David and Goliath - the slingshot that started the neuropeptide revolution.

This review in honor of David de Wied summarizes the work done in my laboratory that first indicated that adrenocorticotropic hormone (ACTH) has a direct effect on the neuromuscular system. Cold stress or ACTH and its related peptides alpha-melanocyte-stimulating hormone (alpha-MSH ) and beta-lipotropin improve the electromechanical characteristics of adrenalectomized and hypophysectomized rats. ACTH-(1-39) accelerates the return of motor and sensory function and improves the morphological characteristics of the motor endplate after peripheral nerve crush. The non-corticotropic fragments ACTH-(4-10), alpha-MSH, the ACTH-(4-9) analogue Organon 2766 (Org 2766) or the ACTH-(4-10) analogue Biomeasure 22015 (BIM 22015) improve electrophysiological and morphological parameters of the regenerating neuromuscular system. ACTH-(4-10) immunoreactivity, present in ventral horn motor neurons in low levels, is decreased ipsilaterally following ipsilateral nerve crush but increases both ipsilaterally and contralaterally if injured animals are treated with ACTH-(4-10) indicating a neuroprotective action. Similarly, Org 2766 appears to have a protective action in the brain following nigrostriatal lesions. In developmental studies, perinatal exposure to ACTH peptides improves the structure of the neuromuscular junction, accelerates the maturation of electromechanical properties and enhances nerve-muscle integration and nerve regeneration. Perinatal exposure to these peptides decreases adult male sexual behavior, a change correlated with increased serotinergic input within the medial preoptic area. Similar changes occur in female rats and appear to be long-lasting. In tissue culture studies, both Org 2766 and BIM 22015 promote neurite outgrowth in the absence of nerve growth factor, indicating a neurotrophic role for these peptides.

New vistas for melanocortins. Finally, an explanation for their pleiotropic functions.

This paper presents a historical overview of melanocortin (MC) research from the early investigations of the many noncorticotropic effects of peptide fragments of adrenocorticotropic hormone to the present focus on the discovery and cloning of the MC receptors (MCRs). Final acceptance of the passage of neuropeptides through the blood-brain barrier provided the scientific basis for the neuropeptide concept, formulated previously by both De Weid and Kastin, that peripherally administered neuropeptides affect neural processes. The discussion includes melanocortin effects on behavior, the cardiovascular system, central and peripheral electrophysiological parameters, food intake, inflammation and analgesia, nerve regeneration and neuroprotection, and development. The localization of specific MCRs in both neural and nonneural tissues is correlated with the pleiotropic effects discussed.

Neonatal ACTH administration elicits long-term changes in forebrain monoamine innervation. Subsequent disruptions in hypothalamic-pituitary-adrenal and gonadal function.

The findings from this study demonstrated that the manipulation of the HPA system resulting from ACTH administration during neonatal development produces long-term, differential effects, not only on adrenocortical activity, but also on the activity and integrity of the forebrain monoamine systems. Increased concentrations of the monoamines within the forebrain regions studied at days 7 and 15, suggest a hastened maturation of these neural systems in animals neonatally treated with ACTH. The observed neurochemical alterations in these animals at one year are suggestive of an accelerated aging in the monoamine systems. A further consequence of these disturbances during development is an altered functioning of the HPG axis, as demonstrated by a delayed onset of puberty as previously reported, as well as significantly decreased proestrus plasma estradiol. Although deficits in sexual behavior also existed, it seems probable that these behavioral changes are a manifestation of altered neural systems regulating the ability to cope with a novel stimulus or situation, rather than a disruption of the "feminization" of the brain during sexual differentiation. This is in contrast to the male rat which exhibits permanent deficits in male typical sexual behavior following developmental ACTH treatment. The clinical relevance of these findings may be extensive. Perinatal exposure to events or agents that markedly increase ACTH and the corticosteroids may cause significant immediate and long-term changes in central monoamine functioning. These changes may constitute some of the most deleterious effects of stress exposure in infants and children. The alterations may be especially devastating in individuals with predispositions to stress-sensitive disorders such as anxiety, depression, and Tourette's syndrome. Finally, the use of ACTH in the treatment of infantile spasms may need to be reassessed in light of the possible long-term effects of ACTH on central monoamine functioning.

Specificity versus redundancy of melanocortins in nerve regeneration.

