Encoding time of day and time of year by the avian circadian system.

The most important zeitgeber for seasonal rhythmicity of physiology and behaviour in birds is the annual cycle of photoperiod. Regulatory mechanisms are less well understood in birds than in mammals since photic information can be perceived by photoreceptors in the retina and the pineal gland, as well as in the brain, and photoperiodic time measurement might be performed with reference to at least three autonomous circadian systems, the retina, the pineal gland and a hypothalamic oscillator. In many bird species, the pineal melatonin rhythm plays a central role in circadian organization. Durations of elevated melatonin in the blood reflect night length when animals are kept under natural photoperiodic conditions, as well as under different light/dark schedules in the laboratory. In the house sparrow, time of year is encoded in a particular melatonin signal, being short in duration and high in amplitude in long photoperiods and being long in duration and low in amplitude in short photoperiods, independent of whether the light zeitgeber is natural or artificial or varies in strength. Specific features of the melatonin signal are retained in vivo as well as in vitro when birds or isolated pineal glands are transferred to constant conditions. To regulate daily and seasonal changes of behaviour and physiology, melatonin may act at various target sites, including a complex hypothalamic oscillator that, unlike that in mammals, is not confined to a single cell group in the house sparrow. There is increasing evidence that interactions between two or more components of the songbird circadian pacemaking system are essential to encode and store biologically meaningful information about time, and thus provide the basis for photoperiodic time measurements and after effects in birds.

Photoperiod affects amplitude but not duration of in vitro melatonin production in the ruin lizard (Podarcis sicula).

The pineal gland and its major output signal melatonin have been demonstrated to play a central role in the seasonal organization of the ruin lizard Podarcis sicula. Seasonal variations in the amplitude of the nocturnal melatonin signal, with high values in spring as compared to low values in summer and autumn, have been found in vivo. The authors examined whether the pineal gland of the ruin lizard contains autonomous circadian oscillators controlling melatonin synthesis and whether previously described seasonal variations of in vivo melatonin production can also be found in isolated cultured pineal glands obtained from ruin lizards in summer and winter. In vitro melatonin release from isolated pineal glands of the ruin lizard persisted for 4 days in constant conditions. Cultured explanted pineal glands obtained from animals in winter and summer showed similar circadian rhythms of melatonin release, characterized by damping of the amplitude of the melatonin rhythm. Although different photoperiodic conditions were imposed on ruin lizards before explantation of pineal glands, the authors did not find any indication for corresponding differences in the duration of elevated melatonin in vitro. Differences were found in the amplitude of in vitro melatonin production in light/dark conditions and, to a lesser degree, in constant conditions. The presence of a circadian melatonin rhythm in vitro in winter, although such a rhythm is absent in vivo in winter, suggests that pineal melatonin production is influenced by an extrapineal oscillator in the intact animal that may either positively or negatively modulate melatonin production in summer and winter, respectively.

Complex bird clocks.

The circadian pacemaking system of birds comprises three major components: (i) the pineal gland, which rhythmically synthesizes and secretes melatonin; (ii) a hypothalamic region, possibly equivalent to the mammalian suprachiasmatic nuclei; and (iii) the retinae of the eyes. These components jointly interact, stabilize and amplify each other to produce a highly self-sustained circadian output. Their relative contribution to overt rhythmicity appears to differ between species and the system may change its properties even within an individual depending, for example, on its state in the annual cycle or its photic environment. Changes in pacemaker properties are partly mediated by changes in certain features of the pineal melatonin rhythm. It is proposed that this variability is functionally important, for instance, for enabling high-Arctic birds to retain synchronized circadian rhythms during the low-amplitude zeitgeber conditions in midsummer or for allowing birds to adjust quickly their circadian system to changing environmental conditions during migratory seasons. The pineal melatonin rhythm, apart from being involved in generating the avian pacemaking oscillation, is also capable of retaining day length information after isolation from the animal. Hence, it appears to participate in photoperiodic after-effects. Our results suggest that complex circadian clocks have evolved to help birds cope with complex environments.

