Multiple forms of aryl acylamidase in regional tissues of developing rat brain

The specific activities of two forms of aryl acylamidase (AAA) were examined in 7 regions of the developing rat brain, plus the remainder of the brain and the whole brain. AAA-1 activity peaked at 15 days old in all brain regions studied except the whole brain where it peaked at 22 days of age. AAA-2 activity peaked between 15 and 29 days old in most brain regions studied except corpus striatum and hippocampus where the AAA-2 activity peaked before 15 days old. In all areas and at all time periods, with the exception of CS after 15 days of age, AAA-2 activity was much higher than that of AAA-1. The developmental pattern of AAA-1 is generally the same in the different brain regions while that of AAA-2 shows more regional specificity. These results indicate that neither AAA-1 nor AAA-2 may be associated with amine N-acetyltransferase in the brain which has an entirely different developmental pattern.     

Failure of urethane anesthetic to block induction of pineal serotonin N-acetyltransferase activity in the rat

SHIVERS, B. D. Failure of urethane anesthetic to block induction of pineal serotonin N-acetyltransferase activity in therat. BRAIN RES. BULL. 3(6) 579–581, 1978.—Ability of urethane anesthetic to block induction of pineal serotonin N-acetyltransferase (SNAT; E.C.2.3.1.5) activity was measured in individual rat pineal glands in animals receiving urethane (25% w/v, IP, 1.2 g/kg) or saline, 6 hr prior to sacrifice. Using a radioenzymatic assay, SNAT determinations were made twice daily (at 1200 or 2400 hr) immediately after the sacrifice of each animal. The results show that urethane had no effect on the induction of SNAT activity: (1) implying that the neural activity of those structures involved in induction of SNAT activity (e.g., suprachiasmatic nucleus) is not substantially altered by this anesthetic and (2) suggesting that the central blockade of ovulation by urethane does not include alterations in suprachiasmatic nucleus activity.     

Calcium channel drugs affect nocturnal serotonin N-acetyltransferase (NAT) activity in rat pineal gland

Summary The effects of different organic compounds that block and increase Ca²⁺ influx through the voltage-sensitive calcium channels (VSCC) on the nocturnal serotonin N-acetyltransferase (NAT) activity was investigated in vivo in rats. Systemic administration of VSCC antagonists, i.e. nimodipine, nifedipine, verapamil and diltiazem, resulted in a marked suppression of the nighttime pineal NAT activity. Bay K 8644, a VSCC agonist, injected to rats before the time of the light offset of the light-dark cycle significantly enhanced the nocturnal increase of the pineal NAT activity. Although Bay K 8644 given during the dark phase of an imposed illumination cycle had little effect on the nocturnal pineal NAT activity, it antagonized the nimodipine- and verapamil-induced decrease in the enzyme activity. These results support the role of Ca²⁺ influx through the VSCC in the nocturnal increase of NAT activity in the pineal gland of rat.     

Extension of the rat pineal N-acetyltransferase rhythm in continuous darkness and on short photoperiod

Extension of the rat pineal N-acetyltransferase rhythm after transition from LD 12:12 to LD 8:16 or to constant darkness proceeds into the morning hours. The rhythm is more extended in continuous darkness than in LD 8:16. After a compression caused by 1 min light pulse at 03.00 h the rhythm gradually decompresses in darkness for more than 4 days.     

Dopamine receptor-mediated inhibition of serotonin N-acetyltransferase activity in retina

The possible involvement of catecholamines in the regulation of serotonin N-acetyltransferase (NAT) activity in retina of the African clawed frog was investigated using an in vitro eye cup preparation. Dopamine (10 microM) and norepinephrine (50 microM) had no significant effect on NAT activity of eye cups incubated in the light. However, dopamine inhibited the increase of retinal NAT activity that occurs in eye cups incubated in darkness; the ED50 for dopamine was 0.3 microM. The effect of dopamine on NAT activity was mimicked by the dopamine receptor agonists apomorphine and bromocriptine, but not by agonists of alpha 1-, alpha 2- or beta-adrenergic receptors. Dopamine-mediated inhibition of NAT activity was antagonized by spiroperidol and by alpha-flupenthixol, but not by beta-flupenthixol, phentolamine or timolol. Benztropine, an inhibitor of dopamine reuptake, also decreased NAT activity in eye cups incubated in the dark. The inhibitory effect of benztropine was antagonized by spiroperidol, suggesting that it was mediated by an increase in the extracellular concentration of endogenous dopamine. These studies indicate that the regulation of NAT activity in the retina is subject to modulation by a dopamine receptor-mediated mechanism and suggest that dopamine may play a role in the inhibition of NAT activity by light.     

