Neonatal 6-hydroxydopamine treatment: noradrenaline levels and in vitro 3Hcatecholamine synthesis in discrete brain regions of adult rats

Endogenous noradrenaline levels are elevated in medulla oblongata, mesencephalon, pons and thalamus of adult rats which had been treated with 6-hydroxydopamine on days 1, 2, 8 and 15 after birth. Levels in spinal cord, cerebellum, hippocampus/amygdala and cortex are depressed, whereas no significant changes are observed in striatum, hypothalamus and medulla spinalis. The rate at which medulla oblongata synthesizes tritiated noradrenaline and dopamine from tritiated tyrosine $\text{in}$$\text{vitro}$ is markedly enhanced. No effect was apparent on catecholamine synthesis in hypothalamus. Tritiated noradrenaline synthesis, but not tritiated dopamine synthesis, in the cortex is depressed. These results support the view that neonatal 6-hydroxydopamine treatment causes a degeneration of noradrenaline nerve terminals in the cortex and induces an increase in noradrenaline terminals in the medulla oblongata.     

Nonacceptance of innervation by innervated neonatal rat muscle

Denervated adult muscle accepts innervation and has high levels of extrajunctional acetylcholine (ACh) receptor, compared to innervated adult muscle. If the high receptor density or any externally oriented part of the receptor molecule permitted or triggered functional synaptogenesis, then innervated neonatal muscle, with its known high extrajunctional sensitivity, should also accept extra synapses from implanted motor nerves. This prediction was tested by implanting the common peroneal nerve into innervated lateral gastrocnemius muscle in 25 neonatal rats and studying the innervation achieved 1–8 weeks later. With one exception, zero or negligible twitch tensions were obtained when the implanted nerve was stimulated. Intracellular recording in two cases showed no evidence of subthresholdevoked potentials in surface muscle fibers. In contrast, when the original motor nerve was cut at the time of common peroneal nerve implantation, reinnervation occurred as soon as 4 days later, and substantial indirect twitches (most observed qualitatively) were invariably found 6–7 days after operation. Four to eight weeks after nerve implantation into denervated muscle, substantial twitch tensions were obtained upon stimulation of the implanted nerve. α-Bungarotoxin binding to extrajunctional ACh receptors per unit surface area was similar in innervated neonatal and denervated adult muscle. Therefore, nonacceptance of additional functional innervation in neonatal muscle implies that a high average density of extrajunctional ACh receptor is not sufficient to permit or trigger functional neuromuscular junction formation.     

Attenuation of noradrenergic neurotransmission by the thromboxane synthetase inhibitor,UK 38,485

The purpose of this study was to determine the effects of chronic administration of the thromboxane synthetase inhibitor, UK 38,485, on noradrenergic neurotransmission. Male Sprague Dawley rats (n=14) were treated once daily with either UK 38,485 (100 mg/kg; n=7) or the vehicle of UK 38,485 (olive oil; n=7) by gavage. The dose of UK 38,485 chosen was sufficient to inhibit $\text{ex vivo}$ platelet TXB₂ production by >90% for 24 hours. One week into the treatment animals were prepared for $\text{in situ}$ perfusion of their mesenteric vascular beds. Vasoconstrictor responses to both exogenous norepinephrine and periarterial nerve stimulation were determined both before and during an infusion of angiotensin II (9ng/min) into the superior mesenteric artery. UK 38,485 significantly (P<0.02) attenuated the vascular response to periarterial nerve stimulation without altering the vascular response to either norepinephrine or angiotensin II. UK 38,485 did not influence the baseline perfusion pressure, the mean arterial blood pressure or the potentiation of neurotransmission by angiotensin II. These data indicate that in the $\text{in situ}$ rat mesentery UK 38,485 attenuates the release of neurotransmitter from sympathetic nerve terminals.     

