Distribution and ovarian control of progestin-metabolizing enzymes in various rat hypothalamic regions

The three principal hypothalamic progesterone metabolizing enzyme activities, namely the progesterone 5 alpha-reductase and 5 alpha-dihydroprogesterone NADH- and NADPH-linked 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities, were examined in discrete rat hypothalamic subsections throughout the estrous cycle and from ovariectomized rats treated with estradiol benzoate or vehicle. The regions studied included the median eminence, the medial preoptic area and the ventromedial and arcuate nuclei. The enzyme assays were performed using radiolabeled steroid substrates and reverse isotopic dilution analysis. While all four hypothalamic regions obtained from intact cycling animals possessed substantial amounts of these three enzyme activities, the median eminence generally had the highest activity levels (2- to 4-fold greater) except during estrus. The other three regions usually had comparable levels. No significant fluctuations were observed in any enzyme activity over the estrous cycle. After ovariectomy, there was a significant decrease (approximately 35%) in the level of the NADPH-linked 3 alpha-HSOR activity in the median eminence compared to the level observed in intact cycling animals, suggesting ovarian control. Estrogen treatment for 3 days did not restore this enzyme level to that observed in intact animals. The NADPH-linked 3 alpha-HSOR activity from the other three hypothalamic regions, as well as the NADH-linked 3 alpha-HSOR and the 5 alpha-reductase activities from all four brain regions, did not change significantly after ovariectomy. These results indicate that the median eminence possesses an increased capacity for progesterone metabolism relative to the other hypothalamic regions tested, and that the NADPH-linked 3 alpha-HSOR activity in this region may be under ovarian control.     

Laminar Distributions of Choline Acetyltransferase and Acetylcholinesterase Activities in the Inner Plexiform Layer of Rat Retina

Choline acetyltransferase and acetylcholinesterase activities were measured in samples taken at 7-micron increments through the inner plexiform layer of rat retina. These enzyme activities were not uniformly distributed through the depth of the inner plexiform layer. Peaks of choline acetyltransferase activity occurred at about one-third and peaks of acetylcholinesterase activity at about one-fifth of the depth into the inner plexiform layer from either side. The positions of the two peaks of choline acetyltransferase activity most likely correspond to the locations of processes from cholinergic amacrine somata in the inner nuclear layer, which spread in sublamina a, and processes from cholinergic amacrine somata "displaced" in the ganglion cell layer which spread in sublamina b of the inner plexiform layer. The peaks of acetylcholinesterase activity may in addition correspond to the processes of cholinoceptive amacrine and ganglion cells. The magnitudes of choline acetyltransferase and acetylcholinesterase activities are as high as found anywhere in rat brain, emphasizing the important role of cholinergic mechanisms in visual processing through the rat inner plexiform layer.     

Testosterone 5 alpha-reductase in discrete hypothalamic nuclear areas in the rat: effect of castration

The conversion of testosterone into 5 alpha-dihydrotestosterone (DHT) has been studied in different hypothalamic nuclear areas and in the superficial layers of the cerebral cortex of normal and castrated male rats. The tissue fragments utilized in each incubation have been punched from frozen brain sections utilizing calibrated needles. Castration has been performed 12 (short term) and 180 (long term) days before sacrifice. The nuclear areas studied include: the medial preoptic nucleus (MPN), the lateral preoptic nucleus (LPN), the anterior hypothalamic nucleus (AHN), the lateral hypothalamic nucleus (LHN), the posterior hypothalamic nucleus (PHN), the nucleus ventromedialis (HVM), the arcuate nucleus (AR), the median eminence (ME), the nucleus paraventricularis (HPV), the supraoptic nucleus (SO) and the suprachiasmatic nucleus (SC). The possible effect of castration on the 5 alpha-reductase, were assessed in the MPN,LPN,AHN,LHN,PHN and in the cerebral cortex. The results indicate that, in the male rat: 1) the lateral preoptic(LPN) and the lateral hypothalamic nuclei(LHN) possess a 5 alpha-reductase activity higher than that present in the cerebral cortex and in the other hypothalamic nuclei considered; 2)the suprachiasmatic nucleus (SC) apparently possesses a testosterone metabolizing activity lower than that found in any other nervous structures studied so far; 3) castration does not seem to influence the 5 alpha-reductase activity either in the hypothalamic nuclear structures considered or in the cerebral cortex.     

