Strain-specific differences in levels of the mRNA for the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase

The activity of the epinephrine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) is 3- to 8-fold greater in rats of the Fischer 344 and Buffalo strains. The biochemical basis for the strain differences has been analyzed at the level of PNMT protein and messenger RNA production. Fischer rat adrenals possess approximately 5-fold more PNMT protein than those of the Buffalo rat as established by Western blotting and immunoprecipitation of adrenal gland homogenates. Poly(A)+ RNAs purified from adrenal glands of each strain were translated in a reticulocyte lysate system, immunoprecipitated with antibody to PNMT and fractionated by SDS-PAGE. A 35S-labelled protein of Mr = 34,000 was immunoprecipitated from adrenals of Fischer and Buffalo rats, indicating that the molecular weights of PNMT do not differ in these strains prior to post-translational processing. Hybridization of a 740 base pair (bp) cDNA for PNMT indicated that the mRNAs for PNMT are the same size in the adrenals of both strains. However, the adrenals of Fischer rats contain 2- to 4-fold more PNMT mRNA than Buffalo rats, as established by quantitative dot blot hybridization and Northern blot analysis. The medulla oblongata, the site of cell bodies of central adrenergic neurons, also contains approximately 2-fold more PNMT mRNA in Fischer rats. The strain specificity in the production of PNMT reflects differences in the expression of the gene for PNMT. Thus, an inherited capacity for PNMT expression may in fact provide the intrinsic determinants responsible for neurotransmitter production. These data provide a direct link between regulation of catecholamine enzyme biosynthesis at the genomic level and the availability of specific catecholamines for neurotransmitter and hormonal functions.     

Increased number of PNMT-immunofluorescent nerve cell bodies in the medulla oblongata of stroke-prone hypertensive rats

An antiserum to bovine adrenal PNMT was used to identify PNMT-containing nerve cell bodies in the medulla oblongata of 4-week-old normotensive Wistar-Kyoto rats and stroke-prone spontaneously hypertensive rats. The regional distribution of PNMT cells and the total number of PNMT cell profiles in tranverse sections of the medulla were examined in each of the two strains. While there was no significant difference in the pattern of distribution of the cells, both the number of PNMT cell profiles per section and the total number seen in all sections of the medulla were significantly higher in the hypertensive rats. The increase in counts of PNMT cell profiles in the medulla suggests that there is a genetic difference in the number of central adrenaline neurons in these hypertensive rats. This is supported by the finding of similar increases of PNMT enzyme activity in the medulla of both stroke-prone spontaneously hypertensive rats and spontaneously hypertensive rats compared with Wistar-Kyoto rats.     

Early increase in phenylethanolamine-N-methyltransferase activity in a new strain of spontaneously hypertensive rats.

By repeated inbreeding, 2 strains of spontaneously hypertensive and normotensive rats have been simultaneously selected. The activities of tyrosine hydroxylase and phenylethanolamine-N-methyltransferase were determined in various central catecholaminergic nuclei (C1, C2, A6 and A9) and in two peripheral tissues (adrenal glands and superior cervical ganglion). These assays were performed on rats belonging to the normotensive or the hypertensive strain at 3 ages which characterize the development of hypertension (5, 9 and 21 weeks). Except for a decrease in the C1 region of 9-week-old rats, no significant change in tyrosine hydroxylase activity occurred in central or peripheral structures of the spontaneously hypertensive rats when compared to the normotensive rats. In contrast, the activity of the phenylethanolamine-N-methyltransferase (PNMT), was increased in the C2 adrenergic group of the medulla oblongata in young spontaneously hypertensive rats: +43% (P less than 0.001) at 5 weeks of age and +32% (P less than 0.001) in 9-week-old rats. However, there was no significant difference between the 21-week-old rats. No modification of the PNMT activity was found in the C1 adrenergic group of the medulla oblongata. PNMT activity was increased significantly in the adrenal glands of 5-week-old hypertensive rats (+22%, P less than 0.001). By 9 weeks, the difference in PNMT activity in the adrenals was no longer significant. Thus, in young rats of the hypertensive strain, there was an increase in the capacity to synthetize adrenaline in the C2 area of the medulla oblongata and in the adrenal glands. While the enzymatic change present in the adrenals seems to be specific to this new strain of hypertensive rats, the elevation of PNMT activity in a specific region of the medulla oblongata (C2 group) is a characteristic common to at least two independently derived strains of genetically hypertensive rats.     

