Monoamines in the hypothalamo-hypophyseal system of the mouse with special reference to the ontogenetic aspects

Summary An accumulation of monoamines is found in the adult eminentia mediana and infundibular stem, close to the capillaries of the primary plexus, where they seem to be stored mainly in closely packed delicate varicose fibres, often with swollen endings, droplets. The first traces of monoamines are visible in the embryonic eminentia of fetuses from the last three days of the gestation period, but typical monoamine-containing fibre endings close to the primary plexus capillaries in zona externa are not found until towards the end of the first post-natal week. The adult condition is attained two weeks later. Nearly all cells in the adult pars intermedia show a specific yellowish fluorescence whose intensity shows a wide individual variation. Such fluorescent cells are sometimes present in the intermedia of the new-born. In some but not all adult specimens, a fluorescent varicose fibre system is visible. Fluorescent fibres were first observed in the intermedia of the new-born. Fluorescent fibres are visible in n. arcuatus and n. paraventricularis in the fetus at the 16th day of the gestation period. Nerve cell bodies storing visible amounts of monoamines are found in n. arcuatus, n. periventricularis anterior, and n. premammillaris ventralis of the adult. This amine storing activity is first visible in n. arcuatus of the embryo at the 17th day of the gestation period. The adult condition of these cells is reached during the first postnatal week.     

A histochemical study of the primary catecholamines in the hypothalamic neurons of the rat in relation to the ontogenetic and sexual differentiation

Summary The alterations in the content of the primary catecholamines in the hypothalamus have been studied with the histochemical technique of para-formaldehyde induced fluorescence.In the adult normal rats, independent of the sex, the fluorescence is located in the cell bodies of a few arcuate neurons, around the perikarya of the arcuate, para-ventricular and supra-optic neurons, and in the nerve endings of the arcuate neurons in the median eminence.The appearance of the primary catecholamines takes place at the 20th day of gestation in the para-ventricular and arcuate-peri-ventricular regions. In the supra-optic nucleus the fluorescent nerve terminals are not seen before birth. In the outer layer of the median eminence the fluorescence develops around the 5th post-natal day. No sexual differences were observed in the maturation of the primary catecholamines during the ontogenic development of the rat.More fluorescent cell bodies and nerve endings are seen in the arcuate neurons during the late diestrus than during estrus. The number and intensity of the catecholamine fluorescent neurons in the arcuate nucleus increases during the pregnancy. Castration increases slightly the number and intensity of the fluorescent cell bodies in the arcuate nucleus, but it diminishes the fluorescence in the median eminence. The changes were compensated by a treatment with testosterone propionate. Hypophysectomy alone has no effect on the fluorescence of the hypothalamic neurons.Supported by a grant from The Finnish Medical Society Duodecim.     

Incorporation of 3H-uridine into the large decidual cells of rats and their differentiation

The antimesometrial part of rat's decidua of the 9th day of gestation was divided into three zones. Cells of either zone display their own morphological and cytochemical properties. Different rates of 3H-uridine incorporation were observed in the cytoplasm and the nucleus in cells of either zone during 5, 30, 60 and 240 minutes after precursor injection. The largest member of silver grain accumulation was observed in the karyoplasm and nucleolus of cells of the transitional zone. The nucleus of basal zone cells had the smallest intensity of 3H-uridine incorporation. The nuclei of the epithelial zone cells are characterized by a lower intensity of 3H-uridine incorporation than those of the transition zone. The intensity to cytoplasmic accumulation of silver grains raised from cells of the basal zone up to cells of the epithelial zone. The largest quantity of cytoplasmic radioactivity was observed 240 minutes after 3H-uridine injection.     

Prenatal development of monoamine neurons in the rat]

The histofluorescence method allows the study of the prenatal development of monoamines neurons in the foetal rat brain. Certain patterns of this development can be defined: the locus niger and the raphe appears fluorescent on the 14th day, the locus coeruleus on the 15th day. These systems differentiate independently in various areas of the neural axis. Their maturation are progressive: first for the DA system, then for the NA and the 5 HT systems. The mechanisms for synthesis and storage of amines precede the complete development of the effector structures.     