AMP deaminase: Stage-specific isozymes in differentiating chick muscle

The molecular basis of the developmental increase in AMP deaminase activity in chick muscle was investigated with a view toward determining whether isozymes of AMP deaminase exist in embryonic avian muscle and, if so, whether a stage-specific isozyme transition occurs during myogenesis in vivo and in vitro. Under specified conditions, AMP deaminase isozymes in adult chicken brain and muscle may be distinguished on the basis of differences in relative substrate specificities for 5′-dAMP and 5′-AMP (expressed as a ratio of the rates observed with these compounds; i.e., $\text{dAMP}\text{AMP}$ ratios), as well as by differential immunoinactivation by antibody directed against breast muscle AMP deaminase. It was found that the AMP deaminase(s) that is (are) present in 6-day embryos is (are) catalytically and immunologically similar to the enzyme in adult brain. With mixtures of known amounts of adult muscle and brain enzymes, values for the $\text{dAMP}\text{AMP}$ ratio (as well as the fraction of uninactivated AMP deaminase at antibody excess) were proportional to the fraction of muscle isozyme present. Standard curves constructed from these data were used to determine that the fraction of adult muscle-like AMP deaminase in developing muscle, as assessed by $\text{dAMP}\text{AMP}$ ratios (and differential immunoinactivation), on days 6, 8, 10, and 15 were 23 (28), 55 (65), 83 (85), and 93% (96), respectively, Thus, parallel results were obtained for the two techniques, and the isozyme transition is virtually complete by the 15th day of incubation. Primary muscle cultures were used to investigate the isozyme transition of AMP deaminase during myogenesis in vitro. Comparison of the data obtained from primary muscle cultures treated with bromodeoxyuridine, cytosine arabinoside, and fluorodeoxyuridine with data from control cultures showed that biochemical differentiation of AMP deaminase in vitro could be attributed to the muscle cell. Also, the isozyme composition changed from a small percentage of adult muscle-like isozyme at the time of plating, to approximately 100% by the 6th day of culture.     

The occurrence of analgesia in an animal model of hypertension

The relationship between blood pressure and pain sensitivity in a spontaneously hypertensive strain of rats (SH) was studied. Both analgesia and systolic blood pressure (SBP) were determined in SH and Wistar-Kyoto (WK) rats between 30 and 70 days of age. The SBP of the 30 day old SH rat was not significantly different from that of the WK rat (SH = 102±10 mmHg; WK = 93±12 mm Hg). Howeverm, the yooung SH rat exhibited considerably more analgesia than corresponding, age matched, WK rats. The SH rat jump latency was 45±10 seconds whereas the WK rat latency was 21±6 seconds. Both naloxone and atropine attenuated the analgesia of the SH rat but did not significantly alter the response of the WK rat. Methyl-alropine did not affect either the SH or WK rat response. These data suggest the presence of higher endorphin-like activity in the SH rat and a possible relationship between endogenous opioid pathways and the development of hypertension.     

Tyrosine's pressor effect in hypotensive rats is not mediated by tyramine

Tyrosine, the amino acid precursor of catecholamines, increases blood pressure (BP) in hemorrhaged hypotensive rats. Since tyrosine may also be decarboxylated to form $\text{tyramine}$^∞, which releases norepinephrine from sympathetic terminals, we tested the hypothesis that $\text{tyramine}$ formation might mediate tyrosine's ability to increase BP. Three lines of evidence indicate that tyrosine does not act via this mechanism: pretreatment with reserpine blocked $\text{tyramine's}$ but not tyrosine's pressor activity; pretreatment with hexamethonium left $\text{tyramine's}$ effect intact but blocked the pressor response to tyrosine; and plasma $\text{tyramine}$ did not increase after an hemodynamically-active dose of tyrosine (100 mg/kg).     

Effect of cell size, age and anatomical location on the lipolytic response of adipocytes

Studies were conducted on the norepinephrine (NE) stimulated lipolytic sensitivity of adipocytes from epididymal (Epi), perirenal (PR), subcutaneous (SC) and mesentric (M) depots from young (7–8 wk.) and adult (14–16 wk.) male rats. In the young rats dose response curves to NE were similar for Epi, PR and M depots whereas adipocytes from the SC depot showed a diminished effect over the mid-portion of the curve. This difference could not be ascribed to differences in cell size. In the adult rats glycerol release in the Epi depot in response to NE was identical to the younger rats which was in marked contrast to the other depots in which glycerol release was decreased in comparison to the younger animals. This decreased responsiveness was probably largely a result of age and not changes vn adipocyte size within a given depot. In these older rats, glycerol release was greatest in the Epi cells, least in the SC and M depots, and intermediate in PR. When young rats were subjected to a 72-hour fast, loss of triglyceride per cell was the same in all depots as predicted by the $\text{in vitro}$ data whereas in old rats (610 g), triglyceride loss was proportional to cell size with Epi ≥ PR > SC ≥ M. This was also essentially in agreement with the $\text{in vitro}$ lipolytic data from adult rats. These data demonstrate lipolytic differences between depots that are minimal in young rats and which are accentuated with age.     