Regulation of phenylethanolamine N-methyltransferase (PNMT) mRNA in the rat adrenal medulla by corticosterone

In the adrenal medulla of adult rat, physiological levels of glucocorticoid hormones are required to maintain the catalytic activity of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT). The present study was undertaken to determine whether glucocorticoid regulation of PNMT occurs at the level of mRNA coding for PNMT. Adult male Sprague-Dawley rats were hypophysectomized (HPX) and killed after 2 weeks; pellets of corticosterone were implanted for 1, 3 or 7 days prior to killing. Determinations were made of plasma corticosterone levels, adrenal PNMT activity and PNMT mRNA levels by Northern gel analysis. HPX resulted in a decrease in plasma corticosterone to undetectable levels, and decreases in PNMT activity and PNMT mRNA levels to 1 and 18% of the levels observed in sham rats, respectively. Corticosterone replacement produced high prolonged plasma levels of corticosterone which were 10 times those of sham rats, and significantly increased levels of PNMT activity and mRNA. However, corticosterone replacement failed to restore PNMT activity and mRNA levels fully. These results suggest that the maintenance of PNMT mRNA levels is dependent on maintaining corticosterone levels and supports the hypothesis that PNMT gene expression in the adrenal medulla is directly regulated by glucocorticoids produced by the adrenal cortex. However, the results also suggest that in the chronically HPX rat, factors in addition to naturally produced glucocorticoids are required for full restoration of PNMT mRNA levels.     

Distribution of PNMT and Epinephrine in the medulla oblongata of normotensive and spontaneous hypertensive rats

Summary The distribution of the Epinephrine forming enzyme (PNMT) activity and Epinephrine (E) levels was investigated in the medulla oblongata of spontaneous hypertensive rats (SH-rats) and in two normotensive strains, namely Wistar Kyoto rats (WK-rats) and Wistar rats. The PNMT activity increases progressively from the caudal to rostral parts in the C₁ and C₂ regions of the medulla oblongata.The enzyme activity and the E levels are in all parts of the C₁ and C₂ regions higher in Wistar rats than in WK-rats. The PNMT activity in all parts of the C₂ region (with the exception of the caudal region), and in the middle part of the C₁ region is higher in SH-rats than in WK-rats. The E levels in the SH-rats are higher than in WK-rats in the mediocaudal parts of the C₂ and C₁ regions.Supported by NIMH and NSF grants.     

Development of phenylethanolamine N-methyltransferase (PNMT) in cultures of dissociated embryonic rat medulla oblongata.

The adrenergic phenotypic marker, phenylethanolamine N-methyltransferase (PNMT) is expressed in a subgroup of catecholaminergic neurons in the brain, as well as in the chromaffin cells of the adrenal medulla. Although PNMT in the rat adrenal is regulated by glucocorticoids, PNMT in the rat brainstem appears not to be regulated by glucocorticoids. Furthermore, little is known about factors required for the differentiation of this specific class of central neuron. The identification of such factors has been hampered not only by the heterogeneity of cell types in the brainstem, of which only a smaller number express PNMT, but also by the lack of a well characterized in vitro system in which the development of these neurons can be studied under defined conditions. The present study addresses this issue by establishing and characterizing a culture system for the study of adrenergic neurons.

Dissociated cultures were prepared from embryonic rat medulla oblongata and the expression and development of PNMT was studied using immunocytochemistry and radioisotopic assay of PNMT activity. The survival of PNMT-immunoreactive (IR) neurons in vitro was found to be critically dependent on embryonic age. Numerous PNMT-IR neurons were observed in cultures prepared only from embryos of 46–51 somites (embryonic day E13–13.5). In contrast, cultures containing numerous neurons immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, could be successfully established from medulla oblongata of any age between E13 and E16. In cultures of the E13 rat, PNMT was found to be catalytically active at 4 days in vitro and the levels of PNMT activity per neuron, as estimated from the number of PNMT-IR neurons in sister cultures, increased 2-fold in cultures grown for 8 days in the presence of 10% fetal bovine serum and 1.5-fold in defined, serum-free conditions. Treatment of cultures with either corticosterone or dexamethasone had no effect on PNMT activity, supporting previous studies suggesting that PNMT in central neurons is not regulated by glucocorticoids.

