Biomedical subjects

L Vollrath

Publications and source records attributed to L Vollrath.

At least 37 records · Page 2Linked to original sources

PubMed1997

Nitric oxide inhibits electrically active units in the rat pineal gland.

Extracellular multiple unit recordings were performed in isolated rat pineal glands to determine a possible effect of nitric oxide (NO) on the spontaneous electrical activity of pinealocytes. Spontaneously active cells forming clusters of 3-5 cells fell into two categories: more or less regularly firing clusters (REG, 64%) and irregularly discharging clusters with periodically repeated bursts (RHY, 36%). The NO-donor sodium nitroprusside (SNP) reduced the discharge rate of the great majority of REG clusters and of all the RHY clusters examined. Moreover, the burst activity of RHY clusters was abolished. These results could be completely reproduced by using another NO-donor, S-nitroso-N-acetyl-penicillamine (SNAP). The NO synthase inhibitor NMLA had no effect on REG and RHY clusters. The results show that spontaneous electrical activity is an intrinsic function of the rat pineal gland. NO can modulate the electrical activity affecting discharge rate and discharge pattern.

Animals↗

PubMed1997

The shape of synaptic ribbons in the rat pineal gland.

Under the transmission electron microscope, synaptic ribbons (SRs) of the mammalian pineal gland appear as rod-like organelles. Their three-dimensional structure is not precisely known. In the present study, pineal SRs were investigated using serial sections obtained from rats killed at noon and midnight. The shape of the SRs was reconstructed based on SR profile length and the number of sections in which the profiles were contained. The results obtained show that SRs are basically flat plate-like structures with polymorphic lateral edges. Reconstructions of SRs revealed that they had average dimensions of 300x150x35 nm and were 19.3% larger at night than at day; the difference in SR size points to perhaps major differences in synaptic function between day and night.

Animals↗

PubMed1997

Adaptation of the disector method to rare small organelles in TEM sections exemplified by counting synaptic bodies in the rat pineal gland.

The disector is the only objective method for quantifying particles of variable size in a given volume. With this method, cell organelles are identified on adjacent sections, but only those present in one section are counted. When counting extremely rare structures in transmission electron microscope sections (physical disector), the usual procedure of counting on electron micrographs is limited for economic reasons (e.g. micrographs highly outnumbering the investigated structures). Hence, to apply this unbiased stereological method, a modification of the physical disector concerning 3 aspects has been developed. (1) The prerequisite of screening large corresponding tissue areas (here approximately 65000 microm2) was fulfilled by examining tissue areas along the edges of ultrathin sections. (2) The size of the counting frame was determined by measuring the lengths of the section margins (minus a guard area) by means of a Morphomat. This value was multiplied by the width of the investigated tissue zone, corresponding to the diameter of the electron microscope viewing screen. (3) Disector counting was carried out simultaneously on both sections (bidirectional disector) to improve efficiency. In the present study tiny synaptic bodies (SBs) were quantitated by disector in a rat pineal gland, yielding approximately 30 SBs/1000 microm3. By contrast, single section profile counts of SBs amounted to 90 SBs/20000 microm2. Since the presently described adaptation of the disector is time-consuming, it is proposed to determine a proportion factor allowing to estimate number of structures per volume based on single section profile counts. This would decrease the evaluation time by more than 50%.

Animals↗

PubMed1997

No short-term effects of high-frequency electromagnetic fields on the mammalian pineal gland.

There is ample experimental evidence that changes of earth-strength static magnetic fields, pulsed magnetic fields, or alternating electric fields (60 Hz) depress the nocturnally enhanced melatonin synthesis of the pineal gland of certain mammals. No data on the effects of high-frequency electromagnetic fields on melatonin synthesis is available. In the present study, exposure to 900 MHz electromagnetic fields [0.1 to 0.6 mW/cm2, approximately 0.06 to 0.36 W/kg specific absorption rate (SAR) in rats and 0.04 W/kg in Djungarian hamsters; both continuous and/or pulsed at 217 Hz, for 15 min to 6 h] at day or night had no notable short-term effect on pineal melatonin synthesis in male and female Sprague-Dawley rats and Djungarian hamsters. Pineal synaptic ribbon profile numbers (studied in rats only) were likewise not affected. The 900 MHz electromagnetic fields, unpulsed or pulsed at 217 Hz, as applied in the present study, have no short-term effect on the mammalian pineal gland.

Animals↗

PubMed1996

Plasticity of retinal ribbon synapses.

