Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: a brain-behavioral study of disconnected mutants

Mutations at the disconnected (disco) locus of Drosophila melanogaster disrupt neural cell patterning in the visual system, leading to the loss of many optic lobe neurons. Drosophila's presumptive circadian pacemaker neurons--the dorsal and ventral lateral neurons--are usually among the missing cells, and most disco flies are behaviorally arrhythmic. In this study, I show that ventral lateral neurons (LNvs) are occasionally present and provoke robust circadian rhythmicity in disco mutants. Of 357 individual disco flies four animals with robust circadian rhythmicity were found. All four retained LNvs together with terminals in the superior protocerebrum. Residual or bi-circadian rhythmicity was found in about 20% of all flies; the remaining flies were completely arrhythmic. One of the flies with residual rhythmicity and two of the arrhythmic flies also had some LNvs stained. However, these flies lacked the LNv fibers in the superior protocerebrum. The results suggest that the presence of single LNvs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LNv terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LNvs indicates that also other cells contribute to the rhythmic control of locomotor activity.     

Neurites of period-expressing PDH cells in the fly's optic lobe exhibit circadian oscillations in morphology

Circadian rhythms have been shown both in the expression of the period (per) gene in 'lateral neurons' and in cells of the outermost neuropil, or lamina, of the fly's optic lobe. Some lateral neurons also exhibit PDH peptide-like immunoreactivity, arborizing widely throughout the optic lobe. Using confocal microscopy in the housefly, we analysed the size and spacing of PDH neurite varicosities, sites of possible peptide release exhibiting circadian rhythmicity. During the subjective day in constant darkness, there were fewer, larger varicosities than during subjective night. The endogenous rhythm was masked by the light exposure that occurred under a day-night cycle and continuous light conditions. Our findings indicate that PDH neurites convey circadian information out from the pacemaker, where they could regulate the circadian rhythms that have been described in the lamina, possibly via cyclical release of their peptide.     

Drosophila photoreceptors contain an autonomous circadian oscillator that can function without period mRNA cycling

Circadian oscillations in period (per) mRNA and per protein (PER) constitute, in part, a feedback loop that is required for circadian pacemaker function in Drosophila melanogaster. Oscillations in PER are required for oscillations in per mRNA, but the converse has not been rigorously tested because of a lack of measurable quantities of per mRNA and protein in the same cells. This circadian feedback loop operates synchronously in many neuronal and non-neuronal tissues, including a set of lateral brain neurons (LNs) that mediate rhythms in locomotor activity, but whether a hierarchy among these tissues maintains this synchrony is not known. To determine whether per mRNA cycling is necessary for PER cycling and whether cyclic per gene expression is tissue autonomous, we have generated per01 flies carrying a transgene that constitutively expresses per mRNA specifically in photoreceptors, a cell type that supports feedback loop function. These transformants were tested for different aspects of feedback loop function including per mRNA cycling, PER cycling, and PER nuclear localization. Under both light/dark (LD) cycling and constant dark (DD) conditions, PER abundance cycles in the absence of circadian cycling of per mRNA. These results show that per mRNA cycling is not required for PER cycling and indicate that Drosophila photoreceptors R1-R6 contain a tissue autonomous circadian oscillator.     

Rhythmic activities of isolated and clustered pacemaker neurons and photoreceptors of Aplysia retina in culture

Each eye of Aplysia contains a circadian clock that produces a robust rhythm of optic nerve impulse activity. To isolate the pacemaker neurons and photoreceptors of the eye and determine their participation in the circadian clock and its generation of rhythmic autoactivity, the retina was dissociated and its cells were placed in primary cell culture. The isolated neurons and photoreceptors survived and vigorously extended neurites tipped with growth cones. Many of the photoreceptors previously described from histological sections of the intact retina were identified in culture, including the large R-type photoreceptor, which gave robust photoresponses, and the smaller tufted, whorled, and flared photoreceptors. The pacemaker neurons responsible for the rhythmic impulse activity generated by the eye were identified by their distinctive monopolar morphology and recordings were made of their activity. Isolated pacemaker neurons produced spontaneous action potentials in darkness, and pacemaker neurons attached to fragments of retina or in an isolated cluster interacted to produce robust spontaneous activity. This study establishes that isolated retinal pacemaker neurons retain their innate autoactivity and ability to produce action potentials in culture and that clusters of coupled pacemaker neurons are capable of generating robust autoactivity comparable to pacemaker neuron rhythmic activity recorded in the intact retina, which was previously shown to correspond to 1:1 with the optic nerve compound action potential activity.     