Seasonal variations of in vivo and in vitro melatonin production in a passeriform bird, the house sparrow (Passer domesticus).

Melatonin, released from the pineal gland, is an important signal within the circadian pacemaking system of passeriform birds. Until now, seasonal variations in melatonin production have only been examined in a few avian species and the role of melatonin in the regulation of annual rhythms in birds is unclear. We investigated plasma melatonin in a group of house sparrows kept in an outside aviary in spring (March/April), summer (May/June), autumn (September/October), and winter (December/January). The durations of elevated melatonin values mirrored the seasonal changes in night length to a certain degree, the melatonin signal being longest in winter and shortest in summer. Additionally, plasma melatonin peak amplitudes differed significantly among seasons, with highest values in spring and summer and lowest values in winter. Cultured explanted pineal glands obtained from animals in winter and summer showed patterns of in vitro melatonin release comparable to in vivo circulating melatonin with different durations of elevated melatonin and peak amplitude values. These data indicate that the circadian pacemaking system of the house sparrow changes properties seasonally, either as a result of endogenous mechanisms or in response to environmental conditions. These properties are maintained in the pineal gland even after isolation from the animal.

Initial demonstration of rhythmic Per gene expression in the hypothalamus of a non-mammalian vertebrate, the house sparrow.

In mammals, the major pacemaker controlling circadian rhythmicity is located in the hypothalamic suprachiasmatic nuclei which are characterized by specific molecular features including the expression of three homologues of the Drosophila clock gene period (per). Until now, no comparable structure has been unambiguously described in the brain of any non-mammalian vertebrate. We cloned the PAS-domain of the Per2 gene in the house sparrow (Passer domesticus), a model organism in circadian research. Hypothalamic expression of passerPer2 (pPer2) showed a marked diurnal rhythm in the suprachiasmatic nucleus, a cell group located in the anterior hypothalamus directly above the optic chiasm and adjacent to the third ventricle. Additionally, pPer2 was diurnally expressed in the lateral hypothalamus. This first demonstration of rhythmic clock gene expression in the hypothalamus of a non-mammalian vertebrate provides basic information for future research on the evolution of circadian pacemaking systems.

Adrenergic and cholinergic regulation of in vitro melatonin release during ontogeny in the pineal gland of Long Evans rats.

Melatonin, produced by the pineal gland, plays an important role in a great variety of neuroendocrine functions. The rhythmic release of melatonin by the mammalian pineal gland is regulated by norepinephrine (NE) acting via alpha- and beta-adrenergic receptors utilizing distinct signal transduction pathways. Acetylcholine has been demonstrated to exert various effects in the mammalian pineal gland, including an inhibitory action on the NE-induced stimulation of melatonin production. However, data obtained by different laboratories on the interaction of adrenergic receptors are not consistent and whether muscarinic and/or nicotinic receptors participate in the various effects of acetylcholine is still contradictory. To investigate noradrenergic as well as cholinergic mechanisms during ontogeny, we have investigated in vitro melatonin release from isolated pineal glands of Long Evans rats of different ages. NE as well as the beta-adrenergic receptor agonist isoproterenol (ISO) significantly elevated the melatonin release in pineal glands from postnatal week 2 on. In pineal glands originating from 2- to 4-week-old rats, simultaneous activation of alpha- and beta-adrenergic receptors by ISO and the alpha-adrenergic receptor agonist methoxamine (MET) or NE resulted in significantly weaker stimulation of melatonin production than beta-receptor activation alone. Acetylcholine evoked a significant increase in melatonin release in pineal glands from 2- to 4-week-old rats. In pineal glands from 8- to 20-week-old animals, ISO, ISO + MET or NE stimulated pineal melatonin release to comparable maxima, whereas acetylcholine was without effect. Our data indicate (1) that the adrenergic stimulation of pineal melatonin production in Long Evans rats is dominated by a beta-adrenergic mechanism, (2) that additional alpha-adrenergic receptor activation is inhibitory and (3) dependent on the developmental status of the animal, and (4) that acetylcholine acting via muscarinic receptors has the capacity to stimulate melatonin release during early ontogeny. These data suggest that the melatonin-generating system of the pineal gland of Long Evans rats undergoes substantial functional changes during early postnatal development, including adrenergic as well as cholinergic mechanisms.