Simultaneous measurement of nitric oxide,blood glucose,and monoamines in the hippocampus of diabetic rat: an in vivo microdialysis study

The present study was undertaken to examine the relationships among the levels of nitric oxide (NO), monoamines, and blood glucose in the diabetic hippocampus. The levels of NO and monoamines (serotonin, 5-hydroxytryptamine [5-HT] and dopamine [DA]) were simultaneously measured in several experiments, using in vivo microdialysis techniques. We used both experimentally and spontaneously diabetic rats as the diabetic animal model, and compared the findings with those obtained from non-diabetic rats. The effects of the changed level of blood glucose due to insulin administration on the levels of NO, 5-HT, and DA were assessed. Total NO metabolite levels (NOx) were calculated as the sum of nitrite (NO2-) and nitrate (NO3-) levels. The results in the present study showed that: (1) the plasma levels of NOx in both diabetic rats were low compared to those in control rats, (2) the hippocampal NOx levels in both diabetic rats were almost the same as those in control rats, while the levels of 5-HT and DA were low in the diabetics, and (3) a sudden decrease in the plasma glucose level due to insulin administration reduced the NOx level as well as enhanced the 5-HT level in the diabetic hippocampus, a finding consistent with the results of 7 days administration of insulin. Taken together, these findings suggest that changes in the plasma glucose level cause, at least in part, the changes in the levels of NOx and monoamines in the diabetic brain.     

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.     

Chromatin matrix activity in hepatocyte nuclei after disturbance of the hepatic vagal innervation

The level of matrix activity of extranucleolar and nucleolar chromatin in hepatocytes of white noninbred rats has been studied in dynamics after disturbance of the vagal innervation. In order to estimate the labelling intensity of nuclear structures, the histoautoradiographic method is used. After bilateral subdiaphragmal vagotomy at early stages (1-2 weeks) the level of the nucleoplasm and nucleolus increases essentially, and by the 30th day a tendency to its normalization is noted. A conclusion is made that the peripheral nervous system influences the intensity of metabolic processes in the nucleus.     

Monitoring membrane potentials in Ehrlich ascites tumor cells by means of a fluorescent dye.

1. The fluorescent intensity of the dye 3,3'-dipropylthiodicarbocyanine iodide was measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potentials under a variety of different ionic and metabolic conditions. 2. In the presence of valinomycin, fluorescent intensity is dependent on log [K+]medium (the fluorescent intensity increased with increasing [K+]medium) where K+ replaced Na+ in the medium. Cellular K+ content also influenced fluorescent intensity in the presence of valinomycin. With lower cellular K+, fluorescent intensity in the presence of valinomycin for any given concentration was increased. 3. In the presence of gramicidin fluorescent intensity was highest in Krebs-Ringer and decreased with the substitution of choline+ for Na+. 4. The observations with ionophores are consistent with the hypothesis that the dye monitors membrane potential in these cells with an increase in fluorescence indicating membrane depolarization (internal becomes more positive). 5. The estimated membrane potentials were influenced by the way in which the cells were treated. Upon dilution of the cells from 1 in 20 to 1 in 300 the initial estimations were between -50 and -60 mV. With incubation at 1 in 300 dilution for 1 h at room temperature or a 37 degrees C, the membrane potentials ranged from -18 to -42 mV. 6. Estimations of membrane potential on the basis of chloride distribution (Cl-cell/Cl-medium) in equilibrated cells ranged from -13 to -32 mV. 7. Addition of glucose to cells equilibrated at 37 degrees C for 30 min in the presence of rotenone led to a decrease in fluorescent intensity indicating hyperpolarization. Addition of ouabain in turn led to a 70 to 100% reversal of fluorescent intensity. This hyperpolarization is therefore probably due to the electrogenic activity of the sodium pump. 8. The addition of amino acids known to require external Na+ for transport increased fluorescent intensity (depolarization) reaching a maximum at higher concentrations of amino acids. Plots of 1/deltafluorescence vs. 1/[glycine] were linear with an apparent Km of 2-3 mM. The increase in fluorescence with amino acids always required external Na+. Plots of 1/fluorescence vs. 1/[Na+]medium were also linear with an apparent Km of 29 mM. These apparent Km values compare favorably with those derived from amino acid transport studies using tracers. These data indicate that the Na+-dependent transport of amino acids in these cells is electrogenic.     

Histochemical distribution of monoamines in the hypothalamo-hypophysial region of the lamprey,Lampetra japonica

The distribution of monoamine fluorescence was studied in the hypothalamohypophysial region of the lamprey. Groups of intensely fluorescent cells were observed in the lateral walls of the caudal part of the third ventricle. The anterior part of the neurohypophysis which is situated over the pars distalis showed weak fluorescence. The posterior part of the neurohypophysis which is contiguous to the pars intermedia contained highly fluorescent material in its rostral part. The distribution of monoamines in the lamprey neurohypophysis is compared with that in the higher vertebrates and their functional significance is discussed.     