The results of the present study demonstrate that administration of the ACTH-(4-9) analogue Org 2766 acutely enhances behavioral, morphological, and biochemical recovery after nigrostriatal destruction. Animals treated with Org 2766 (10 micrograms/kg every 24 hr) demonstrated an acceleration of denervation supersensitivity and a significantly decreased ipsilateral rotational response, as compared to their saline counterparts. Upon evaluation of the mesolimbic DA system using open field behavior, peptide-treated rats demonstrated a compensatory response in their rearing behavior. Furthermore, tyrosine hydroxylase immunocytochemical analysis indicated an enhanced staining in the Org 2766-treated groups. This evaluation was confirmed and quantified using specific high-affinity dopamine uptake. The brains of animals treated with Org 2766 maintained higher uptake levels, suggesting a greater fiber density than the saline-treated animals. Although recovery via reinnervation is very unlikely in this short period of time, improved recovery may be the result of a protective effect of Org 2766 after administration of 6-OHDA into the substantia nigra. Thus, it appears that Org 2766 provides the rapid effects in this system, by both accelerating some compensatory mechanisms necessary for functional recovery and promoting cell survival by providing neuronal protection. However, it does not appear that this protection is due to NMDA receptor manipulation. Org 2766 neither mimicked the NMDA antagonist MK-801 behaviorally nor biochemically in binding displacement studies. Interestingly, other studies have suggested that only the full ACTH molecule, and fragments larger than ACTH-(1-17), demonstrated binding activity at micromolar concentrations, whereas the shorter, noncorticotropic fragments were either less active or inactive (Table 2). As for ACTH-(4-10) immunoreactivity, it appears that this neurotrophic fragment of ACTH reappears in adults following injury to the nigrostriatal system. In addition, the systemically administered ACTH-(4-9) analogue, Org 2766, seems to be gaining access to the CNS, but is only effective in the injured system. Therefore, based on the immunocytochemical localization of the ACTH-(4-10) fragment in neonatal brains and in the injured adult rat CNS, the interesting possibility may be raised that endogenous ACTH peptides appear during both ontogeny and regeneration. These studies demonstrate once again that biological responses to the family of ACTH/MSH peptides depend on the specific peptide fragment administered, its dosage, and the timing of the administration. Consequently, since early intervention is of vital importance in CNS recovery processes, synergistic administration of ACTH fragments and other neurotrophic agents may offer a viable approach with which to combat degeneration in the CNS.

Sex hormones affect muscle contractility and motor functional recovery following peroneal nerve crush.

This study was performed to investigate possible sex differences in the contractile responses of the reinnervated extensor digitorum longus (EDL) muscle. Electromechanical studies on muscle stimulated indirectly through its nerve provided information on muscle contractility during early reinnervation. Locomotor functional recovery was followed by analyzing changes in footprint parameters over time. At 9 days postsurgery, nerve crush affected negatively many isometric contractile parameters, including twitch peak rate and contraction amplitude, as well as tetanic peak rate, contraction amplitude, and half relaxation time (HRT), regardless of sex. Female EDLs had a greater tetanic peak contraction amplitude and fully recovered twitch HRT levels. While this sex difference is clearly apparent in muscle contractility, it was abolished when considering motor functional recovery. Locomotor tests were less sensitive in demonstrating sex differences during recovery from nerve crush. Peroneal functional index (PFI), a parameter well-described by Bain in 1989, is a paradigm which compares all footprint changes incurred in a rat after peroneal nerve crush, using data obtained from uncrushed side to normalize values. PFI indicated that removal of the sex steroids seriously hindered recovery of motor function regardless of gender.

Melanocortins as factors in somatic neuromuscular growth and regrowth.

Melanocortins, non-corticotropic fragments of adrenocorticotropic hormone, accelerate growth of the developing neuromuscular system and regrowth of damaged neurons, both in the adult and neonatal rat. Morphological, electrophysiological and behavioral characteristics are all improved by melanocortins, which, however, vary in potency, with alpha-MSH being the most effective. Tissue substrate, dosage, critical time periods and pattern of neuropeptide administration are all important variables. Melanocortins protect central neurons affecting motor behavior during development or following neuronal damage in the adult brain. Possible mechanisms of melanocortin action are discussed.

Rapid neurotrophic actions of an ACTH/MSH(4-9) analogue after nigrostriatal 6-OHDA lesioning.