In vitro effects of putative neurotransmitters on synaptic ribbon numbers and N-acetyltransferase activity in the rat pineal gland

Summary The pineal contains a large number of classical transmitters and neuropeptides. Some of these neurochemicals are involved in the regulation of serotonin N-acetyltransferase (NAT) activity and hence in melatonin synthesis. Synaptic ribbons present in the pineal gland also exhibit a numerical day/night rhythm parallel to that of NAT activity. There is scarcity of information regarding the regulation of synaptic ribbon (SR) numbers. In the present study, we have investigated in vitro effects of a number of classical neurotransmitters and neuropeptides. NAT activity was used to monitor melatonin synthesis under the experimental conditions used. Norepinephrine (NE), Delta sleep-inducing peptide (DSIP), vasoactive intestinal polypeptide (VIP), adenosine and N-acetylasp-glu (NAAG) significantly increased NAT activity in rat pineal. DSIP and VIP also increase the stimulatory effect of NE on NAT activity. These neurochemicals had no effect on SR numbers. Gamma aminobutyric acid (GABA), serotonin and taurine affected neither NAT activity nor SR. Somatostatin increased SR numbers significantly, without having any effect on NAT activity. The effect of somatostatin is regarded to be pharmacologic, since rather high dosages (10^–4 M) were required to obtain a significant effect. Although somatostatin is present in the pineal and may change rhythmically, the inconsistency of the day/night rhythmicity and the lack of such a rhythm in female rats and male gerbils speaks against an important physiological role of somatostatin in regulating SR numbers.     

Expression of plasmolipin in the developing rat brain.

Plasmolipin is an hydrophobic plasma membrane proteolipid present in both kidney and brain. The protein consists of two subunits of 17-18.5 kD, which together form K+ selective voltage-dependent channels. In this report, we define the embryonic and postnatal expression of plasmolipin in the developing rat brain. Plasmolipin was found to be essentially restricted to the postnatal period increasing eight-fold between the first to fourth week after birth. A fetal plasmolipin immunoreactive protein (FPIP) was identified in embryonic brain and also during the early postnatal development of the cerebellum. The expression of FPIP was biphasic with an initial transient increase between E15-E20 followed by a decrease in its levels. FPIP was not detected in the developed rat CNS. FPIP was found in a variety of dividing and immature cells including cultured astrocytes and embryonic neurons, neuroblastoma cells, and rat thymus. In contrast, plasmolipin was restricted to oligodendrocytes of the neural cells tested and to renal tubular epithelial cells.     

Light affects neonatal rat pineal gland N-acetyltransferase activity by an extra-retinal mechanism

Summary To determine whether extra-retinal mechanisms mediate photoperiodic changes in neonatal rat pineal gland N-acetyltransferase activity, 4-day-old intact or bilateral orbital enucleated rats were killed during the dark phase of the lighting cycle, either in darkness or following 4 hr exposure to fluorescent light. Light suppressed the high nighttime N-acetyltransferase activity equally in intact and enucleated pups. Subsequent studies showed that at least 0.5 hr exposure and nocturnal illuminances of 109 W/cm² or greater were required to cause statistically significant reductions in the activity of the enzyme in 4-day-old rats. Taken together, these data indicate that relatively intense environmental light can affect neonatal rat pineal gland biochemistry via extra-retinal mechanisms.     

Aromatase in developing sensory systems of the rat brain

Sex differences in the rat brain are dependent, in part, on oestrogen exposure during specific developmental perinatal periods. The availability of oestrogen requires precursor androgen and the presence of intraneuronal aromatase. To examine sites of oestrogen formation and action in the brain, immunocytochemical and biochemical localization of aromatase in the rat brain were determined between embryonic day 14 and postnatal day 20. Aromatase-immunolabelled neuronal profiles were present in hypothalamic, cortical and limbic regions. Surprisingly, aromatase immunoreactivity was also observed in non-limbic regions of the immature brain where it was previously unsuspected. Among these regions, aromatase staining was robust in developing sensory systems, including primary afferents of the olfactory, trigeminal, vestibulocochlear, and visual systems. To determine whether this aromatase is functional in these systems, i.e. converts testosterone to estradiol, the trigeminal nerve was dissected from the hindbrain of perinatal animals and studied for enzyme activity by the tritium release method. The dpm/mg protein/h tritium release in these tissues equalled that of hypothalamic or limbic controls, indicating that these sensory areas are sites of in-situ estradiol synthesis. Our data suggests that aromatase (estradiol)-dependent mechanisms may play a role in the differentiation and maturation of sensory pathways, which, in turn, may contribute to sex differences in the activity of these systems.     