These studies provide a convenient and defined in vitro system for studying factors that influence the development of central adrenergic neurons. The differential development of PNMT-IR and TH-IR neurons in these cultures suggests that the factors involved in adrenergic neuron survival and/or differentiation are not common to catecholaminergic neurons in general.     

Effect of a conditioned aversive stimulus on the immune response in three strains of rats

In the present study we investigated the effect of a brief exposure (15 s) to a conditioned aversive stimulus (CS) on the proliferative response of spleen and peripheral blood lymphocytes (PBL) in Lewis, Fischer 344 and Sprague-Dawley rats. Plasma levels of ACTH and corticosterone were also measured. For conditioning, rats were exposed to 10 presentations of a 5 s duration foot-shock (1.6 mA) preceded by a 15 s tone. Seven days later, animals were exposed to the auditory signal without electric shock. Significant differences were found in both the kinetics and the magnitude of altered mitogenic responsiveness of PBL between the different strains of rats. Enhancement of PBL responsiveness to mitogens was observed in Fischer and Sprague-Dawley rats immediately after exposure to the CS. A significant decrease in the response of PBL to mitogens was found in Lewis and Sprague-Dawley rats 10 min after exposure to the CS. The PBL response of Sprague-Dawley and Fischer rats returned to baseline at 30 min, but not in Lewis rats.

Proliferative activity of spleen lymphocytes in response to the CS was suppressed from baseline in all rat strains, but the timing and degree of suppression differed. Fischer rats had the largest percentage of suppression. The earliest suppression of spleen mitogenic function after exposure to the CS was in Fischer rats, while the Lewis rats had the latest onset of suppression, with the Sprague-Dawley rats being intermediate. Plasma levels of ACTH and corticosterone peaked at 10 min in all strains of rats. The magnitude of hormonal elevation differed in the different rat strains, suggesting that corticosterone may not have a variable immunomodulatory role in each strain. These data suggest that a brief psychological stressor results in activation of the HPA axis and is associated with strain-dependent alterations of lymphocyte responsiveness to non-specific mitogens. The short-term exposure to a CS which produces different parameters of lymphocyte functional modulation, provides a useful tool to study the mechanisms of stressor-induced immune alteration.     

Development of phenylethanolamine N-methyltransferase (PNMT) in cultures of dissociated embryonic rat medulla oblongata

The adrenergic phenotypic marker, phenylethanolamine N-methyltransferase (PNMT) is expressed in a subgroup of catecholaminergic neurons in the brain, as well as in the chromaffin cells of the adrenal medulla. Although PNMT in the rat adrenal is regulated by glucocorticoids, PNMT in the rat brainstem appears not to be regulated by glucocorticoids. Furthermore, little is known about factors required for the differentiation of this specific class of central neuron. The identification of such factors has been hampered not only by the heterogeneity of cell types in the brainstem, of which only a smaller number express PNMT, but also by the lack of a well characterized in vitro system in which the development of these neurons can be studied under defined conditions. The present study addresses this issue by establishing and characterizing a culture system for the study of adrenergic neurons.Dissociated cultures were prepared from embryonic rat medulla oblongata and the expression and development of PNMT was studied using immunocytochemistry and radioisotopic assay of PNMT activity. The survival of PNMT-immunoreactive (IR) neurons in vitro was found to be critically dependent on embryonic age. Numerous PNMT-IR neurons were observed in cultures prepared only from embryos of 46–51 somites (embryonic day E13–13.5). In contrast, cultures containing numerous neurons immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, could be successfully established from medulla oblongata of any age between E13 and E16. In cultures of the E13 rat, PNMT was found to be catalytically active at 4 days in vitro and the levels of PNMT activity per neuron, as estimated from the number of PNMT-IR neurons in sister cultures, increased 2-fold in cultures grown for 8 days in the presence of 10% fetal bovine serum and 1.5-fold in defined, serum-free conditions. Treatment of cultures with either corticosterone or dexamethasone had no effect on PNMT activity, supporting previous studies suggesting that PNMT in central neurons is not regulated by glucocorticoids.These studies provide a convenient and defined in vitro system for studying factors that influence the development of central adrenergic neurons. The differential development of PNMT-IR and TH-IR neurons in these cultures suggests that the factors involved in adrenergic neuron survival and/or differentiation are not common to catecholaminergic neurons in general.     