Neural substrates of Drosophila rhythms revealed by mutants and molecular manipulations

In the fruit-fly Drosophila, rhythmic expression of the clock gene period is detected in cells throughout the body. Whereas these cells could be pacemakers for circadian rhythms of unknown physiological processes, the brain pacemakers are known to be responsible for circadian behavior. Recent progress in genetic and molecular studies of clock genes in Drosophila has permitted the identification of brain pacemakers at the cellular level and their output pathways to rhythmic behavior. Similar studies in other insect species have suggested considerable diversity in the anatomical and neurochemical properties of pacemaker cells, as well as in the mechanisms of clock-gene regulation.     

Growth hormone prevents human monocytic cells from Fas-mediated apoptosis by up-regulating Bcl-2 expression

Voltage-sensitive dyes NK 2761 and RH 155 were employed (in conjunction with a 12 x 12 photodiode array) to study membrane potential transients in optic lobe neuropils in the eye stalk of the crayfish Procambarus clarkii. By this means we investigated a pathway linking deutocerebral projection neurons, via hemiellipsoid body local interneurons, to an unidentified target (most likely neurons processing visual information) in the medulla terminalis. Rapid (10- to 20-ms duration), transient changes in absorption with the characteristics of action potentials were recorded from the optic nerve and the region occupied by deutocerebral projection neurons after stimulation of the olfactory globular tract in the optic nerve and were blocked by 1 microM tetrodotoxin. Action potentials appeared to propagate to the glomerular layer of the hemiellipsoid body where synaptic responses were recorded from a restricted region of the hemiellipsoid body occupied by dendrites of hemiellipsoid body neurons. Action potentials were also recorded from processes of hemiellipsoid body neurons located in the medulla terminalis. Synaptic responses in the hemiellipsoid body and medulla terminalis were eliminated by addition to the saline of 500 microM Cd2+ or 20 mM Co2+, whereas the action potential attributed to branches of deutocerebral projection neurons in the hemiellipsoid body remained unaffected. Action potentials of hemiellipsoid body neurons in the medulla terminalis evoked postsynaptic potentials (50- to 200-ms duration) with an unidentified target in the medulla terminalis. Transient absorption signals were not detected in either the internal or external medulla nor were they recorded from other parts of the optic lobes in response to electrical stimulation of axons of the deutocerebral projection neurons. Functional maps of optical activity, together with electrophysiological and pharmacological findings, suggest that gamma-aminobutyric acid affects synaptic transmission in glomeruli of the hemiellipsoid body. Synapses of the olfactory pathway located in the medulla terminalis may act as a "filter," modifying visual information processing during olfactory stimulation.     

Nitric oxide as a putative messenger molecule in the crayfish olfactory midbrain

NADPH-d histochemistry was used to investigate presumptive nitric oxide synthase (NOS)-containing neurons in the crayfish olfactory midbrain. Three anatomically different types of local olfactory interneurons exhibiting NADPH-d activity were observed: two pairs of large interneurons as well as positively stained globuli cells. Branches derived from the large interneurons were confined to the ipsilateral olfactory lobe and accessory lobe, but only a few branches innervated the olfactory lobe glomeruli. Local field potential recordings on the olfactory lobe showed that administration of SNP or SIN-1 (10-4 M) into the brain had reversible inhibitory effects on electrically-evoked responses of unidentified neuronal cell populations.     

The site of action of general anaesthetics in insect olfactory receptor neurons

The effect of volatile anaesthetics such as N2O, Xe, short-chain alkanes and cyclopropane, at pharmacologically relevant concentrations, on olfactory receptor neurons of insects was tested in electrophysiological recordings. CO2-receptor neurons in moths and flies respond with increased action potential activity, whereas in honeybees the effect is inhibitory. With increasing chain length of the alkanes, the effectiveness increases initially, in adherence to the Meyer-Overton rule; alkanes of a chain length of 5 and above are less effective or evoke suppression of action potentials. In olfactory receptor neurons sensitive to benzoic acid in female moths of Bombyx mori and in pheromone receptor neurons of male moths of Antheraea polyphemus, anaesthetics are ineffective if applied alone; if superimposed on an excitatory olfactory stimulus, an inhibitory effect occurs. Local stimulation of only part of a sensory dendrite reveals that the anaesthetics are effective only if applied at the same location as the excitatory stimulus. This indicates that the anaesthetics reversibly block the reception of pheromone or its effect on the conductance of the receptor cell membrane. The observed interactions are consistent with the hypothesis that the anaesthetics do not interact with the primary transduction process, but rather affect a later stage such as the activation of ion channels.     