Photoperiodic information acquired and stored in vivo is retained in vitro by a circadian oscillator, the avian pineal gland.

Endogenous circadian rhythms have been described in a wide range of organisms from prokaryotes to man. Although basic circadian mechanisms at the molecular level are genetically fixed, certain properties of circadian rhythms at the organismic level can be modified by environmental conditions and subsequently retained for some time, even in organisms shielded from 24-hr environmental variations. To investigate the capacity of animals to acquire and store photoperiodic information, we examined activity and melatonin rhythms in house sparrows during synchronization to two different photoperiods and during subsequent prolonged darkness. Under constant environmental conditions, intact animals continued to have long feeding activity times when previously exposed to long days and short feeding activity times when previously exposed to short days. Correspondingly, significantly different durations of elevated melatonin in the plasma directly reflected the differences in night length during synchronization as well as during prolonged darkness. Additionally, we found a significant difference in the amplitude of the nocturnal melatonin signal, which also was conserved in prolonged darkness. To investigate whether the photoperiodic experience of an intact animal can be "memorized" by an isolated component of its circadian pacemaking system, we have investigated in vitro melatonin release during continuous darkness from explanted pineal glands of house sparrows after in vivo synchronization to two distinct photoperiods. Differences in the durations of elevated melatonin occurred during the first two cycles in culture and a difference in melatonin amplitude was detectable during the first night in culture. Our data indicate that photoperiodic patterns imposed on sparrows during in vivo synchronization can be maintained as an internal representation of time within the isolated pineal gland. Hence, the pineal gland, as one of the most significant components of the songbird circadian pacemaker, not only has the capacity to autonomously produce circadian rhythms of melatonin release but also is capable of storing biologically meaningful information experienced during previous cycles.

Evidence for an endogenous clock in the retina of rainbow trout: II. Circadian rhythmicity of serotonin metabolism.

The purpose of the present study was to investigate patterns of circadian rhythmicity in the retina of a salmonid fish, the rainbow trout (Oncorhynchus mykiss). Our data present the first demonstration of intraretinal variations of serotonergic substances under light/dark-conditions (LD) and during continuous darkness (DD). All substances examined (serotonin, N-acetyl serotonin, 5-hydroxytryptophol, 5-hydroxyindole acetic acid) were rhythmic in LD. Serotonin, N-acetyl-serotonin, and 5-hydroxyindole acetic acid showed a preservation of specific features of rhythmicity in DD indicating the involvement of an endogenous pacemaker in the regulation of serotonin metabolism in the rainbow trout eye.

Evidence for an endogenous clock in the retina of rainbow trout: I. Retinomotor movements, dopamine and melatonin.

The purpose of the present study was to investigate patterns of circadian rhythmicity in the retina of a salmonid fish, the rainbow trout (Oncorhynchus mykiss). In a first attempt to show both morphological and neurochemical variations during light/dark conditions (LD) and during continuous darkness (DD), we investigated retinomotor movements and the associated regulatory transmitters, dopamine and melatonin. All parameters studied showed patterns of rhythmicity in LD and DD, clearly indicating the presence of an endogenous clock in the rainbow trout eye. The most salient variations of all parameters studied were found at the transitions from light to dark and vice versa in LD and from subjective day to subjective night and vice versa in DD. The amplitudes of rhythms compared between LD and DD were similar in pigment index, whereas a clear reduction was found for cone index, dopamine and melatonin.