Ribbon synapses differ from conventional chemical synapses in that they contain, within the cloud of synaptic vesicles (SV's), a specialized synaptic body, most often termed synaptic ribbon (SR). This body assumes various forms. Reconstructions reveal that what appear as rod- or ribbon-like profiles in sections are in fact rectangular or horseshoe-shaped plates. Moreover, spherical, T-shaped, table-shaped, and highly pleomorphic bodies may be present. In mammals, ribbon synapses are present in afferent synapses of photoreceptors, bipolar nerve cells, and hair cells of both the organ of Corti and the vestibular organ. Synaptic ribbons (SR's) are also found in the intrinsic cells of the third eye, the pineal gland, and in the lateral line system. The precise function of SR's is enigmatic. The prevailing concept is that SR's function as conveyor belts to channel SV's to the presynaptic membrane for neurotransmitter release by means of exocytosis. The present article reviews the evidence that speaks for a plasticity of these organelles in the retina and the third eye, as reflected in changes in number, size, shape, location, and grouping pattern. SR plasticity is especially pronounced in the mammalian and submammalian pineal gland and in cones and bipolar cells of teleost fishes. Here, SR number and size wax and wane according to the environmental lighting conditions. In the pineal SR numbers increase at night and decrease during the day. In teleost cones, SR's are in their prime during daytime and decrease or disappear at night, when transmitter release is enhanced. In addition to numerical changes, SR's may also exhibit changes in size, shape, grouping pattern, and location. In the mammalian retina of adults, in contrast to the developing retina, the reported signs of SR plasticity are subtle and not always consistent. They may reflect changes in function or may represent signs of degradation. To distinguish between the-two, more detailed studies under selected experimental conditions are required. Probably the strongest evidence for SR plasticity in the mammalian retina is that in hibernating squirrels SR's leave the synaptic site and accumulate in areas as far as 5 microns from the synapse. Changes in shape include the occurrence of club-shaped SR's and round SR's or synaptic spheres (SS's). SS's may represent a special type of synaptic body, yet belonging to the family of SR's, or may be related to the catabolism of SR's. SR number is regulated by Ca2+ in teleost cones, whereas in the mammalian pineal gland cGMP is involved. An interesting biochemical feature of ribbon synapses is that they lack synapsins. The presently reviewed results suggest to us that SR's do not primarily function as conveyor belts, but are devices to immobilize SV's in inactive ribbon synapses.

Animals↗

PubMed1996

Expression of brain nitric oxide synthase in the developing rat pineal gland coincides with the appearance of the adrenergic cyclic guanosine 3',5'-monophosphate response.

In the developing rat pineal gland, norepinephrine (NE)-stimulated cyclic guanosine 3',5'-monophosphate (cGMP) formation appears 2 weeks later than that of cyclic adenosine 3',5'-monophosphate (cAMP) formation. Since NE-stimulated cGMP formation requires formation of nitric oxide (NO) in the adult rat pineal, we investigated the developmental appearance of the NO-forming enzyme brain NO-synthase (NOS) to find out whether the delayed cGMP response results from the late appearance of NOS. In this study, we investigated pineal glands of rats aged 8-21 days and 8 weeks. Western blot analysis revealed that NOS-immunoreactivity (NOS-IR) was barely detectable until day 12. On day 15 NOS-IR exhibited the same intensity as in adult rats. Expression of NOS at this postnatal stage appears to be responsible for the developmental appearance of the adrenergic cGMP response.

Adrenergic alpha-Agonists↗

PubMed1996

Morphometry of pineal synaptic ribbon profile numbers after cytochalasin D treatment.

Synaptic ribbons (SRs) are electron-dense, plate-shaped synaptic organelles, to which electron-lucent synaptic vesicles (SVs) are attached by tiny stalks. In the mammalian pineal gland SRs are dynamic organelles, waxing and warning in number under different physiological and experimental conditions. The way in which SRs are formed, or catabolized, is not known. Since it has been suggested that actin may be part of SRs, in the present study the effect of the actin-disrupting drug cytochalasin D (CD, 1 microgram/ml, for 4 h) was examined in cultured guinea pig and rat pineal glands. The glands were preincubated for 48 h so that intra-pineal sympathetic nerve fibres degenerate and no longer release noradrenaline which may distort the results. CD had no effect on SR profile numbers in guinea pigs, but decreased them in rats (p > 0.05). The nonsignificant depressive effect of CD in rats was verified in a second experiment. To clarify the issue, acutely cultured rat pineal glands were treated with CD for 4 h, without effect. The results taken together suggest to us that CD has no major effect on pineal SR profile numbers, but that in rats preincubation for 48 h makes them vulnerable to catabolic processes. In all the experiments, the electron-dense plate of the SRs was qualitatively unaffected. However, the SVs were often larger and more irregular in shape and the stalks linking the SVs with the SRs were less frequently seen in CD-treated glands. In guinea pig pineals, in which SRs frequently lie in groups and parallel to each other, neither the distance between neighboring SRs nor the thickness of individual SRs were affected by CD. It is concluded that actin is not a major component of the SRs and the connecting stalks.