ACTH peptide fragments demonstrate potent neurotrophic effects on peripheral nerves in situ, central neurons in culture, and have been implicated to have effects on central neurons in vivo. Neurotoxic lesioning of the nigrostriatal system, which depletes the striatum of dopamine, provides a feasible model of central regeneration in which to test these peptides. Male Sprague-Dawley rats were lesioned unilaterally with 6-hydroxydopamine (8 micrograms/4 microliters), infused into the substantia nigra. They were subsequently treated with 10 micrograms/kg IP of Org 2766 [ACTH/MSH(4-9) analogue] or saline every 24 h starting immediately after the infusion and were observed for 2 weeks. Rotational behavior data indicate that Org 2766 significantly decreases ipsiversive turning (p < 0.05), induced by amphetamine (2 mg/kg), as well as accelerating the onset of denervation supersensitivity induced by apomorphine (0.05 mg/kg). Evaluation of dopamine immunohistochemistry, using an anti-tyrosine hydroxylase antibody, demonstrates an enhanced intensity of staining in the ORG 2766-treated tissue compared to its saline counterpart. This difference is confirmed and quantified through specific high-affinity dopamine uptake. Dopamine uptake is about 17% higher in the striata of animals treated with Org 2766. Higher dopamine uptake levels in these ACTH-treated animals correlate with greater fiber density in this group. Therefore, it appears that treatment with the ACTH/MSH(4-9) analogue Org 2766 (10 micrograms/kg/24 h) offers a protective effect from 6-OHDA lesions in the substantia nigra as well as accelerating various compensatory mechanisms involved in functional recovery.

Non-corticotropic ACTH peptides modulate nerve development and regeneration.

Short peptide sequences of ACTH 1-39 (the ACTH 4-9 analog Org 2766, ACTH 4-10 and its analog BIM 22015, and ACTH 1-13 [alpha-MSH]), which do not stimulate the adrenal cortex, have profound effects on the developing and regenerating neuromuscular system, in neonatal and in adult rats. Both development and regeneration are accelerated, as indicated by improved morphological, electrophysiological, behavioral and biochemical parameters. Regeneration in the central nervous system is problematic but the ACTH peptides may provide protection for CNS neurons, enhance denervation sensitivity or permit compensatory processes which facilitate functional recovery. Neuronal cells in culture respond to ACTH peptides by greater neurite outgrowth, and in some cell types, by increased B-50 expression. In all cases, susceptibility to ACTH peptide treatment varies with cell type, age, the specific peptide administered, its dosage and pattern of administration. External stress and the gender of the animal are additional factors that interact with the neurotrophic actions of the melanocortins.

Melanotropins as growth factors.

Peptides that regulate the growth of tissues, whether in a positive or negative manner, are termed growth factors. The melanocortins, neurotrophic sequences that correspond to peptide fragments contained within ACTH-(1-13), beneficially affect neural growth during development and regeneration. Analogues of ACTH-(4-9) (Org 2766) and ACTH-(4-10) (BIM 22015) are capable of sustaining neurite outgrowth from cultured dorsal root ganglion and spinal cord cells in the absence of nerve growth factor. The development of sexually dimorphic behavior in both male and female rats is influenced by perinatal administration of ACTH. This change appears to be correlated with changes in the growth and metabolism of developing serotonergic and dopaminergic systems in the hypothalamic nuclei associated with male and female sexual behavior. Similar melanotropic influences are found in the developing neuromuscular system. Neuromuscular development is accelerated by perinatal administration of melanocortins, provoking both nerve and muscle to attain early maturation. However, the responding tissue varies pivotally with age: early in gestation, embryonic muscle is acutely sensitive to peptide exposure; but once innervation has occurred, only the developing nerve reacts to melanocortin treatment. Melanocortins have little if any effect on the normal, adult neuromuscular system. Following peripheral nerve injury or pathology, melanotropins once again become effective growth factors, accelerating and enhancing nerve regeneration and muscle reinnervation. Electrophysiological, morphological, biochemical, and functional tests all indicate that ACTH-(4-10), Org 2766, BIM 22015, and alpha-MSH improve various facets of nerve regeneration, the degree to which the specific parameter is improved being dependent on the peptide fragment, its dosage, and pattern of administration. BIM 22015, while less effective as a neurotrophic factor, has potent myotrophic effects that the other peptides lack. Org 2766 may provide some protective action to the injured CNS as demonstrated by tests of cognitive function following brain lesions, although evaluation of recovery is sometimes enigmatic. Recovery from destruction of the nigrostriatal system is more easily measured through tests of motor function and open field behavior, both of which support a protective role for Org 2766. Compensatory mechanisms, including the presence of increased tyrosine hydroxylase and greater density of dopaminergic fibers, may be involved. Melanocortins are effective growth factors in sciatic nerve regeneration in neonatal rats. Both alpha-MSH and ACTH-(4-10) favor the formation of morphologically normal end plates despite the trauma following nerve crush at postnatal day 2.(ABSTRACT TRUNCATED AT 400 WORDS)

ACTH/MSH(4-10) analog BIM 22015 aids regeneration via neurotrophic and myotrophic attributes.