Lewis and Fischer rat strains display differences in biochemical, electrophysiological and behavioral parameters: studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals

In previous studies, we demonstrated that tyrosine hydroxylase and neurofilament proteins are regulated by chronic morphine and chronic cocaine treatments in the ventral tegmental area in Sprague-Dawley rats and that the imbred Lewis and Fischer 344 rat strains, under drug-naive conditions, show different levels of these proteins specifically in this brain region. In the current study, we compared Lewis and Fischer rats with respect to levels of adenylate cyclase, cyclic AMP-dependent protein kinase and G-proteins in the nucleus accumbens (NAc) and locus coeruleus (LC), brain regions in Sprague-Dawley rats where these proteins are regulated by chronic exposure to morphine or to cocaine. We found that levels of adenylate cyclase and cyclic AMP-dependent protein kinase activity are higher in the NAc and LC of Lewis rats compared to Fischer rats, whereas levels of G_iα and G_β were lower. These strain differences were not seen in several other brain regions analyzed and no strain differences were detected in levels of other G-protein subunits. Lewis and Fischer rats also differed in the ability of chronic morphine to regulate adenylate cyclase and cyclic AMP-dependent protein kinase in the NAc and LC. In the NAc, chronic morphine increased levels of the two enzymes in the Fischer strain only, whereas in the LC chronic morphine increased levels of the enzymes in both strains, with more robust effects seen in the Lewis rat. To understand possible physiological consequences of these strain differences in the cyclic AMP pathway, we studied LC neuronal activity under basal and chronic morphine-treated conditions. LC neurons of Lewis rats showed higher spontaneous firing rates in brain slices in vitro than those of Fischer rats and also showed greater morphine-induced increases in responsiveness to bath-applied 8-bromo-cyclic AMP. These electrophysiological findings are generally consistent with the biochemical observations. Moreover, Lewis and Fischer rats displayed very different opiate withdrawal syndromes, with different types of behaviors elicited upon precipitation of opiate withdrawal with the opiate receptor antagonist, naltrexone. The possible relationship between these behavioral findings and the biochemical and electrophysiological data is discussed. These studies provide further support for the possibility that Lewis and Fischer rat strains provide a useful model system in which some of the genetic factors that contribute to drug-related behaviors can be investigated.     

Glucocorticoid effects on phenylethanolamine N-methyltransferase (PNMT) in explants of embryonic rat medulla oblongata

Although glucocorticoid hormones have important roles in the development of neurotransmitter systems in cells derived from the neural crest, it is not known whether they have parallel effects on neuronal development in the brain. To address this issue, we have established an in vitro system of fetal medulla oblongata (MO) to follow development of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT). Embryonic MO was explanted from E13 or E18 embryos and maintained for up to 3 weeks. Successful culture of adrenergic neurons was possible only in explants taken from young embryos, since E18 explants failed to develop. In E13 explants, immunoreactivity to both PNMT and tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis, was observed. PNMT catalytic activity which was barely detectable at the time of explanation increased markedly during the first week in vitro. To study the effects of glucocorticoids on PNMT development in central neurons, MO explants were grown in glucocorticoid deficient medium in which rat serum from adrenalectomized rats was substituted for human placental serum. Addition of natural glucocorticoids, cortisol or corticosterone, or the mineralcorticoid, deoxycorticosterone, during the third culture week had no effect on PNMT activity. Dexamethasone (DEX), a synthetic glucocorticoid, also had no effect on PNMT during the first or second weeks in culture. However, addition of DEX during the third culture week resulted in a doubling of PNMT activity. However, attempts to block the DEX effect during the third week or to block the increase in PNMT activity during the first week in control cultures with the glucocorticoid receptor antagonist, dexamethasone 21-mesylate, were unsuccessful. These results suggest that PNMT in central neurons does not require glucocorticoids for ontogeny during the embryonic period. This is in contrast to PNMT in adrenal medulla which requires glucocorticoids for normal development during both the embryonic and postnatal periods. More generally, these studies suggest that development of the same neurotransmitter phenotype in brain and periphery may be differentially regulated.     