Ontogenetic changes in pacemaker activity in chick heart

Pacemaker activity of the isolated chick heart changed considerably during development. The spontaneous impulse frequency increased up to the 11th–15th incubation day. This period of acceleration was followed by a marked deceleration around the time of hatching. Both acceleration and deceleration appeared to be caused by changes intrinsic to the pacemaker. Deceleration was related to a decrease in rate of slow diastolic depolarization. The results were compared with well-known ontogenetic changes in heart rate $\text{in vivo}$. This comparison indicates that the deceleration of pacemaker firing around hatching is counteracted by the development of sympathetic cardiac reflexes $\text{in vivo}$.     

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix: II. Chondroitin sulfate synthesis and reaction to exogenous glycosaminoglycans

In the first paper in this series (Nathanson, M. A., and Hay, E. D. (1980). Develop. Biol. 78, 301–331), we described the ultrastructural alterations that take place when embryonic skeletal muscle is induced to form hyaline cartilage by demineralized bone matrix in vitro. In this paper, we analyze the pattern of appearance of chondroitin sulfates and dermatan sulfate in injured muscle in situ and in explants of muscle cultured either on bone matrix or on collagen gel. We also investigate the effects of exogenous glycosaminoglycans on the cultures to determine whether chondroitin sulfate (Ch-S) and hyaluronic acid (HA) can enhance or inhibit the biochemical differentiation of cartilage under these conditions. Our results indicate that during the first morphological phase, 1–3 days in vitro, there is an increased sulfate uptake, a shift in the relative abundance of Ch-S, and an increase in the ratio of chondroitin-4-sulfate (Ch-4-S) to chondroitin-6-sulfate (Ch-6-S); this change is correlated with the transformation of myoblasts to fibroblast-like cells in both types of cultures. A similar increase in the $\text{Ch-4-S}\text{Ch-6-S}$ ratio occurs in injured muscle in situ, suggesting that phase I is a regenerative response. Explants on bone matrix sustain Ch-4-S levels between 4 and 5 days (phase II) and show a large increase in Ch-4-S and sulfate incorporation when they form cartilage at 6–10 days (phase III). Explants on collagen gels regenerate muscle at 4–10 days with decreasing $\text{Ch-4-S}\text{Ch-6-S}$ ratios and decreasing sulfate incorporation. The data demonstrate that an environmental influence, such as trauma, is sufficient to alter the biosynthetic expression of skeletal muscle and that under appropriate conditions (such as the presence of bone matrix) this response may be augmented, leading to the synthesis of extracellular matrix components at ratios characteristic of cartilage. Exogenous Ch-S and HA did not significantly effect this overall pattern. These results are discussed in relation to the morphological observations presented in the preceding paper.     

Increased phosphorylation of a specific nuclear protein in rat superior cervical ganglia in response to nerve growth factor.

The addition of nerve growth factor to the media of cultures of sympathetic ganglia produces an increase in the phosphorylation of a specific nuclear protein. Similar data are obtained when nerve growth factor is administered $\text{in vivo}$. A comparable effect is produced by analogs of cyclic AMP.     

Induction of tyrosine hydroxlase synthesis in rat superior cervical ganglia in vitro by nerve growth factor and dexamethasone.

The addition of dexamethasone and nerve growth factor to organ cultures of superior cervical ganglia from young rats induces the synthesis of tyrosine hydroxylase. The combination of nerve growth factor and dexamethasone $\text{in vitro}$ produces a differential rate of tyrosine hydroxylase synthesis which approaches that obtained by the $\text{in vivo}$ administration of nerve growth factor.     