Animals↗

PubMed1995

In rat pinealocytes the cyclic GMP response to NO is regulated by Ca2+ and protein kinase C.

There is ample evidence that beta-adrenergic stimulation of cyclic GMP formation is potentiated by alpha1-adrenergic mechanisms, the latter leading to elevation of intracellular Ca2+ concentration ([Ca2+]i) and protein kinase C (PKC) activation. Recent studies have shown that nitric oxide synthase (NOS) and nitric oxide (NO) are a component of the adrenoceptor-cyclic GMP signalling pathway. The aim of the present investigation was to study the roles of alpha1-adrenergic mechanisms, Ca2+ and PKC on NO-stimulated cyclic GMP formation. To this end suspension cultures of rat pinealocytes were treated with the NO donor sodium nitroprusside (SNP) in the presence of alpha1-adrenergic agonists, [Ca2+]i-elevating substances, PKC inhibitors, followed by measurement of cyclic GMP accumulation. It was found that alpha1-adrenergic stimulation did not affect NO-activated cyclic GMP synthesis. Therefore alpha1-mechanisms act prior to NO induction of cyclic GMP. Agents, which elevate [Ca2+]i depressed NO-induced cyclic GMP formation. Since literature data show that Ca2+ stimulates pineal NO formation it is apparent that Ca2+ has antagonistic effects in the pineal adrenoceptor-cyclic GMP signalling pathway. The inhibitory effect of Ca2+ was unchanged after inhibition of phosphodiesterases suggesting that it may interfere with cytosolic guanylyl cyclase activation. Inhibition of PKC, but not of other protein kinases, decreased NO-activated cyclic GMP formation. Therefore it appears that non-alpha1-adrenergic-regulated PKC possesses a regulatory rote in NO-induced cyclic GMP formation.

Animals↗

PubMed1995

In the rat pineal gland, but not in the suprachiasmatic nucleus, the amount of constitutive neuronal nitric oxide synthase is regulated by environmental lighting conditions.

To investigate whether the expression of neuronal isoform of nitric oxide synthase is photoneurally regulated, we examined the amount of the enzyme by means of Western Blot analysis under different environmental lighting conditions in two photoneuronally regulated tissues, the pineal gland and the suprachiasmatic nucleus of the rat. In the pineal gland nitric oxide synthase immunoreactivity was strikingly decreased when rats had been exposed for 8 days to light:dark 20:4 conditions or to constant light. The decline in nitric oxide synthase immunoreactivity following LL reversed after 4 days under light:dark 12:12. We conclude that in the rat pineal the amount of neuronal nitric oxide synthase is controlled by environmental lighting conditions. That it is not justified to extrapolate from the pineal to other photoneuronally regulated centers is illustrated by the present finding that the suprachiasmatic nucleus did not reveal changes in the amount of neuronal nitric oxide synthase.

Amino Acid Oxidoreductases↗

PubMed1995

Down-regulation of the nocturnally elevated guanylyl cyclase activity in the rat pineal gland.

Previous studies have shown that in the rat pineal, the cytosolic and the particulate forms of guanylyl cyclase (GC) activity undergo a biphasic 24-h rhythm with two prominent peaks, one in the middle of the light phase and the other in the middle of the dark phase. In this study we investigated whether the well established photo-neural adrenergic regulatory processes identified for pineal melatonin synthesis also apply to the nocturnal elevation of GC activity. A 10-min light pulse given in the middle of the dark phase decreases the cytosolic and the particulate forms of GC. Administration of the beta-receptor blocker propranolol did not depress the nocturnally elevated GC activity. Sympathetic denervation of the pineal gland by means of superior cervical ganglionectomy did not noticeably affect nocturnal GC activity studied 6 days and 2 months after surgery. In vitro, administration of the nitric oxide synthase blocker NG-mono-methyl-L-arginine (NMMA) for 10 min did not change the cytosolic form of GC activity. The results obtained reveal that in the rat pineal, the down-regulation of the nocturnally elevated GC activity does not appear to be adrenergically mediated.