Experimental allergic neuritis. I. Rat strain differences in the response to bovine myelin antigens

Strain differences among rats to the induction and severity of experimental allergic neuritis (EAN) in response to whole PNS myelin were observed. Lewis rats were highly susceptible and developed severe EAN without central nervous system lesions (EAE), while Brown Norway rats were most resistant. Wistar, Sprague-Dawley, and Buffalo rats were susceptible but developed less severe disease than Lewis rats. Only Lewis rats consistantly developed EAN in response to isolated P₂ with mercaptoehtanol prior to injection. None of the strains developed EAN in response to galactocerebroside and none developed the lesions of EAE in response to any of the bovine myelin antigens tested. Myelin protein profiles from these rat strains were similar which suggests that factors other than target tissue differences, such as genetically determined immune responses to bovine myelin antigens, must be involved in these differing responses.     

Fischer (F-344) rats have different morphology,sensorimotor and locomotor abilities compared to Lewis,Long-Evans,Sprague-Dawley and Wistar rats

Locomotor and/or sensory behaviour is commonly evaluated in laboratory rats in the field of neuroscience. Many strains of rats, however, have been propagated through intensive breeding programs. With any breeding program, traits are selected purposefully or inadvertently. We set out to investigate whether differences in morphology, sensory or motor behaviours exist using five age-matched strains of laboratory rats. Personal observations of morphological differences between different strains of rats led us to hypothesize that Fischer rats were dissimilar to the other strains in each of the parameters investigated. Evaluation of morphology involved measuring long-bone lengths and body weights of each strain. Motor skills were evaluated by measuring paw preferences while rearing, abduction of the distal portion of hindlimbs during locomotion, footfalls through a horizontal ladder during locomotion, and ground reaction forces generated during trotting. Sensory ability was assessed by von Frey testing. Fischer rats had shorter long-bone lengths, weighed less, and had significantly abducted distal portion of their hindlimbs during locomotion compared to the other strains. Lewis and Sprague-Dawley rats were less sensitive to mechanical pedal stimulation compared to Fischer rats. While rearing, all strains of rats tended to use individual forelimbs 25% of the time for each right and left limbs, and both forelimbs together 50% of the time. There were no significant differences in the number of footfalls during the ladder task. Ground reaction force determination revealed that Fischer and Sprague-Dawley rats bore more weight on their hindlimbs compared to forelimbs during locomotion, Long-Evans and Lewis rats bore more weight on their forelimbs compared to their hindlimbs, while Wistar rats distributed weight evenly between forelimbs and hindlimbs during trotting. We conclude that morphologic, sensory and motor differences exist between the five strains of laboratory rats examined and several of these differences are most pronounced in the Fischer strain.     

Strain differences in the expression of corticotropin-releasing hormone immunoreactivity in nerves that supply the spleen and thymus

The existence of nerve fibers containing corticotropin-releasing hormone (CRH) immunoreactivity in primary and secondary lymphoid organs from three strains of young adult male rats was examined. Spleens and thymuses from Fischer 344 (F344), Sprague-Dawley (SD) and Lewis (LEW) rats were prepared for immunocytochemistry using antisera directed against CRH. In F344 and SD rats, we were unable to demonstrate CRH-immunoreactive nerves in either the thymus or the spleen. Despite the lack of CRH-containing nerves, CRH immunoreactivity was present in pleotropic cells in the septum, cortex and medulla of the thymus, and in the red and white pulp of spleens from F344 and SD rats. In contrast, CRH+ nerves were found in thymuses and spleens from LEW rats. CRH+ nerves coursed in the interlobular septa, capsule, cortex and medulla of the LEW rat thymus. Large CRH-immunoreactive nerve bundles were present in the hilar region of the LEW rat spleen, and individual CRH+ fibers coursed in the capsule, trabeculae, red pulp, venous sinuses and marginal zone of the white pulp of the spleen. These findings indicate strain differences in neurotransmitter-specific nerves that innervate the rat spleen and thymus under basal conditions.     

Running and cocaine both upregulate dynorphin mRNA in medial caudate putamen

Physical activities such as long-distance running can be habit forming and associated with a sense of well-being to a degree that justifies comparison with drug-induced addictive behaviours. To understand molecular similarities and dissimilarities controlling these behaviours in humans we compared the effects of running in running wheels to the effects of chronic cocaine or morphine administration on mRNA levels in brain reward pathways in the inbred Fischer and Lewis rat strains. These strains are both inbred from the Sprague-Dawley strain; Lewis rats display a higher preference towards addictive drugs and running than do Fischer rats. After chronic cocaine or running a similar increase of dynorphin mRNA in medial caudate putamen was found in the Lewis rat, suggesting common neuronal adaptations in this brain region to both cocaine and running. Fischer and Lewis rats both responded to cocaine with increased dynorphin mRNA levels in medial caudate putamen. However, only Lewis rats increased dynorphin mRNA after running, possibly reflecting the much higher degree of running by the Lewis strain as compared to the Fischer strain. Moreover, the running-induced upregulation of dynorphin mRNA was blocked by the opioid receptor antagonist naloxone. We suggest that running increases dynorphin mRNA by a mechanism that involves endogenous opioids. The voluntary wheel-running model in rats might be used to study natural reward and compulsive behaviours and possibly also to screen candidate drugs for treatment of compulsive disorders.     