Modulation of ganglion cell activity in the pineal gland of the rainbow trout: effects of cholinergic, catecholaminergic, and GABAergic receptor agonists.

Second order neurons within intact isolated pineal glands of the rainbow trout were explored by extracellular recordings to investigate modulatory effects of putative intrapineal neurotransmitters. Acetylcholine, dopamine, and norepinephrine were found to increase ganglion cell activity in a majority of cells tested. The excitatory effects of acetylcholine, dopamine, and norepinephrine were mimicked by muscarinic, dopamine D2, and beta-adrenergic receptor agonists and significantly increased with the applied light intensity, resulting in an attenuation of the ganglion cell response to light. GABA decreased discharge activity in most cells tested. This effect, which could be mimicked with the GABAA receptor agonist piperidine, was independent from the adaptive status. Acetylcholine and GABA were still active if applied during synaptic blockade with low Ca++ high Mg(++)-perfusion medium, whereas dopamine and norepinephrine exhibited no effects if applied during synaptic blockade, suggesting a differential cellular distribution of neurotransmitter receptors in the trout pineal gland. These data demonstrate that ganglion cell activity in the trout pineal gland is under the influence of several neurotransmitters, including acetylcholine, dopamine, norepinephrine, and GABA, which is in contrast to the originally proposed simple bineuronal transduction pathway from photoreceptors onto ganglion cells. Since the above-mentioned neurotransmitters are believed to be released from pineal interneurons, we may conclude that ganglion cell activity in the teleost pineal gland is, similarly to the retina, the product of photoreceptor signals and a modulatory active interneuronal system.

gamma-Aminobutyric acid enhances the light response of ganglion cells in the trout pineal organ.

Electrical recordings from achromatic second order neurons of intact superfused pineal organs of the rainbow trout were used to investigate the role of gamma-aminobutyric acid (GABA) in the transmission of photoreceptor signals onto centrally projecting ganglion cells. Bath-applied GABA decreased the spike discharge rate of 98% of achromatic ganglion cells in a dose-dependent manner. GABA was also active if applied during synaptic blockade, demonstrating the presence of GABAergic receptors at the ganglion cell level. Responsiveness of ganglion cells to light was reversibly enhanced by GABA. The light response curve of ganglion cells, which was obtained by plotting spike rate versus light intensity, was significantly shifted to lower frequencies by GABA, indicating that GABA is an important inhibitory modulator of ganglion cell activity in the trout pineal organ.

Signal transmission in the photosensitive pineal organ of the rainbow trout: modulation of ganglion cell activity by intrinsic dopamine.

The photosensitive pineal organ of the rainbow trout (Oncorhynchus mykiss) transduces photic information into nycthemeral neuronal signals. To investigate origin, cellular localization, and functional significance of pineal catecholamines, we performed HPLC-analysis of catecholamines and tyrosine hydroxylase (TH) activity, as well as immunocytochemical and electrophysiological studies. In biochemical and immunocytochemical investigations, pineal cells were found to contain endogenous TH. Using HPLC-analysis, the presence of a catecholamine precursor (L-dopa), catecholamines (dopamine, norepinephrine, epinephrine), and a metabolite (DOPAC) was demonstrated. The release of L-dopa, dopamine and DOPAC from isolated pineal organs was shown by superfusion experiments. Extracellular recordings were used to monitor the action of dopaminergic drugs on electrical activity of ganglion cells. Dopamine increased the discharge activity of action potentials, whereas dopamine receptor antagonists resulted in a reduction of ganglion cell activity. Our data provide evidence for establishing dopamine as an intrinsic neurotransmitter or neuromodulator in the photosensitive pineal organ of the rainbow trout.

Acetylcholine modulates ganglion cell activity in the trout pineal organ.