Modulation of swimming activity in the medicinal leech by serotonin and octopamine

1. The monoamines serotonin (5-HT) and octopamine (OA) enhance the expression of swimming activity in the medicinal leech (Willard, 1981; Belanger and Orchard, 1988). We explored further the effects of these monoamines and related agents on swimming activity observed in isolated leech nerve cords. 2. We confirmed that swimming activity is induced reversibly following exposure of the nerve cord to 5-HT (50 microM); the half-maximal rate of swimming activity develops in about 15 min. Swimming activity returns to control levels about 30 min after drug washout. 3. Swim-induction by 5-HT is blocked by the presence of 10 microM cyproheptadine (a 5-HT antagonist). 4. Although apparently less effective than 5-HT, OA application to nerve cords also induced swimming activity. 5. Depletion of endogenous amines from nerve cords by acute exposure to reserpine (10-150 microM) blocked stimulus-evoked swimming activity within 4 hr. 6. Subsequent application of 5-HT (50 microM) or OA (100 microM) reinstated stimulus-evoked swimming and induced repeated episodes of non-triggered swimming activity. 7. Application of cAMP and cAMP analogs, as well as phosphodiesterase inhibitors (theophylline and IBMX), mimicked the effects of the monoamines, suggesting that 5-HT and OA may activate swimming activity by increasing neuronal cAMP. 8. We obtained episodes of swim-like activity from individual, isolated ganglia exposed to 5-HT or OA. Such episodes were usually brief, with variable cycle period. 9. We conclude that individual nerve cord ganglia contain the complete neuronal circuitry required to generate the rudiments of swimming activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative histochemical studies of changes in the intensity of formaldehyde-induced fluorescence and autofluorescence during storage of tissue sections

Summary Changes are reported in (a) the fluorescence intensity of specific formaldehydeinduced fluorescence derived from monoamines, and (b) autofluorescence, during storage of tissue sections of the caudate nucleus and cerebral cortex of the rat brain. Storage as ribbons over 13 weeks was accompanied by a marked increase in autofluorescence, while storage as ribbons for 3 days after sectioning was associated with a marked increase in the specific formaldehyde-induced fluorescence of the striatal neuropil, which was prevented by mounting the sections in Entellan.     

Signal stability of Cy3 and Cy5 on antibody microarrays

Background  

The antibody microarray technique is a newly emerging proteomics tool for differential protein expression analyses that uses fluorescent dyes Cy 3 and Cy 5. Environmental factors, such as light exposure, can affect the signal intensity of fluorescent dyes on microarray slides thus, it is logical to scan microarray slides immediately after the final wash and drying processes. However, no research data are available concerning time-dependent changes of fluorescent signals on antibody microarray slides to this date. In the present study, microarray slides were preserved at -20°C after regular microarray experiments and were rescanned at day 10, 20 and 30 to evaluate change in signal intensity.     

Assessment of mitochondrial function in the surviving cytoplasts of cultured cells of pig embryonic kidney by using ethylrhodamine

A study was made of the functional state of chondriome in cytoplasts previously cultivated for a long time under vital staining with fluorescent dye ethylrhodamine (10 micrograms/ml) which is known to accumulate preferentially in mitochondria. The energization was estimated by the intensity of mitochondrial fluorescence. It was realized that 30 minutes after enucleation cytoplasts retained the same fluorescence as did the untreated cells, and that the mitochondrial distribution within the cell was very similar. Such a high intensity of fluorescence seen within one day after enucleation gives a strong evidence on the high degree of independence on the cell nucleus of organelles that provide the energy to metabolic processes. In the course of survival in the cultivation medium for 1-4 days the intensity of fluorescence is shown to fall, especially on the second day after enucleation. All these changes coincide with the changes in cytoplast shape: originally well spread bodies transform into squeezed, ball-shaped or strongly deformed ones. The adhesive ability is going down, and in result only single units of cytoplast can hardly be found on the cover slips by the 4th day after enucleation. These changes give evidence on the enhanced degeneration of cultured cytoplasts.     

Establishment of the afferent function of the striatum in the rabbit during early ontogenesis

The study is dedicated to electrophysiological analysis of development of the caudate nucleus afferent function in rabbits in early postnatal ontogenesis. By amplitude-temporal parameters of the evoked potentials (EPs) of the caudate nucleus, recorded in response to stimulation of afferent inputs from the cortical regions (motor and limbic) and substantia nigra, similar dynamics of these afferent pathways functional maturation is revealed from the beginning of functioning (in the age of 3-5 days) to the definitive level (by the 30th day of life). The most significant changes of the amplitude-temporal parameters of the evoked potential, particularly of latencies are observed during the 3d week of postnatal life. On the basis of the obtained data, the conclusion is made on the formation of a common system of striatum sensory integration in the 3d-4th week of rabbits life. This age period is considered as critical.     