Acquisition and maintenance of intravenous cocaine self-administration in Lewis and Fischer inbred rat strains

Lewis and Fischer inbred rat strains differ in behavioral and biochemical responses to psychoactive drugs: Lewis rats show greater behavioral responses to psychoactive drugs than Fischer rats and they fail to show biochemical adaptations in the mesolimbic dopamine system after chronic drug exposure, in contrast to Fischer and outbred rats. This suggests that Fischer and Lewis rats may differ in the initial, reinforcing effects of psychoactive drugs, but not in responses seen after the exposure that occurs with maintenance of drug-reinforced behavior. Thus, the present study tested whether these strains differ in acquisition or maintenance of intravenous cocaine self-administration. Acquisition of cocaine self-administration was examined in separate groups that were allowed 15 days to acquire the operant at one of three cocaine doses (0.25, 0.5, or 1.0 mg/kg/infusion). Compared to Fischer rats, Lewis rats acquired cocaine self-administration after fewer training trials and at lower doses. After maintenance, both strains showed characteristic extinction responding with saline substitution and dose-related responding to cocaine, although Fischer rats tended to show higher response rates. Finally, cocaine plasma levels, obtained after an intravenous cocaine infusion (1.0 mg/kg), showed no strain differences suggesting that the strain difference in acquisition was not due to cocaine pharmacokinetics. These strain differences in acquisition of cocaine self-administration may be related to reported strain differences in the mesolimbic dopamine system. Further, because acquisition of drug self-administration is an animal model of vulnerability to drug addiction, these inbred strains may be useful to study factors underlying such vulnerability.     

Distribution of PNMT-immunoreactive neurons in the cat medulla oblongata

The distribution and morphology of presumptive adrenaline neurons in the cat medulla oblongata were studied in conjunction with the indirect immunoperoxidase technique utilizing antibody raised against bovine adrenal phenylethanolamine-N-methyltransferase (PNMT), an enzyme which converts noradrenaline to adrenaline. Small PNMT-immunoreactive (IR) perikarya are located in the dorsomedial part of the medulla (C2 group) extending from P17 to P12, and larger ones in the ventrolateral part (C1 group) from P11 to P9 of the caudorostral extent. In the rostral medulla (P11-10), larger cells extended more dorsally in the lateral tegmental field, where only a few catecholamine cells are localized. No midline PNMT-IR cells were identified corresponding to C3 cell group in the rat. Distinct longitudinal PNMT-IR axon bundles can be traced through the dorsal part of the lateral tegmental field. Comments are made on the relative localizations of the PNMT-IR and DBH-IR cells, as well as on species differences in comparison with the results reported in the rat.     

Role of N-acetylamino acids in cerebral protein synthesis. I. Incorporation of radioactivity from labelled acetyl and aminoacyl moieties into protein

With the help of immunohistochemical studies using antibodies against bovine adrenal phenylethanolamine-N-methyltransferase (PNMT), the enzyme converting noradrenaline (NA) to adrenaline (A), the cellular localization of PNMT in the rat central nervous system has been demonstrated.The brains were fixed by perfusion with ice cold 4% formalin and sectioned on a cryostat, after which the sections were stained using the indirect immunofluorescence technique. Preimmune serum and PNMT antiserum adsorbed with PNMT served as control sera.Specific immunofluorescence was localized in two groups of reticular nerve cell bodies in the medulla oblongata and in nerve terminals in special nuclei of the brain stem and the spinal cord. A PNMT positive axon bundle was also observed in the reticular formation of the pons-medulla oblongata. The distribution and morphology of these PNMT containing neurons was such that they probably are identical with some catecholamine nerve terminals and cell bodies previously demonstrated with the Falck-Hillarp technique (see Dahlstro¨m and Fuxe¹⁸). The hypothesis is therefore given that the PNMT containing neurons represent A containing neurons, and that A may act as a neurotransmitter in the rat brain.The reticular A neurons appear to have similar morphological characteristics as the NA neurons with long ascending and descending fibers to the brain stem and spinal cord. The A terminals are mainly found in certain visceral afferent and efferent nuclei of the lower brain stem, in the locus coeruleus, in certain nuclei or the hypothalamus and in the periventricular grey. The well defined distribution of the A networks to these nuclei underline the view that the A neuron may participate in the control of oxytocin secretion, food and water intake, body temperature, gonadotrophin secretion, blood pressure and respiration and sleep and wakefulness.     