Structural differences between noncorticotropic ACTH peptides result in marked differences in their effects on regenerating nerve and muscle in rats. The ACTH/MSH(4-10) analog BIM 22015 was administered IP in dosages from 0.1 to 40 micrograms/kg/48 h for 5, 7, or 11 days after peroneal nerve crush, and characteristics of extensor digitorum longus (EDL) muscle were studied and compared with ACTH/MSH(4-10). Eleven days postcrush 40 micrograms/kg BIM 22015 increases rate of development of tetanic tension and amplitude of contraction of indirectly stimulated EDL. In a 21-day study, reinnervated BIM 22015-treated muscles retain tetanic strength, whereas ACTH/MSH(4-10)-treated muscles are significantly weakened. Both peptides show neurotrophic characteristics in their stimulation of endplate nerve terminal branching. However, in contrast to ACTH/MSH(4-10), BIM 22015 also prevents denervation atrophy of the EDL. This dual neurotrophic and myotrophic role for BIM 22015 accords it a clinical potential for degenerative myopathies of either pure or mixed origin, such as muscular dystrophy, infantile spinal atrophy, and hypotonia.

Neonatal ACTH and corticosterone alter hypothalamic monoamine innervation and reproductive parameters in the female rat.

Female Sprague-Dawley rat pups were injected SC with either ACTH(1-24) (0.5 mg/kg) or saline vehicle once daily from postnatal day 1 (day of birth) to day 7. Plasma corticosterone (CORT) levels were recorded via radioimmunoassay (RIA) on day 4 to measure adrenal response to these treatments. Hypothalamic 5-HT and DA fiber densities were assessed using high-affinity specific 3H-5-HT and 3H-DA uptake at days 7, 25, and at adulthood (80-90 days). Animals were checked daily for vaginal opening (starting on day 30) as a sign of sexual maturation and later tested for sexual behavior as virgins (60-70 days of age). Plasma estradiol and progesterone levels were measured via RIA. Plasma CORT levels were greatly increased among ACTH-treated animals during the treatment period. The 5-HT uptake was significantly increased in ACTH-treated animals at day 7 (p < 0.01) and at adulthood (p < 0.02) compared to controls. The DA uptake was significantly higher among ACTH-treated animals at day 7 (p < 0.01). The sexual maturation of ACTH-treated animals was delayed when compared to control animals (p < 0.01). The ACTH-treated animals displayed slight deficits in female sexual behavior compared to control animals (p < 0.05). No significant changes in plasma sex steroid levels were found. Based on this study, we suggest that monoamine innervation into the developing female hypothalamus is susceptible to early postnatal manipulation with ACTH and CORT, and that the resulting changes in these monoamine fiber densities may be responsible for the observed deficits in reproductive maturation and behavior.

Sexual behavior of male rats is differentially affected by timing of perinatal ACTH administration.

The laboratory rat was used as a model to investigate the effect of pre- and/or postnatal ACTH administration on sexual differentiation of the brain. Pregnant Sprague-Dawley rats were injected with ACTH 1-24 (10 micrograms/kg/2x/day or 500 micrograms/kg/2x/day); postnatally treated neonates were injected with the above dosages once a day. Perinatal treatment with ACTH (10 micrograms/kg/2x/day) altered several sexual behavior measurements, but did not have an overall effect on the number of males that exhibited sexual behavior. At a higher dose (500 micrograms/kg/2x/day) prenatal ACTH administration decreased sexual behavior in male rats, as measured by an increase in the percent of males that did not mount or intromit. In contrast, all males treated postnatally with ACTH (500 micrograms/kg/2x/day) completed 2 ejaculatory series and initiated a third series. No significant differences were observed in adult plasma testosterone or prolactin levels; however, serotonin levels in the preoptic area of adult male rats treated prenatally with ACTH (500 micrograms/kg/2x/day) were significantly higher than in prenatally treated saline males. In addition, an increase in plasma ACTH in adulthood was observed in animals injected postnatally with saline. This study indicates that the decrease in sexual behavior observed in males treated prenatally with ACTH is associated with increased serotonin levels in the preoptic area, which suggests that ACTH may act as a neuromodulator during sexual differentiation of the brain. It also demonstrates that the effect of perinatal manipulations on the development of male sexual behavior may vary depending on the ontogenetic period of the brain.

A sensitive period in gestation for nicotine acceleration of neuromuscular maturation.