In the teleost pineal organ light activates functional photoreceptors, which transmit electrical activity to the brain via axons of intermediate ganglion cells. To investigate whether acetylcholine plays a role in the transduction of pineal photoreceptor signals, extracellular recordings were performed from ganglion cells of intact superfused pineal organs of the rainbow trout. Bath-applied acetylcholine increased the spike discharge rate of 96% of achromatic ganglion cells in a dose-dependent manner. The light response curve of ganglion cells, which was obtained by plotting spike rate vs light intensity, was significantly shifted by acetylcholine to higher frequencies. Acetylcholine was also active if applied during synaptic blockade with low Ca2+/high Mg(2+)-medium, demonstrating the presence of cholinergic receptors at the ganglion cell level. These data represent the first demonstration of acetylcholine constituting a postsynaptic modulation of photoreceptor signals in the trout pineal organ.

Brain growth patterns in four European cyprinid fish species (Cyprinidae, Teleostei): roach (Rutilus rutilus), bream (Abramis brama), common carp (Cyprinus carpio) and sabre carp (Pelecus cultratus).

This study compares brain growth in 4 species of cyprinids, each distinctly different in adult brain morphology: roach have generalized brains; bream are characterized by well-developed visual, octavolateralis and gustatory brain regions; common carp show chemosensory (gustatory)-dominated brains, and sabre carp octavolateralis-dominated brains. The growth patterns of 16 regions relative to total brain volume were investigated by computer-aided quantitative histology to illustrate internal brain allometries. In all species the tectum opticum decreases in relative size during growth, whereas the corpus cerebelli increases. In bream and common carp, primary taste centers steadily increase in relative size during growth. In most if not all fish, the brain attains no definite final morphology. Lifelong, growth-related shifts in relative sizes of primary sensory regions may reflect lifelong shifting sensory capabilities.

Life history of roach, Rutilus rutilus (Cyprinidae, Teleostei). A qualitative and quantitative study on the development of sensory brain areas.

The present study deals with aspects of the brain development in the roach, Rutilus rutilus, a common mid-European cyprinid fish. The morphogenesis of selected brain areas from hatching to early juveniles was examined on serial paraffin cross-sections. From early juveniles to large adults, brain growth was quantitatively analyzed by computer-aided planimetry. The hatchlings of roach show a cytologically distinct optic tectum, but a poorly differentiated brainstem, reflecting the predominance of the optic sense during the larval planktivorous period. The differentiation and outgrowth of chemosensory brainstem centers is related to the onset and development of benthivorous feeding in juveniles. The optic tectum decreases in size relative to the total brain volume from juveniles through adults. The corpus cerebelli increases in relative size, whereas chemosensory and acousticolateral centers grow isometrically with the brain as a whole.

Identification of individuals at increased risk for AIDS among clinically asymptomatic homosexuals and abusers of intravenous drugs.

Immunological parameters previously shown to constitute an increased risk for progression towards AIDS have been observed in clinically asymptomatic individuals considered to be at risk for this syndrome. These parameters include severely decreased numbers of T helper cells (count below 400/mm3 blood for CD4+ cells were detected in 7 our of 33 HIV antibody-positive, asymptomatic homosexuals and in 3 out of 29 HIV antibody-positive, asymptomatic drug abusers) and elevated serum IgA and IgM levels (found in 7 to 30 percent of these subjects). Furthermore, up to 60% of risk group members showed a decreased lymphoproliferative response to tetanus toxoid as compared to only 11% of so-called low responders in the simultaneously tested healthy controls. Finally, the capacity to mount an immune response to viral glycoproteins was found to be impaired in individuals at risk for AIDS, as indicated by a low serum level of antibodies to tick-borne meningoencephalitis virus antigen in recently vaccinated subjects.

[Thoracic internal bleeding following subclavian puncture in a child]. / Thoracica Interna-Blutung nach Subclaviapunktion beim Kind.

We report the case of a four year old female patient who underwent an unsuccessful puncture of the subclavian vein in another department with an adult subclavian catheter. Unilateral haematothorax with massive bleeding from the right Art. thoracica interna occurred as a sequel of this attempt and was successfully treated in our department.