Allatostatin-like-immunoreactive neurons of the tobacco hornworm, Manduca sexta, and isolation and identification of a new neuropeptide related to cockroach allatostatins

The YXFGLamide C-terminus serves to define most members of a family of structurally related neuropeptides, the YXFGLamides. These peptides have been identified from the nervous system of various insects and include the allatostatins of cockroaches and crickets, the schistostatins of locusts, and the callatostatins of blowflies. The YXFGLamides have been shown to have various functions, including inhibition of juvenile hormone biosynthesis in cockroaches and crickets and inhibition of contraction of certain insect visceral muscles. We wanted to know if these peptides occur in Manduca sexta and what functions they might have. A new peptide, AKSYNFGLamide, was isolated and identified from M. sexta and has been named "lepidostatin-1"; this is the first YXFGLamide to be found in a lepidopteran, and there are indications that additional YXFGLamides occur in M. sexta. An antiserum to cockroach allatostatins (YXFGLamides) was shown to recognize lepidostatin-1 of M. sexta and was used to map YXFGLamide-immunoreactive neurons in larvae. Because immunoreactive interneurons were found to form an extensive neuropil, YXFGLamides probably function as neuromodulators in M. sexta. Neuroendocrine cells in the brain, abdominal ganglia, and their respective neurohemal organs were YXFGLamide immunoreactive and appear to release YXFGLamides as neurohormones. Immunoreactivity to YXFGLamides and M. sexta diuretic hormone were found to be colocalized and appear to be coreleased in these neuroendocrine cells, indicating that YXFGLamides may be involved in regulation of fluid transport. Innervation of the corpora allata by YXFGLamide-immunoreactive processes was very sparse, suggesting that this innervation does not play an important role in allatostasis. Many thoracic motor neurons were YXFGLamide immunoreactive, suggesting that YXFGLamides may have a myomodulatory or myotrophic function in larvae. However, this immunoreactivity disappeared early in metamorphosis and did not reappear in the adult. The YXFGLamide-immunoreactive neurons in the terminal abdominal ganglion were found to innervate the hindgut, indicating that YXFGLamides may be involved in the control of the rate of myogenic contractions of the larval hindgut.     

The timSL mutant affects a restricted portion of the Drosophila melanogaster circadian cycle

The circadian rhythm genes period (per) and timeless (tim) are central to contemporary studies on Drosophila circadian rhythms. Mutations in these genes give rise to arrhythmic or period-altered phenotypes, and per and tim gene expression is under clock control. per and tim proteins (PER and TIM) also undergo circadian changes in level and phosphorylation state. The authors previously described a period-altering tim mutation, timSL, with allele-specific effects in different per backgrounds. This mutation also affected the TIM phosphorylation profile during the mid-late night. The authors show here that the single amino acid alteration in TIM-SL is indeed responsible for the phenotype, as a timSL transgene recapitulates the original mutant phenotype and shortens the period of perL flies by 3 h. The authors also show that this mutation has comparable effects in a light-dark cycle, as timSL also accelerates the activity offset during the mid-late night of perL flies. Importantly, timSL advances predominantly the mid-late night region of the perL phase response curve, consistent with the notion that this portion of the cycle is governed by unique rate-limiting steps. The authors propose that TIM and PER phosphorylation are normally rate determining during the mid-late night region of the circadian cycle.     

GABAergic modulation of optic nerve-evoked field potentials in the rat suprachiasmatic nucleus

The suprachiasmatic nuclei (SCN) at the base of the hypothalamus are known to be the site of the endogenous circadian pacemaker in mammals. The SCN are innervated by the retinohypothalamic tract, which conveys photic information to the SCN. GABA is one of the most abundant neurotransmitters in the SCN, and has been implicated in the modulation of photic responses of the SCN circadian pacemaker. This study sought to examine the effect of GABAergic compounds on optic nerve-evoked SCN field potentials recorded in rat horizontal hypothalamic slices. The GABAA agonist muscimol (10 microM) potentiated SCN field potentials by 23%, while application of the GABAA antagonist bicuculline (10 microM) inhibited SCN field potentials by a similar amount, (22%). Conversely, the GABA, agonist baclofen (1.0 microM) inhibited SCN field potentials by 48%, while the GABAB antagonist phaclofen (0.5 mM) augmented SCN field potentials by 62%. Recordings performed at both day and night times indicate that there were no qualitative day-night differences in GABAergic activity on SCN field potentials. This study concludes that, in general, GABAA activity tends to increase, and GABAB activity tends to decrease the response of SCN neurons to optic nerve stimulation.     