Animals↗

PubMed1995

The response to isoproterenol of synaptic ribbon numbers in the rat pineal gland changes during postnatal development.

In the mammalian pineal gland synaptic ribbons (SRs) are dynamic organelles of pinealocytes undergoing a day/night rhythm, with small numbers during daytime and significantly higher numbers at night, similar to the formation of the pineal hormone melatonin. Whereas the day/night rhythm of melatonin synthesis is adrenergically regulated, data on the adrenergic regulation of SR numbers in the rat pineal gland are at variance. While some authors have demonstrated that isoproterenol (ISO) stimulates SR numbers, others could not find any effect. To clarify the issue, we carried out identical experiments in two age groups. It was found that in male Sprague-Dawley rats, administration of the beta-adrenergic agonist ISO (2.5 or 10 mg/kg body weight) increased SR profile numbers in one age group (3 weeks), but not in the other (8 weeks). These findings resolve the apparent discrepancy reported in the literature, by showing that the response to ISO of SR profile numbers is apparently lost during postnatal differentiation of the pineal gland.

Animals↗

PubMed1995

p-Chlorophenylalanine treatment depresses the number of synaptic ribbon profiles in the rat pineal gland, but does not abolish their day-night rhythm.

It is largely unknown as to how the day/night rhythm of pineal synaptic ribbon number is regulated. Transcriptional events related to the nocturnal formation of new synaptic ribbons occur early in the morning, when pineal serotonin levels begin to increase. The present study was carried out in order to elucidate as to how altered serotonin levels affect the day/night changes in the number of synaptic ribbon profiles. To this end, male Sprague-Dawley rats received a single dose of p-chlorophenylalanine (pCPA, 300 mg/kg body weight, 72 hours before sacrifice), which depresses tryptophan hydroxylase activity and hence pineal serotonin levels. Control animals received saline injections. Experimental and control animals were killed in the middle of the light period and in the middle of the dark period, respectively. The pineal glands were removed and bisected. One half was used for electron microscopic quantification of synaptic ribbon profile numbers, and the other half for determination of several biochemical parameters (see below). pCPA decreased pineal serotonin levels to c. 30% of the controls and synaptic ribbon profile numbers by c. 30%, both at day and night. The day/night rhythm of the two parameters did not appear to be affected. The rate-limiting enzyme of melatonin synthesis, serotonin N-acetyltransferase, which is only measurable at night, and pineal melatonin levels were not affected at night. The intermediary product of melatonin formation, N-acetylserotonin, was significantly depressed at night. It is concluded that serotonin plays a more important modulatory role on pineal synaptic ribbons than does melatonin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Lack of effect of oxytocin on the numbers of "synaptic" ribbons, cyclic guanosine monophosphate and serotonin N-acetyltransferase activity in organ-cultured pineals of three strains of rats.

In addition to the stimulating influence of the sympathetic system on the function of the mammalian pineal gland, neuropeptides such as neuropeptide Y, vasoactive intestinal polypeptide and arginine-vasopressin (AVP) are thought to function as modulators. Since AVP has been shown to influence pineal melatonin synthesis, the aim of the present study was to investigate the possible effects of the second hypothalamic nonapeptide oxytocin (OT), which likewise has been detected in the pineal gland. We therefore studied "synaptic" ribbon (SR) numbers, N-acetyltransferase (NAT) activity and the intracellular concentration of cyclic guanosine monophosphate (cGMP) following in vitro incubation of rat pineals in media containing OT (10(-5) M), noradrenaline (NA, 10(-5) M) or both NA and OT. Pineal glands were derived from rats of three different strains (Sprague-Dawley, Long-Evans and the AVP-deficient strain Brattleboro). Neither morphological nor biochemical analyses showed a difference between control and OT-incubated organs in any of the strains tested. In Brattleboro rats, but not in the other strains, noradrenaline slightly increased the number of SR which was not observed when NA and OT were combined. The addition of NA resulted in distinct augmentation of NAT activity and cGMP content, which were not affected by additional OT application. These results suggest that oxytocin is not crucially involved in the regulation of pineal gland function.

Animals↗

PubMed1993

Protein gene product (PGP) 9.5 immunoreactivity in nerve fibres and pinealocytes of guinea-pig pineal gland: interrelationship with tyrosine- hydroxylase- and neuropeptide-Y-immunoreactive nerve fibres.