A regional study of electrophoretically separated proteins from the crude synaptosomal fractions of the developing rat brain.

Albino rats of 1, 5, 9, 16, and 25 days of age were sacrificed, and six brain regions were dissected free. Crude synaptosomal preparations from each brain region and age were obtained by differential centrifugation. Protein content in these fractions from the six brain regions increased almost threefold between 1 and 25 days of age. The preparations were delipidated, solubilized, and subjected to polyacrylamide gel electrophoresis. The densitometric patterns of five samples from each age and brain region were analyzed for changes in the relative amount of stained protein bands. Of the polyacrylamide gel-separated protein bands, 16 regional differences at specific ages and 20 developmental changes in specific brain regions were found to be statistically significant. The hippocampal region showed the greatest number of statistical changes in the protein pattern as a function of age, whereas the cerebellum showed none. The results suggest the importance of considering regional differences in studies of biochemical developmental changes.     

Incorporation of radiolabelled alphafetoprotein in the brain and other tissues of the developing rat

The time course of uptake and autoradiographic localization of alphafetoprotein (AFP) were studied in the brain and other organs of fetuses, neonates and young rats injected with homologous radiolabelled AFP. Comparative data of radioactivity accumulation in the brain relative to that of several tissues (blood, liver, tongue, small intestine) showed bimodal patterns reflecting two periods of more active incorporation, with a maximum before the 16th day of fetal development. In brain autoradiographs, the strongest labelling was observed in 17-day-old fetuses 24 h after injection into the mother of [125I]AFP. The labelling included all regions of the brain. The results presented here give experimental support to the hypothesis that the presence of AFP in the developing nervous system of mammals and birds is primarily due to protein uptake rather than in situ synthesis.     

Expression of connexin36 in the adult and developing rat brain

The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.     

The regional distribution of kassinin-like immunoreactivity in central and peripheral tissues of the cat

The regional distribution of kassinin-like immunoreactivity (KLI) was investigated by radioimmunoassay in central and peripheral tissues of the cat. In the cat brain KLI was found to have a widespread distribution with the highest concentrations present in the substantia nigra followed by the hypothalamus and caudate nucleus. Moderate levels were detected in the spinal cord, brainstem and thalamus but only low levels were found in the frontal cortex and cerebellum. In the periphery KLI was present in moderate amounts in the pituitary but in only low amounts in the dorsal root ganglion. KLI was present throughout the gastrointestinal tract with the highest concentration in the duodenum. The distribution of KLI was found to resemble closely that of substance P-like immunoreactivity (SPLI) although the ratio of SPLI to KLI varied widely throughout the tissues tested. In the frontal cortex and cerebellum the concentration of SPLI was at least 5-fold higher than that of KLI whereas in the pituitary gland, caudate nucleus and terminal ileum the concentrations of SPLI were only 50% higher. High pressure liquid chromatographic analysis of individual tissue extracts was used in order to characterize the KLI. In the caudate nucleus, substantia nigra and hypothalamus the major immunoreactive peak co-eluted with synthetic substance K. In contrast, in the spinal cord and terminal ileum the major peak of KLI co-eluted with synthetic neuromedin K. These results demonstrate that KLI is widely distributed throughout cat tissues and that this distribution closely resembles that of SPLI. However, it also appears that there are important qualitative differences in the KLI between the areas tested.     

Regional distribution of kassinin-like immunoreactivity in rat central and peripheral tissues and the effect of capsaicin

The regional distribution of kassinin-like immunoreactivity in rat central and peripheral tissues was investigated by radioimmunoassay and found to resemble closely that of substance P-like immunoreactivity. Neonatal capsaicin treatment caused a similar decrease to both kassinin-like and substance P-like immunoreactivity in primary sensory areas. These results suggest that more than one member of the tachykinin family of neuropeptides exist within the same neuron.