Ontogeny of adrenergic fibers in rat spinal cord in relationship to adrenal preganglionic neurons

Adrenergic neurons in the C1 cell group in the rostral ventrolateral medulla oblongata contain epinephrine, as well as its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). These neurons send axons to regions of the central nervous system known to regulate autonomic function, including the sympathetic preganglionic nuclei of thoracic and upper lumbar spinal cord. Previous studies have shown that PNMT is expressed in neurons located in the medulla oblongata on embryonic day 14; however, the development of the projections from these cells has not been studied. With the aid of high-performance liquid chromatography (HPLC) to determine levels of catecholamines and immunocytochemistry to demonstrate PNMT, the ontogeny of the adrenergic bulbospinal pathway in the embryonic, postnatal, and adult rat has been studied. In addition, the relationship between PNMT-immunoreactive (IR) fibers and retrogradely labeled sympathetic preganglionic neurons projecting to adrenal medulla are described. PNMT-IR fibers were first observed in the caudal medulla oblongata and lateral funiculus of spinal cord on gestational day 15(E15). On E16, PNMT-IR fibers in the thoracic spinal cord were observed in the intermediate gray matter at the level of the lateral horn. Epinephrine was measureable in spinal cord on E20. Both the density of PNMT-IR fibers and the levels of epinephrine increased to a maximum during the second postnatal week and then declined to adult levels. These observations suggest that a period of adrenergic hyperinnervation of spinal sympathetic nuclei occurs during the neonatal period. PNMT-IR terminals in spinal cord were observed, primarily, although not exclusively, in sympathetic nuclei of thoracic cord and parasympathetic nuclei of upper sacral cord. Adrenergic fibers in the intermediolateral nucleus (IML) and the central autonomic nucleus (CAN) dorsal to the central canal were particularly dense during the second postnatal week in both midthoracic and upper sacral segments. In the neonate, a "ladder-like" pattern of PNMT-IR fiber staining was observed which represented transverse fiber bundles connecting IML with CAN and extensive longitundinal fiber bundles along the border of the funiculus in IML. At all spinal levels, adrenergic fibers were also observed adjacent to the ependyma dorsal or lateral to the central canal. The relationship between adrenal preganglionic neurons and PNMT-IR fibers in IML was examined on postnatal days 4, 15, and 60. With retrograde labeling from adrenal medulla, it was demonstrated that PNMT-IR fibers are associated with adrenal preganglionic neurons throughout postnatal development.(ABSTRACT TRUNCATED AT 400 WORDS)     

Do glucocorticoids induce adrenergic differentiation in adrenal cells of neural crest origin?

The chromaffin cells of the adrenal medulla originate in the neural crest and migrate to populate the emerging adrenal gland. When differentiated, the adrenal medulla is formed by two populations of cells: the norepinephrine (NE) cells, which contain the first 3 enzymes of the catecholamine pathway, and the epinephrine (Epi) cells, which contain all 4 enzymes. It has been suggested that in rat, the last enzyme, phenylethanolamine-N-methyltransferase (PNMT), appears in NE cells that are exposed to very high levels of fetal glucocorticoids (GCs), such as those present in the adrenal gland. If so, PNMT would appear during development after the initiation of fetal GC synthesis by the adrenal cortex at E18. In this study we examined the time of appearance and the relative level of PNMT mRNA and protein in rat embryos. We found (a) PNMT protein and mRNA are present at E16. Moreover, (b) the proportion of NE and Epi cells is already similar to that of adults and (c) the adult proportion of steady-state PNMT mRNA is also achieved prior to E18. We conclude that the appearance of PNMT is not affected by the surge of fetal GCs. Questions are raised as to the identity of the cues, genetic and/or epigenetic, which determine the differentiation of NE and Epi cells in the adrenal gland.