, particularly the rat. The sympathetic nervous system of adult rabbit immunized against mouse NGF underwent degeneration with up to an 85% decrease in neuronal numbers in the superior cervical ganglion after 10 months of immunization, thus providing further evidence that NGF is required for the survival of mature sympathetic neurons. Despite the fact that newborn rabbits born to anti-NGF producing mothers had much higher titers of anti-NGF than did rats, the effects on the developing sympathetic and sensory nervous systems were not found to be any greater than in rats. Reductions in norepinephrine levels in the heart and spleen of adult rabbits born to anti-NGF producing mothers were greater than in small intestine. Prenatal exposure to maternal anti-NGF caused reductions (up to 70%) in the number of neurons in the dorsal root ganglia. Substance-P immunoreactivity was reduced in the substantia gelatinosa of the spinal cord of rabbit exposed to maternal anti-NGF. These changes, however, were not greater than seen in the rat. We conclide that although the rabbits offers some advantage in the study of the effects of NGF deprivation in the adult animal, it appears less well suited than the rat or guinea pig to the study of the effects of NGF deprivation on development.     

Role of nerve growth factor in ethylnitrosourea-induced neural carcinogenesis

The sensitivity of the rat nervous system to malignant transformation by ethylnitrosourea (ENU) is a function of age at treatment. From late gestation, nervous structures decrease in sensitivity with age as non-neural structures increase in susceptibility. There is a decrease in the proportion of neural tumors induced by ENU and an increase in survival time when nerve growth factor (NGF) levels are elevated in the fetal or neonatal stage. If antibodies directed against mouse beta-NGF (anti-NGF) are administered prior to neonatal ENU treatment, neural tumors appear earlier, although in the same proportion as with treatment by ENU alone. This effect is not observed if the ENU is administered first. This phenomenon seems to be attributed to an increased number of trigeminal nerve neurinomas, which have a shorter latent period than other nervous system tumors. The induced neurological tumors in rats treated neonatally with anti-NGF prior to ENU seem to be almost exclusively neurinomas in the peripheral nervous system. Fetal anti-NGF treatment leads to an increased number of intracerebral gliomas and a longer survival time, which corresponds to the longer latent period of these tumors. The role of NGF in the sensitivity of the rat nervous system to carcinogenesis by ENU and its possible implications in the development of the nervous system are discussed.     

Persistence of anti-NGF induced dorsal root axons: possible penetration into the mammalian spinal cord

Neonatal rats were given daily injections of antisera to nerve growth factor protein (anti-NGF) for a period of 1 month and then allowed to survive 17 more months. The number of neurons in dorsal root ganglia (DRG) and axons in the dorsal root (DR) were determined in the anti-NGF rats and compared to similar numbers from untreated littermates. We found a 32% decrease in DRG neuron number and 32 and 34% increases in myelinated and unmyelinated DR fibers, respectively, in the anti-NGF rats. The sensory cell bodies in the anti-NGF rats were on the average 23% larger than in the normal rats. We conclude that in an NGF deprived environment a population of DRG neurons dies, principally the small neurons, and in response the surviving neurons emit extra processes which persist for most of the life of the rat. This suggests that the anti-NGF induced axons enter the spinal cord and synapse.     

Effects of injecting antibodies to mouse nerve growth factor into the chick embryo

The function of NGF in chick embryos was studied by injecting antibodies to mouse nerve growth factor (NGF). The uptake of mammalian antibodies into the 8- to 15-day-old chick embryo was followed by an enzyme-linked immunoassay. Normal rabbit antibodies (250 μg) were administered to the yolk, of which less than 5% was found in the embryo (300 ng of IgG per g wet wt of embryo). The concentration was proportionally lower when 100 μg anti-NGF antibodies were injected (40 ng/g). The concentration of anti-NGF antibodies was 1.5 times higher following injection directly into the body of the embryos. The effects of injecting antibodies at days 3–7 were studied in 10-day-old embryos by measuring the diameter frequencies of neurons in sympathetic and sensory ganglia. In comparison with controls, significantly smaller neurons were found in the sympathetic ganglia in embryos directly injected with anti-NGF. In the spinal ganglia, distribution of neuron diameters did not differ between anti-NGF-treated embryos and controls. Finally, the ability of different antibodies to mouse NGF to inhibit the in vitro activity of recombinant chick NGF was investigated. Total block was found at 1000–2000 ng of IgG per ml for most of the antibodies tested, levels not reached when injecting the embryo with antibodies to NGF. We conclude that the main reason for the limited effects in chick embryos by injection of NGF antibodies is due to the low levels of penetration of the anti-NGF IgG into the embryo.     