THE TOXIC EFFECT OF SODIUM GLUTAMATE ON RAT RETINA: CHANGES IN PUTATIVE TRANSMITTERS AND THEIR CORRESPONDING ENZYMES

Abstract– Subcutaneous administration of high doses of sodium glutamate to rats during their first week after birth produced an almost total loss of choline acetyltransferase, a 90% reduction in glutamate decarboxylase and 70% reductions in acetylcholinesterase and DOPA decarboxylase activities in the adult retina. In addition there was a 70% decrease in GABA and 35-55% decrease in aspartate, glutamate, glycine, alanine and glutamine. No reduction in taurine was observed. The results support the view that the enzymes are mainly localized in the interneurons of retina and that taurine is present in the photoreceptor cells.
Glutamate treatment was also followed by a small reduction in choline acetyltransferase and glutamate decarboxylase of the superior colliculus and in choline acetyltransferase of hippocampus, whereas no changes could be detected in the lateral geniculate body of the adult rat. Unilateral enucleation performed on 1-day-old animals did not alter choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase and DOPA decarboxylase activities in the superior colliculus and in the lateral geniculate body of the adult rat.     

The regional distribution of adenylate cyclase in individual nuclei of rat hypothalamus

The regional distribution of adenylate cyclase (AC) activity in separated individual hypothalamic nuclei was studied. The highest activity was found in the paraventricular nucleus and the lowest activity in the median eminence. In several other hypothalamic nuclei, there was also found higher activity of AC than in the total hypothalamus. These data show that in the individual hypothalamic nuclei there exist considerable differences in the activity of adenylate cyclase that may indicate their different function in regulation of neuroendocrine phenomena.     

CHOLINE ACETYLTRANSFERASE LEVELS IN DIENCEPHALIC NUCLEI OF THE RAT

Choline acetyltransferase levels have been measured in specific nuclei of the diencephalon. On the whole, the thalamic nuclei contain higher concentrations of this enzyme than do the hypothalamic and preoptic nuclei. Those nuclei which seem to receive the most dense cholinergic innervation contain the highest levels of choline acetyltransferase.     

Noncorrelation of Choline Kinase or ATP Citrate Lyase with Cholinergic Activity in Rat Brain

Abstract: The activity of choline acetyltransferase was used as an index of cholinergic structures in regions of rat brain. The activities of ATP citrate lyase and choline kinase correlated poorly with cholinergic activity in whole tissue fractions, contrasting with the good correlation between acetylcholinesterase and choline acetyltransferase. Choline acetyltransferase was preferentially localised in synaptosomes prepared from regions of high (striatum) or intermediate (cortex, medulla oblongata/pons) cholinergic activity. In general, this was not true for either choline kinase or ATP citrate lyase.     

Subcellular and regional distribution of 125I-labeled alpha-bungarotoxin binding in rat brain and its relationship to acetylcholinesterase and choline acetyltransferase.

1. The subcellular distribution of binding sites for 125I-labeled alpha-bungarotoxin was studied in rat cerebral cortex. Primary fractions showing higher specific activity than homogenate were P2 (crude mitochondria and nerve endings) and P3-P2 was subfractionated on a Ficoll gradient with the P2B (nerve ending) subfraction exhibiting the greatest recovery (65%) and enrichment of toxin binding. Toxin binding showed a distribution similar to that of acetylcholinesterase, choline acetyltransferase, and sodium and potassium ion-activated ATPase. 2. P2B and P3 were subfractionated on five-step discontinuous sucrose gradients. The highest specific activity of toxin binding and acetylcholinesterase was associated with fractions of relatively low buoyant density, while choline acetyltransferase activity was associated with fractions of higher density. 3. Toxin binding, acetylcholinesterase, and choline acetyltransferase activities were relatively high in olfactory lobes, cerebral cortex, thalamic region, caudate nucleus, and brain stem; intermediate in hippocampus; low in cerebellum. 4. The relationship of toxin binding to the putative acetylcholine receptor in brain is discussed.     

Thyrotropin-releasing hormone (TRH)-immunoreactive structures in the brain of the domestic mallard