Developmental changes of glycine transport in the dog

The renal clearance of amino acids was measured in canine pups between 5 days and 12 weeks of age. The reabsorption of glycine was incomplete at 5 and 21 days, indicating a physiologic aminoaciduria of immaturity. An adult pattern of 97–100% reabsorption appeared by 8 weeks of age. The uptake of glycine by isolated renal tubules from 5-day-old, 3-month-old and adult dogs was examined towards an understanding of the events underlying this aminoaciduria. The initial uptake of 0.042 mM glycine by isolated tubules from the newborn was lower than that of the adult, but after 30 min of incubation the newborn surpassed the adult. A steady state of uptake was not achieved by the newborn even after 90 min of incubation, while it was achieved in the adult after 30 min. The uptake by the 3-month-old tubules resembled the adult at the early time points and the newborn at later points. With 1.032 mM glycine, a similar relationship of uptake between adult and newborn tubules was found, except with this concentration, the uptake by both the newborn and adult tubules reached a steady state. The concentration dependence of glycine uptake showed two saturable transport systems with similar apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ values after 30 min of incubation for all three age groups. Determination of glycine flux by compartmental analysis revealed decreased influx and efflux in the newborn, but with a greater decrease in efflux, compared to adult. These changes of influx and efflux which accompany renal tubule maturation could contribute to the increased intracellular amino acid levels and decreased reabsorption of amino acids seen in the immature dog.     

Differential effects of oestrogen on developing and mature uterine sympathetic nerves

Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.     

Proctolin: a peptide transmitter candidate in insects.

The slow, striated muscles of the proctodeum (hindgut) of the cockroach, $\text{Periplaneta americana}$ (L.), were examined pharmacologically with reference to the responses evoked by nerve stimulation, glutamate, 5-HT, and proctolin, a myotropic peptide from $\text{Periplaneta}$ recently isolated and identified. The graded contractions evoked by repetitive nerve stimulation were simulated by 5-HT and proctolin at threshold concentrations of about 10⁻⁷ and 10⁻⁹ M respectively; responses to glutamate (∼10⁻⁴ M) were not similarly graded. The 5-HT receptors are distinct from other receptors, including the post-synaptic receptors, since they were specifically blocked by bromolysergic acid diethylamide. Proctolin was fully active on TTX-treated or surgically denervated muscle indicating that the proctolin receptors are located on the muscle fibre membrane. Tyramine, at threshold levels 5×10⁻⁸ M, reversibly antagonized the responses evoked by proctolin and by nerve stimulation but was without effect on the 5-HT and glutamate responses. Neurally evoked responses were potentiated by subthreshold concentrations of proctolin but not by glutamate. Pharmacologically, the proctolin and post-synaptic receptors appear to be identical and distinct from the glutamate and 5-HT receptors. Since proctolin is known to be a constituent of an efferent pathway of the proctodeal nerves, the evidence suggests that it may function as an excitatory transmitter substance. Peptidergic transmission is discussed in relation to the ultrastructural organization of the proctodeal nerve terminals which contain neurosectory granules in addition to electron-lucent, synaptic vesicles.     

Topology of membrane sulfhydryl groups in the human erythrocyte Demonstration of a non-reactive population in intrinsic proteins

A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, $\text{N-}\text{ethylmaleimide}$. About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations.These ‘non-reacting’ SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labelled $\text{N-}\text{ethylmaleimide}$ or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with $\text{N-[ethyl-2-}{}^3\text{H]}\text{ethylmaleimide}$.The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive.After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards $\text{N-}\text{ethylmaleimide}$. It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane.     

A new photoaffinity-labeled derivative of mitochondrial cytochrome c

Three lysine residues of horse heart cytochrome $\text{c}$ were modified by reaction with methyl-4-mercaptobutyrimidate hydrochloride and the free SH group of the latter was covalently linked to p-azidophenacyl bromide yielding a photoaffinity-labeled cytochrome $\text{c}$. The photoaffinity-labeled cytochrome $\text{c}$ was bound by irradiation into a covalent complex with cytochrome $\text{c}$ oxidase.     