Nerve growth factor depletion by autoimmunization produces thermal hypoalgesia in adult rats

Nerve growth factor (NGF) plays a role in mechanisms of inflammation and hyperalgesia in adult animals. We sought to determine if NGF depletion produced by autoimmunization of adult rats altered their thermal sensitivity to an acute noxious thermal stimulus. Anti-NGF IgG was not detected in the cerebrospinal fluid of any tested samples. Only those rats with the highest anti-NGF serum titers showed significant (P<0.05) thermal hypoalgesia measured using the hot plate test (52°C): the mean (±S.D.) hind paw lick latency of rats in the high anti-NGF titer group was 18.0±4.6 s compared to means of 10.8±4.3 s, 9.2±2.6 s and 10.1±3.0 s in the medium, low and control groups, respectively. Thus, NGF depletion by autoimmunization is a useful model for investigating the role of NGF in behavioral responses of adult rats to noxious stimuli, providing high titers of antibody are present.     

Antiserum to nerve growth factor does not prevent the increase of brain stem noradrenaline after neonatal 6-hydroxydopa

Antiserum to nerve growth factor (anti-NGF) given intraventricularly to newborn rats systemically injected with 6-hydroxydopa (6-OH-DOPA), did not prevent the long-term increase of brain stem noradrenaline produced by 6-OH-DOPA when given alone. Since the anti-NGF was biologically active and penetrated into the brain parenchyma, the role played by NGF in the outgrowth of central noradrenergic neurons, responsible for the elevation of brain stem noradrenaline, does not seem to be important.     

Effects of long-term nerve growth factor deprivation on the nervous system of the adult rat: An experimental autoimmune approach

Adult rats immunized with 2.5S mouse nerve growth factor (NGF) produced antibodies which cross-reacted with rat NGF. By the criterion of ammonium sulfate precipitation followed by Sephadex G-200 chromatography, all serum anti-NGF activity was retained in the IgG fraction. Animals which developed and maintained chronic (5–6 months) serum titers of anti-NGF demonstrated a pronounced biochemical and morphological atrophy of the superior cervical ganglion which was accompanied by a 35–40% reduction in neuronal number. Norepinephrine was reduced by approximately 90% in heart and brown fat. The extent of biochemical atrophy correlated well with serum titers of anti-NGF. No effects were observed on the short adrenergic neurons of the vas deferens, adrenal medullary chromaffin cells, central adrenergic neurons, or peripheral sensory neurons. These results strongly suggest that mature peripheral sympathetic neurons remain dependent on NGF for survival as well as for maintenance.     

Chronic 1,25-dihydroxyvitamin D3-mediated induction of nerve growth factor mRNA and protein in L929 fibroblasts and in adult rat brain

We have proposed that elevating levels of nerve growth factor (NGF) in the CNS is a rational strategy for treating certain neurodegenerative disorders. The present studies were conducted to determine: (1) if pharmacologically induced levels of NGF could be sustained for an extended time, and (2) if correlations exist between increases in NGF mRNA and NGF protein in L929 cells and in vivo. Short-term treatment of L929 cells with 1,25-dihydroxyvitamin D₃ resulted in a two-fold increase in both NGF mRNA and NGF protein. These increases were sustained for up to 48 h with continuous exposure to 1,25-dihydroxyvitamin D₃. In rats, 1,25-dihydroxyvitamin D₃ (2.5 nmol; i.c.v.) induced NGF mRNA transiently, with peak two-fold increased observed 4 h post-injection. In contrast to L929 cells, 1,25-dihydroxyvitamin D₃ did not elicit an increase in NGF protein after a single administration in vivo. However, consistent with long-term exposure in L929 cells, chronic 6 day infusion of 1,25-dihydroxyvitamin D₃ resulted in induction of both NGF mRNA and NGF protein in the brain. These results indicate that 1,25-dihydroxyvitamin D₃-mediated NGF induction in cultured L929 cells may predict of NGF induction in vivo, suggesting that L929 cells may have utility in studying underlying mechanisms of NGF induction by 1,25-dihydroxyvitamin D₃. On the basis of NGF's ability to increase cholinergic function in animal models of cholinergic degeneration, these results are supportive of a role for NGF inducers as potential drugs for neurodegenerative disorders.