Summary The distribution of immunoreactive thyrotropin-releasing hormone (TRH) in the central nervous system of the domestic mallard was studied by means of the peroxidase-antiperoxidase technique. After colchicine pretreatment, the highest number of TRH-immunoreactive perikarya was found in the parvocellular subdivision of the paraventricular nucleus and in the preoptic region; a smaller number of immunostained perikarya was observed in the lateral hypothalamic area and in the posterior medial hypothalamic nucleus. TRH-immunoreactive nerve fibers were detected throughout the hypothalamus, forming a dense network in the periventricular area, paraventricular nucleus, preoptic-suprachiasmatic region, and baso-lateral hypothalamic area. TRH-containing nerve fibers and terminals occurred in the organon vasculosum of the lamina terminalis and in the external zone of the median eminence in juxtaposition with hypophyseal portal vessels. Scattered fibers were also seen in the internal zone of the median eminence and in the rostral portion of the neural lobe. Numerous TRH-immunoreactive fibers were detected in extra-hypothalamic brain regions: the highest number of immunoreactive nerve fibers was found in the lateral septum, nucleus accumbens, olfactory tubercle, and parolfactory lobe. Moderate numbers of fibers were located in the basal forebrain, dorsomedial thalamic nuclei, hippocampus, interpeduncular nucleus, and the central gray of the mesencephalon. The present findings suggest that TRH may be involved in hypophysiotropic regulatory mechanisms and, in addition, may also act as neuromodulator or neurotransmitter in other regions of the avian brain.     

Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum

The distribution of growth hormone releasing factor (GHRF) immunoreactive structures in the rat hypothalmus was studied after colchicine treatment with PAP immunocytochemistry in vibratome sections using an antiserum directed to rat hypothalamic GHRF. The majority of the GHRF-immunoreactive cell bodies were found in the arcuate nucleus, the medial perifornical region, and the ventral premammillary nuclei of the hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Immunoreactive fibers were observed in all the regions mentioned above. GHRF terminals were located in the central region of the median eminence. In addition, GHRF-immunoreactive neuronal processes were seen in the ventral region of the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, dorsal portion of the suprachiasmatic nucleus, bed nucleus of the stria terminals and the hypothalamic portion of the stria terminals. The localization of GHRF-immunoreactive terminals in the median eminence reinforces the view that GHRF plays a physiological role in the regulation of pituitary function. In addition, the localization of GHRF-immunoreactive structures in areas not usually considered to project to the median eminence suggest that GHRF may act as a neuromodulator or neurotransmitter.     

Cholinergic systems in muscle and brain in vitamin E-deficient rats

Rats fed a vitamin E-deficient diet for 7–8 weeks postweaning showed no change in brain weight or the activity in brain of various enzymes involved in neurotransmitter synthesis and metabolism. Body and muscle weights were markedly reduced. Muscle choline acetyltransferase and acetylcholinesterase activities were significantly elevated on a protein basis, but the total amount of choline acetyltransferase/muscle was essentially normal and total acetylcholinesterase activity was slightly reduced. Total carnitine acetyltransferase and butyrylcholinesterase activities were markedly decreased. The results are quite different from those found in hereditary murine muscular dystrophy and suggest a myogenic etiology for the vitamin E-deficiency-induced condition.     

Ascending projections to the hypothalamus and limbic nuclei from the dorsolateral pontine tegmentum: a biochemical and electron microscopic study.

Axons arising from the dorsolateral pontine tegmentum of the rat were traced in various hypothalamic and limbic nuclei by the electron microscopic degeneration method (0.5-8 day survival times) and by measuring regional norepinephrine (NE) concentrations after 12 days of survival using a radioenzymatic method. Significant reductions (41-85%) in NE contents were observed in the supraoptic, arcuate, basal and lateral amygdaloid nuclei and in the hippocampus 12 days after the bilateral electrolytic lesions of the locus coeruleus. No changes in NE concentrations were observed in the ventromedial, septal, central amygdaloid nuclei, in the median eminence and olfactory tubercle. Parabrachial lesions resulted in a decrease of NE content only in the olfactory tubercle. By means of electron microscopy terminal degeneration was found in the hypothalamic paraventricular, dorsomedial nuclei, in the median eminence, in the bed nucleus of the stria terminalis, in the central, lateral and basal amygdaloid nuclei, in the hippocampus and in the anterior ventral thalamic nucleus.     

Cholinesterase activity of the hypothalamo-hypophysial neurosecretory system in rats during the ontogenetic development

Summary The hypothalamic region and the neural lobe of rats from the 16th foetal day to adult animals have been studied for acetylcholinesterase and cholinesterase activity after Karnovsky. The attention was focused on the magnocellular nuclei-supraoptic and paraventricular, the median eminence and the neural lobe. Acetylcholinesterase activity appears in the paraventricular nucleus on the 18th foetal day, i.e. prior to that in the supraoptic nucleus. Heterochronic development and heteromorphism of paraventricular neurosecretory cells have been noticed. The median eminence shows no clear acetylcholinesterase activity. There are acetylthiocholine and butyrylthiocholine positive structures in the posterior pituitary. These structures are especially pronounced in 30–47-day rats. The cholinergic mechanism of release of neurohormones from the neural lobe is suggested. The results are discussed in functional and phylogenetic aspects.     