Nicotine, administered to pregnant Sprague-Dawley rat dams during gestation, altered the maturation of the developing extensor digitorum longus (EDL) muscle-peroneal nerve complex of the 2-week-old offspring. Initial isometric twitch time to peak and rate of rise of tension and tetanus time to peak tension of the group treated with nicotine during gestational (G) days G3-G8 were faster than controls indicating accelerated maturation of EDL muscle. Contractile parameters of the group treated with nicotine during G9-G13, a period immediately prior to muscle innervation, differed little from control. Nicotine administered during G14-G21 significantly increased twitch and tetanus tension and twitch rate of rise. There was little to no effect on motor unit size or number of motor units with nicotine during any of the three prenatal periods. Thus, while late prenatal nicotine administration primarily altered the strength of muscle, early gestational administration of nicotine increased the rate of muscle development suggesting a possible sensitive period for the accelerative action of nicotine on muscle maturation at this time.

Differential effects of prenatal and postnatal ACTH or nicotine exposure on 5-HT high affinity uptake in the neonatal rat brain.

These studies were designed to examine the differential effects of prenatal or postnatal administration of ACTH 1-39 and nicotine, on 5-HT high affinity uptake in brainstem and hippocampal synaptosomes. ACTH was administered prenatally (to pregnant dams) and postnatally to the neonates. Postnatal administration of ACTH significantly increased high-affinity 5-HT uptake in the hippocampus and especially the brainstem at both 7 and 21 days after birth. Prenatal ACTH, on the other hand, transiently increased 5-HT uptake in only the brainstem at 7 days, a change that was reversed at 21 days. While the effects of postnatal nicotine administration were essentially the same as those of postnatal ACTH treatment, prenatal nicotine, unlike ACTH, did not alter 5-HT uptake in 7-day-old rats but did reduce uptake in both tissues at 21 days. The observation that postnatal nicotine mimics the effects of postnatal ACTH and that nicotine stimulates ACTH release, suggests that the postnatal effects of nicotine may be exerted through ACTH.

ACTH/MSH(4-10) peptide increases postnatal metabolic activity of EDL muscle following early prenatal administration.

ACTH/MSH(4-10) (10 micrograms/kg/b.i.d.; IP), administered to pregnant Sprague-Dawley rats during gestational days (GD) 3 to 12, significantly increased the metabolic activity of extensor digitorum longus (EDL) muscle at postnatal day 14. ACTH/MSH peptide, administered from day of birth to postnatal day 13, had no effect on EDL muscle metabolic activity using the 2,3,5-triphenyltetrazolium chloride indicator. By postnatal day 30, no differences were seen between the early prenatally treated group and saline controls. These results confirm our previous electrophysiological studies that showed that early prenatal ACTH/MSH(4-10) administration accelerates EDL muscle maturation.

Accelerated neuromuscular development in the rat with prenatal exposure to nicotine.

Nicotine, administered to pregnant Sprague-Dawley rats either throughout gestation or during two selective prenatal periods before or after muscular innervation (i.e. before or after gestational day 13), accelerated the maturation of developing nerve and muscle as viewed in 2-week-old offspring. Prenatal treatment of rats with nicotine (tartrate salt; 0.25 mg/kg twice a day; i.p.) induced marked changes in the contractile force and speed of the developing extensor digitorum longus (EDL) muscle when tested in situ on 14 to 15-day-old pups. In all nicotine treatment groups isometric twitch and tetanus half contraction durations were shorter than controls. Use of 2,3,5-triphenyltetrazolium chloride, a biochemical indicator for cellular respiration, showed that totigestational nicotine administration increases cellular metabolic activity of EDL muscle. Hypertrophy of adrenal glands in these animals also was noted. While each of the three courses of nicotine administration accelerated muscle development and maturation, nicotine during the first half of prenatal development, prior to EDL muscle innervation, produced a more dramatic effect than nicotine during the second half of prenatal life.

Melanocortins and fast axonal transport in intact and regenerating sciatic nerve.

The effect of ACTH/MSH peptides on fast axonal transport along intact or regenerating sciatic nerve was examined following injection of tritiated leucine into the rat lumbar spinal cord. The rate of fast axonal transport was not significantly changed by treatment with ACTH/MSH(4-10), the ACTH(4-9) analog ORG 2766, hypophysectomy, or adrenalectomy. Fast axonal transport was unchanged in regenerating nerves and in regenerating, ACTH(4-10)-treated nerves. However, treatment with ORG 2766 in dosages of either 1 or 10 micrograms/kg/day IP for seven days significantly reduced (62% and 64%, respectively) the crest height of the fast axonal transport curve of intact sciatic nerve. The results suggest that the reported peptide-induced enhancement of nerve regeneration is not due to changes in the rate of fast axonal transport.