The distribution of monoamines in the hypothalamus of the Japanese quail Coturnix coturnix japonica

Summary The distribution of monoamines in the hypothalamus of the Japanese quail (Coturnix coturnix japonica) has been studied using a histochemical fluorescence technique. In the posterior hypothalamus catecholamine-containing nerve fibres are localised in the nucleus tuberis and nucleus hypothalamicus posterior medialis and are linked by fluorescent tracts running in the stratum cellulare internum. Further tracts may be traced from the nucleus tuberis around the base of the third ventricle to the sub-ependymal layer of the median eminence, where they then appear to pass through the hypothalamo-hypophysial neurosecretory tract to terminate in the palisade zone on the portal vascular bed. The innervation of the palisade layer by catecholamines is sparse. The fluorescent terminals are spread evenly throughout both the anterior and posterior divisions of the median eminence. There is no monoamine innervation of the pars nervosa. The paraventricular organ has both 5-hydroxytryptamine- and catecholamine-containing cell bodies and axons may be traced into the region of the nucleus hypothalamicus posterior medialis. In the anterior hypothalamus the neurosecretory paraventricular nucleus contains many catecholamine nerve fibres and terminals. These are linked by fibre tracts to the nucleus basalis and to the nucleus hypothalamicus posterior medialis. The supraoptic nucleus is less well innervated although a dense accumulation of fibres lies in the preoptic recess. The latter is thought to give rise to long axons which pass in association with the neurosecretory tract to end in the nucleus tuberis.Supported by a Grant (AG 24/36) from The Agricultural Research Council. We are indebted to Dr. G. A. Clayton, Institute of Animal Genetics, University of Edinburgh, for supplying the birds.     

Monoamines in the C cells of the thyroid gland of callithricid primates

Summary The thyroid gland of three species of marmosets were studied with the Falck and Hillarp fluorescence technique for monoamines. Two types of fluorescent C cells were observed. The predominant type displayed the greenish (or blue) fluorescence characteristic of catecholamines. The other type displayed a yellowish fluorescence most probably due to 5-HT. The presence of monoamines in C cells now reported for the first time in Primates offers an interesting possibility for studying their presumed role in calcitonin secretion.     

Respiration and glycolysis in the liver of developing chick embryos and chicks]

Oxygen uptake in liver slices remains constant between the 12th and the 17th days of embryonic development, being equal to that in 30-60-day chicks. During the transition from allantoic respiration to the pulmonary one, oxygen consumption decreases, the decrease being observed up to the end of embryonic period. After hatching, oxygen consumption increases 4-5-fold to the 6-7th and decreases up to the initial level at the 10th day. Respiration of mitochondria isolated from the liver and concentration of cytochromes in mitochondria remain constant. The value P/O is the lowest, whereas catalase activity is the highest during hatching. The intensity of anaerobic glycolysis changes similarly to that of respiration.     

Spectrofluorimetric determination of 5-hydroxytryptamine using chlorosulphonylthenoyltrifluoroacetone-europium probe.

A new spectrofluorimetric method was developed for the determination of trace amounts of 5-hydroxytryptamine (5-HT). Using chlorosulphonylthenoyltrifluoroacetone (CTTA)-europium (Eu(3+)) ion as a fluorescent probe in a buffer solution at pH 11.0, 5-HT can remarkably enhance the fluorescence intensity of the CTTA-Eu(3+) complex at lambda = 612 nm; the enhanced fluorescence intensity of Eu(3+) is proportional to the concentration of 5-HT. Optimum conditions for the determination of 5-HT were also investigated. The linear range and detection limit for the determination of 5-HT were 1.0 x 10(-7)-1.2 x 10(-5) mol/L and 8.5 x 10(-8) mol/L, respectively. This method is simple, practical and relatively free of interference from coexisting substances, and can be applied to assess 5-HT in urine samples.     

龙虾肌羧甲基甘油醛-3-磷酸脱氢酶NAD~+荧光衍生物的生成与性质

龙虾肌甘油醛-3-磷酸脱氢酶与兔肌酶一样,用碘代乙酸修饰后,在NAD~+存在下经紫外光照射也能形成荧光衍生物。 pH对荧光衍生物的生成和稳定性有很大影响,同时,异类离子的不同影响也很明显。 定磷分析法测定荧光衍生物上的NAD~+含量,同位素示踪法观察衍生物生成过程的脱羧,都证明此光化学反应为半位反应。