Cholinergic development in chick optic tectum and retina reaggregated cell cultures

The developmental profiles of acetylcholinesterase and choline acetyltransferase in chick optic tectum and retina cell aggregates, over a 30-day period, have been determined and compared with the corresponding developmental curves obtained in vivo. Both acetylcholinesterase and choline acetyltransferase activities in retina cell aggregates and the acetylcholinesterase activity in optic tectum cell aggregates usually lie between 40 and 90% of the values measured in vivo for the same cell (tissue) type and developmental age. However, the choline acetyltransferase activity in tectum aggregates increases only during the first 7 days of culture, and then decreases to reach a low value of 8% of that measured in vivo, by day 24. This fact, which is associated with widespread degeneration and cell death, could be attributed to the condition of natural deafferentiation occurring in a tectum cell aggregate. A parallel has been drawn between this behavior of a tectum cell aggregate and the effect of early embryonic eye removal on the development of the contralateral optic tectum in vivo. Thus, the tectum may have a biphasic pattern of development, with an autonomous period of growth of about 2 wk, followed by an afference-dependent phase, while the retina behaves, from a cholinergic point of view, as a relatively self-sufficient structure.Abbreviations AChE acetylcholinesterase - ChAT choline acetyltransferase - ACh acetylcholine - BW284 C51 dibromide 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide     

Cholinergic development in chick brain reaggregated cell cultures

Reaggregated cell cultures from dissociated 7-day-old chick embryo whole brains were prepared, and the developmental profiles of acetylcholinesterase and choline acetyltransferase, in the aggregates, determined over a 30-day period. Enzyme activities in vitro, at different times of culture, typically lie between 30 and 60% of the values obtained for embryos or chicks of the same developmental age, up to day-10 posthatching. The increase in acetylcholinesterase activity over a 24-day period of culture/incubation is fourfold in the aggregates vs. sixfold for embryos, while the choline acetyltransferase values increase, during the same period of time, 32-fold in the aggregates vs. 17-fold in vivo. Choline acetyltransferase activity seems to be more dependent on good cell-to-cell contact than acetylcholinesterase activity. On the other hand, morphological studies on the aggregates with light and electron microscopy reveal a number of structural features characteristic of well-developed nervous tissue. It is suggested that aggregate cultures of chick brain cells are an adequate model system that is especially useful in analyzing developmental phenomena requiring free tridimensional interaction.Abbreviations AChE acetylcholinesterase - ChAT choline acetyltransferase - BW284 C51 dibromide 1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide - ACh acetylcholine     

Localization of hypophysiotropic neurohormones by assay of sections from various brain areas.

The results of studies of the localization of the hypothalamic hypophysiotropic factors based on their direct determination in sections or nuclear punches are described. Luteinizing hormone-releasing hormone was found in high concentrations in the median eminence-arcuate nucleus complex, in lower concentrations in the mediobasal zone of the preoptic area. In addition to these hypothalamic sites, it is present in all four periventricular organs, especially in the organum vasculosum laminae terminalis. Thyrotropin releasing hormone has a widespread distribution. High concentrations are in the median eminence, arcuate nucleus, dorsomedial nucleus, and anterior part of the ventromedial nucleus. Lower concentrations are in several other structures of the hypothalamus, preoptic area and septum, and low but measurable quantities are found in most of the structures of the brain. Somatostatin is also present in most structures of the central nervous system, with highest concentrations in the median eminence, arcuate nucleus, ventromedial nucleus and periventricular nucleus. There are indications that the ventromedial nucleus or its immediate vicinity contains growth hormone releasing factor. Prolactin releasing activity was present in the median eminence and mediobasal parts of the anterior hypothalamus, whereas prolactin inhibitory activity was in the dorsolateral parts of the anterior hypothalamus and/or preoptic area.     

Histochemically demonstrable esterase activity in the hypothalamus of the developing rat