Development of gonadotropin-releasing hormone (GnRH) neuron regulation in the female rat

The central projections of the cold receptor axons were examined by filling two types of cold receptive sensilla with cobalt lysine--a cold and hygroreceptive (C/H) sensillum and a cold receptive and olfactory (C/O) sensillum--on the antennae of the cockroach, Periplaneta americana L. When the dye filled a single C/H sensillum, four axons were stained. Three of these axons terminate in the ipsilateral antennal lobe, while the other branches in the ipsilateral dorsal lobe. One of the branches passed through the tritocerebrum to terminate in the suboesophageal ganglion, while the other branches end in the lobe. When a single C/O sensillum is dye filled, all axons of the four receptor neurons terminate exclusively in the ipsilateral antennal lobe. One axon from the C/H sensillum and one axon from the C/O sensillum terminate in a particular glomerulus in the ventroposterior region of the antennal lobe. Each of these axons also has a tuft in separate glomeruli situated just dorsal to the glomerulus in which both axons terminate. This set of three glomeruli have indistinct boundaries and appear to form a complex of glomeruli similar to the macroglomerular complex of male moths. Assuming modality-specific convergence of antennal afferents, these axons appear to belong to the cold receptor neurons, and the set of glomeruli appear to function in cold reception. Two other neurons stained from C/H sensilla always terminate in the glom-eruli distinct from the set of glomeruli mentioned earlier. These neurons are assigned to the pair of hygroreceptor neurons, and their glomeruli are thought to function in hygroreception.     

Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies

Circadian (approximately 24-hr) rhythms in Drosophila melanogaster depend upon cyclic expression of the period (per) and timeless (tim) genes, which encode interacting components of the endogenous clock. The per gene has been isolated from other insects and, more recently, a per ortholog was found in mammals where its expression oscillates in a circadian fashion. We report here the complete sequence of a tim gene from another species, Drosophila virilis. TIM is better conserved than the PER protein is between these two species (76 vs. 54% overall amino acid identity), and putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. We show here that deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene displayed rhythms with approximately 24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the segment mentioned above resulted in very long activity rhythms with periods ranging from 30.5 to 48 hr.     

Neuronal correlates of sleep, wakefulness and arousal in a diurnal insect

The discovery that various states of sleep, rest, wakefulness and arousal in man can be correlated with specific forms of the electroencephalogram1 has led to intensive studies of these states, mostly in mammals2-5. Today it is generally accepted that circadian sleep-wakefulness cycles occur in mammals and birds2,3,6. Behavioural observations on sleep in moths have also been published7; many other invertebrates demonstrate rest/activity cycles8. Circadian sensitivity fluctuations in both central9 and peripheral10-15 components of the visual system of various nocturnal arthropod species have been demonstrated. We now report that long-term, extracellular, single-unit recordings from optomotor interneurones in the optic lobes of forager honey bees reveal an oscillation in their sensitivity to moving visual stimuli16, 17. The oscillation displays properties typical of a circadian rhythm6, 18. The sensitivity of the neurons is higher during the subjective day than during the subjective night. The locomotor activity of individual, fixed walking forager bees shows a similar circadian oscillation and is also higher during the subjective day. Visual and mechanical stimuli can act directly on the interneurones and restore their sensitivity during times of reduced neuronal responsiveness. A comparison with results available for mammals makes it likely that the neuronal phenomena presented here are correlates of the bee's circadian sleep-wakefulness rhythm.     

Morphology, axonal projection pattern, and response types of tectal neurons in plethodontid salamanders. II: intracellular recording and labeling experiments

In the plethodontid salamanders Plethodon jordani and P. glutinosus, the morphology and axonal projections of 140 tectal neurons and their responses to electrical optic nerve stimulation were determined by intracellular recording and biocytin labeling. Six types of neurons are distinguished morphologically. TO1 neurons have wide dendritic trees that arborize mainly in tectal layers 1 and 3; they project bilaterally to the tegmentum and contralaterally to the medulla oblongata. TO2 neurons have very wide dendritic trees that arborize mainly in layers 2 and 3; axons project bilaterally or unilaterally to the pretectum and thalamus and ipsilaterally to the medulla oblongata. TO3 neurons have very wide and flat dendritic trees confined to layers 3-5; some have the same axonal projection as TO2 neurons, whereas others have descending axons that reach only the level of the cerebellum. TO4 neurons have narrower dendritic trees that arborize in layers 2 and 3; they project to the ipsilateral pretectum, thalamus, and medulla oblongata. TO5 neurons have dendritic trees that arborize in layers 1 and 2 or 1-3 and project bilaterally or unilaterally to the pretectum and thalamus. TO-IN are interneurons, with a number of subtypes with respect to variations in dendritic arborization pattern. TO1-TO5 neurons generally have short latencies of 2-16 ms (average = 8.4 ms) at electrical optic nerve stimulation; first responses are always excitatory, often followed by inhibition. They are likely to be mono- or oligosynaptically driven by retinal afferents. TO-IN interneurons have long latencies of 20-80 ms (average = 38.6 ms) and appear to receive no direct retinal input. With their specific dendritic arborization, consequent dominant retinal input, specific axonal projections, the different types of tectal projection neurons constitute separate ascending and descending visual pathways. Hypotheses are presented regarding the nature of the information processed by these pathways.