Comparison of the effects of botulinum toxin in adult and neonatal rats: neuromuscular blockade and toxicity

A single dose of botulinum toxin (BoTX) was injected subcutaneously to induce neuromuscular blockade in the triceps surae muscles of the hindlimbs of neonatal and adult rats. The efficacy of the toxin in producing complete neuromuscular blockade of the lower limb muscles, assessed by blockade of (a) postural and flexor reflexes and (b) muscle contraction in response to nerve stimulation, was dose dependent at all ages over a BoTX dose range of 10-60 ng/kg. However, BoTX was dramatically more toxic in adult animals resulting in a decline in body weight and lethal consequences in 25% of adult animals 1 week after administration of BoTX doses as low as 40 ng/kg. In contrast, neonatal animals, given the same dosage, continued to grow and no mortalities were observed. The differences in toxicity of BoTX in adult and neonatal rats are readily accounted for by the short duration of effect in the younger animals, which, in turn, is probably the result of more rapid generation of new and functional nerve terminals.     

Appearance of contractile activity in muscular dysgenesis (mdgmdg) mouse myotubes during coculture with normal spinal cord cells

Muscular dysgenesis (mdg) in the mouse is an autosomal recessive mutation expressed in the homozygous mutant as lack of skeletal muscle contraction. To test the ability of normal neurons to form neuromuscular contacts with, and/or possibly induce contractions in $\text{mdg}\text{mdg}$ muscle, dispersed cell cultures of normal and dysgenic muscle from newborn mice were cocultured with normal embryonic rat, mouse, and chick dissociated spinal cord cells. Contraction was induced in $\text{mdg}\text{mdg}$ muscle 1 to 10 days (depending upon the species of the neuronal source) following establishment of the cocultures. Control experiments indicated that the dispersed spinal cord preparations were free of myoblasts capable of fusing with $\text{mdg}\text{mdg}$ muscle. The establishment of neuromuscular contacts in the rat neuron cocultures was monitored by cytochemical staining of acetylcholinesterase (AChE), autoradiography of ¹²⁵I-α-bungarotoxin-bound acetylcholine receptors (AChR), and electrophysiological study of muscle membrane activity. Patches of high AChE activity were similar in size and distribution to high-density clusters of AChR on both control and $\text{mdg}\text{mdg}$ myotubes cocultured with rat neurons. The resting membrane potentials of normal myotubes and those of $\text{mdg}\text{mdg}$ myotubes in the presence of neurons were similar (? ?52 mV). The mepp frequency and the mepp amplitude distribution were the same for both control and mutant cocultured muscle. Thus, normal rat spinal cord neurons were capable of forming normal, functional neuromuscular junctions with $\text{mdg}\text{mdg}$ myotubes, and contractions were induced under coculture conditions, in otherwise noncontracting mutant muscle.     

Infant mortality in Macaca nemestrina: Neonatal and post-neonatal mortality at the Regional Primate Research Center Field Station,university of washington, 1967–1974

One-thousand-one-hunderd-seventy-four $\text{Macaca nemestrina}$ live births that were recorded at the Regional Primate Research Center Field Station, 1967–1974, were used to calculate neonatal and postneonatal infant mortality rates. The neonatal mortality rate (death on or before the thirtieth day of life) was 15.72%, and the post-neonatal mortality rate (death between 31 and 183 days of life) was 15.53%. Virtually all births occurred in harem groups housed indoors at the RPRC Field Station.     

Functional significance of alpha-stimulation and alpha-blockade on responses to cardiac nerve stimulation in anesthetized dogs

Experiments were conducted to determine if α-stimulants could inhibit responses to sympathetic nerve stimulation $\text{via}$ a feedback inhibition loop mediated by prejunctional α-receptors. Responses to cardiac nerve stimulation in anesthetized dogs were compared before, during the peak effect of a drug infusion, and during a second drug infusion subsequent to the administration of phentolamine (5 mg/kg i.v.). The drugs infused were norepinephrine, phenylephrine, clonidine, naphazoline - all α-stimulants - and guanethidine. All drugs caused marked elevations of blood pressure, an indication of α-stimulation, but only guanethidine caused significant blockade of responses to sympathetic nerve stimulation. In addition, phentolamine, an α-receptor blocker, and desipramine, an inhibitor of amine uptake, did not potentiate responses to sympathetic nerve stimulation. These results do not support the hypothesis that sympathetic nerves are under a functionally significant feedback loop mediated by α-receptors.