Summary The distribution of the acetylcholinesterase, non-specific cholinesterase and non-specific esterase activity has been investigated histochemically in the hypothalamic neurons during the ontogenic development of the rat.Acetylcholinesterase activity is located in the supra-optic and para-ventricular nuclei mostly, but some activity is present in the other nuclei and in the median eminence of the adult rat, as well. The supra-chiasmatic neurons are always negative. The activity of non-specific cholinesterase was encountered in the endothelial cells of the capillaries, in the glia and in the ependymal cells especially around the supra-optic and para-ventricular neurons. The localization of the non-specific esterase was similar to that of the non-specific cholinesterase, but in addition activity is seen in the supra-optic and para-ventricular perikarya, in the parvo-cellular neurons of the tuberal area and in the median eminence. No sexual differences were seen in the distribution of the estrase activity.The appearance of acetylcholinesterase took place already before birth. At about the 16th post-coital day the area from which the arcuate and ventro-medial nuclei will differentiate was positive for acetylcholinesterase. A strong activity in these nuclei was observed during the critical period of the sexual differentiation of the rat hypothalamus (0–10 postnatal days). In the development of the non-specific cholinesterase and esterase no similar variation was seen. Acetylcholinesterase and non-specific esterase were seen in the neurosecretory nuclei before birth, non specific cholinesterase after birth, and non-specific esterase in the parvo-cellular neurons during the first post-natal week.Supported by a grant from The Finnish Medical Society Duodecim.     

Structural localization of the elements of the acetylcholine system in the rat cerebellum

The elements of the acetylcholine system (choline acetyltransferase, acetylcholinesterase, acetylcholine receptors) were analysed in the cerebellum of the rat by histochemical and biochemical means. All the elements were found. Besides the granular layer, the molecular layer exerts CAT activity. The 3H-QNB binding site is highest in the molecular layer and lowest in the intracerebellar nuclei, while the 125I-alpha-bungarotoxin distribution in these structures is the opposite. The results support the view that cholinergic transmission may be present in the rat cerebellum.     

Immunoreactivity to vasoactive intestinal polypeptide (VIP) and thyreotropin-releasing hormone (TRH) in hypothalamic neurons of the domesticated pigeon (Columba livia). Alterations following lactation and exposure to cold

Summary The distribution of VIP- and TRH-immunoreactivity in neurons and processes within the hypothalamus of the pigeon was investigated with light-microscopic immunocytochemical techniques. Most of the VIP-containing neurons are concentrated in the middle and caudal parts of the hypothalamus, with the greatest concentration of perikarya occurring in the medial and lateral part of the ventromedial hypothalamic nucleus and the infundibular nucleus. These cells give rise to axons that seem to extend into the median eminence. An extensive network of VIP-immunoreactive fibers and varicosities occupy the external layer of the median eminence. The majority of TRH-containing neurons is found in the anterior hypothalamus with the greatest concentration of cells in the magnocellular preoptic, medial preoptic, suprachiasmatic and paraventricular nuclei. TRH-immunoreactive fibers and varicosities form a dense arborization in the external layer of the median eminence. Lactation seems to induce substantial changes in VIP as well as in TRH-immunostaining in the median eminence and other hypothalamic regions as compared to control, sexually active animals. Furthermore, TRH-immunoreactivity decreased in the median eminence following 60-min exposure to cold. These results suggest that VIP- and TRH-containing pathways in the pigeon hypothalamus are involved in the mediation of neuroendocrine responses.     

DISTRIBUTION OF ACETYLCHOLINE, CHOLINE, CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN REGIONS AND SINGLE IDENTIFIED AXONS OF THE LOBSTER NERVOUS SYSTEM

Abstract— Acetylcholine, its precursor (choline), and the enzymes of its biosynthesis and degradation (choline acetyltransferase and acetylcholinesterase, respectively) have been studied and quantified in extracts of several regions of the nervous system of the lobster and in single, isolated axons of identified efferent excitatory, efferent inhibitory and afferent sensory neurons. The choline acetyltransferase is a soluble enzyme similar to that from other species. The predominant acetylcholine-hydrolysing enzyme is largely membrane-bound and has been characterized as a specific acetylcholinesterase. A single peak of acetylcholinesterase activity can be detected upon velocity sedimentation analysis of Triton X-100-treated extracts of all regions of the nervous system. Choline acetyltransferase distribution parallels that of sensory neural elements, and its specific activity shows nearly a 500-fold difference from the richest to the poorest neural source. Acetylcholinesterase levels span only a 23-fold range, and activity is found in all neural regions, including those free of known sensory components. A radiochemical microassay for choline and acetylcholine in the range of 20–2000 pmol is described in detail. All 3 types of axons contain comparable levels of choline ( ca. 2 pmol/μg protein), but acetylcholine is asymmetrically distributed. Efferent axons contain no detectable acetylcholine, while sensory axons from abdominal muscle receptor organs have an average of 1·9 pmol/μg protein. Choline acetyltransferase is similarly distributed; sensory axons show at least 500-fold greater activity than efferent axons. Acetylcholinesterase is nearly uniformly distributed among the three types of fibres. These results are discussed in terms of a general view of transmitter accumulation in single neurons.