This light-microscopic (LM) immunohistochemical study has evaluated the presence and distribution of the pan-neural and neuroendocrine marker protein gene product (PGP) 9.5 in pinealocytes and nerve fibres of guinea-pig pineal gland. The pattern of PGP 9.5-immunoreactive (ir) nerve fibres has been compared with that of fibres staining for tyrosine hydroxylase (TH) or neuropeptide Y (NPY). The vast majority of pinealocytes stained for PGP 9.5, although with variable intensity. PGP 9.5 immunoreactivity was localized in pinealocytic cell bodies and processes. Double-immunofluorescence revealed that PGP 9.5 immunoreactivity was absent from glial cells identified with a monoclonal antibody against glial fibrillary acidic protein (GFAP). PGP 9.5 immunoreactivity was also present in a large number of nerve fibres and varicosities distributed throughout the pineal gland. The number of TH-ir and NPY-ir nerve fibres was lower compared with those containing PGP 9.5 immunoreactivity. All fibres staining for NPY also stained for TH. NPY-ir nerve fibres were found to be much more numerous than previously reported for this species. The double-immunofluorescence analysis indicated that almost all TH-ir nerve fibres of the pineal gland contained PGP 9.5 immunoreactivity. However, few PGP 9.5-ir nerve fibres, located in the periphery and the central part of the gland, were TH-negative. A large number of PGP 9.5-ir fibres was concentrated in the pineal stalk. In contrast, TH-ir and NPY-ir nerve fibres were rare in this part of the pineal gland. Our data provide evidence that immunohistochemistry for PGP 9.5 may be a useful tool further to differentiate central and peripheral origins of pineal innervation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Strain differences in the ratio of synaptic body types in photoreceptors of the rat retina.

In the retinal outer plexiform layer of seven different rat strains, synaptic bodies (SB) were counted and, according to their morphology, characterized as synaptic ribbons (SR), synaptic spheres (SS) or intermediate structures. It was found that absolute SB numbers showed relatively small variations while SR/SS ratios differed considerably between the strains investigated. These results are discussed with respect to retinal pigmentation and to formation and degradation, respectively, of synaptic ribbons.

Animals↗

PubMed1993

Muscarinic agonists have no measurable effect on cGMP formation of rat pinealocytes.

Previous studies have shown that muscarinic agonists stimulate cGMP formation in various tissues including rat brain. As in the pineal gland cGMP formation varies considerably under various experimental conditions, in the present investigation the effects of muscarinic agonists were tested. Muscarinic agonists neither stimulated pineal cGMP formation nor affected cGMP accumulation, resulting from administration of phosphodiesterase (PDE) inhibitors, norepinephrine (NE), or sodium nitroprusside (SNP). Because muscarinic agonists are known to stimulate pineal inositol phosphate (Ip) formation we suspect that muscarine-related Ip formation does not affect cGMP formation in rat pineal gland.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Adrenergic stimulation of cyclic GMP formation requires NO-dependent activation of cytosolic guanylate cyclase in rat pinealocytes.

Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving beta- and alpha 1-adrenergic receptors. The effects of NG-monomethyl-L-arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited beta-adrenergic stimulation of cGMP formation as well as alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of L-arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.

Animals↗

PubMed1992

Involvement of cyclic guanosine monophosphate (cGMP) and cytosolic guanylate cyclase in the regulation of synaptic ribbon numbers in rat pineal gland.

In the rat pineal gland N-acetyltransferase (NAT) activity and synaptic ribbon (SR) numbers display a circadian rhythm. It is well-known that NAT activity is regulated by adrenergic mechanisms involving cyclic adenosine monophosphate (cAMP) as a second messenger. However, the mechanism involved in the regulation of SR numbers has not been established so far. In the present in vitro study, we have investigated the effects of 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP), a cyclic guanosine monophosphate (cGMP) analog, and stimulation of guanylate cyclase on SR numbers. Incubation with 8-bromo-cGMP increased SR numbers in a dose- and time-dependent manner. Further, stimulation of the cytosolic guanylate cyclase also resulted in increased SR numbers. Adrenergic agonists stimulated cGMP but did not alter SR numbers. These findings suggest that cGMP is involved as a second messenger in the regulation of SR numbers. Since the adrenergically stimulated increase in cGMP did not influence SR numbers, a non-adrenergic cGMP metabolic pathway seems to be involved in the regulation of SR numbers in the rat pineal gland.

Animals↗