Tectal CRFR1 receptor involvement in avoidance and approach behaviors in the South African clawed frog, Xenopus laevis.

Animals in the wild must balance food intake with vigilance for predators in order to survive. The optic tectum plays an important role in the integration of external (predators) and internal (energy status) cues related to predator defense and prey capture. However, the role of neuromodulators involved in tectal sensorimotor processing is poorly studied. Recently we showed that tectal CRFR1 receptor activation decreases food intake in the South African clawed frog, Xenopus laevis, suggesting that CRF may modulate food intake/predator avoidance tradeoffs. Here we use a behavioral assay modeling food intake and predator avoidance to test the role of CRFR1 receptors and energy status in this tradeoff. We tested the predictions that 1) administering the CRFR1 antagonist NBI-27914 via the optic tecta will increase food intake and feeding-related behaviors in the presence of a predator, and 2) that prior food deprivation, which lowers tectal CRF content, will increase food intake and feeding-related behaviors in the presence of a predator. Pre-treatment with NBI-27914 did not prevent predator-induced reductions in food intake. Predator exposure altered feeding-related behaviors in a predictable manner. Pretreatment with NBI-27914 reduced the response of certain behaviors to a predator but also altered behaviors irrelevant of predator presence. Although 1-wk of food deprivation altered some non-feeding behaviors related to energy conservation strategy, food intake in the presence of a predator was not altered by prior food deprivation. Collectively, our data support a role for tectal CRFR1 in modulating discrete behavioral responses during predator avoidance/foraging tradeoffs.

GABA- and serotonin-expressing neurons take part in inhibitory as well as excitatory input pathways to the circadian clock of the Madeira cockroach Rhyparobia maderae.

In the Madeira cockroach, pigment-dispersing factor-immunoreactive (PDF-ir) neurons innervating the circadian clock, the accessory medulla (AME) in the brain's optic lobes, control circadian behaviour. Circadian activity rhythms are entrained to daily light-dark cycles only by compound eye photoreceptors terminating in the lamina and medulla. Still, it is unknown which neurons connect the photoreceptors to the clock to allow for light entrainment. Here, we characterized by multiple-label immunocytochemistry the serotonin (5-HT)-ir anterior fibre fan and GABA-ir pathways connecting the AME- and optic lobe neuropils. Colocalization of 5-HT with PDF was confirmed in PDF-ir lamina neurons (PDFLAs). Double-labelled fibres were traced to the AME originating from colabelled PDFLAs branching in accessory laminae and proximal lamina. The newly discovered GABA-ir medial layer fibre tract connected the AME to the medulla's medial layer fibre system, and the distal tract fibres connected the AME to the medulla. With Ca2+ imaging on primary cell cultures of the AME and with loose-patch-clamp recordings in vivo, we showed that both neurotransmitters either excite or inhibit AME clock neurons. Because we found no colocalization of GABA and 5-HT in any optic lobe neuron, GABA- and 5-HT neurons form separate clock input circuits. Among others, both pathways converged also on AME neurons that coexpressed mostly inhibitory GABA- and excitatory 5-HT receptors. Our physiological and immunocytochemical studies demonstrate that GABA- and 5-HT-immunoreactive neurons constitute parallel excitatory or inhibitory pathways connecting the circadian clock either to the lamina or medulla where photic information from the compound eye is processed.

Candidates for the light entrainment pathway to the circadian clock of the Madeira cockroach Rhyparobia maderae.

The circadian pacemaker controlling locomotor activity rhythms in the Madeira cockroach is located at the accessory medulla (AMe). The ipsi- and contralateral compound eyes provide light input to the AMe, possibly via the γ-aminobutyric acid (GABA)-immunoreactive (-ir) distal tract, which connects the glomeruli of the AMe to the ipsilateral medulla and lamina. To identify possible light-entrainment pathways, double-label immunocytochemistry was performed employing antibodies against GABA, myoinhibitory peptide (MIP), allatotropin (AT) and orcokinin (ORC). While all antisera tested, except the anti-ORC, prominently stained the glomeruli of the AMe, colocalization with anti-GABA was detected neither in the glomeruli nor in the distal tract. However, one median neuron that colocalized GABA-, AT- and MIP-immunoreactivity appeared to connect all glomeruli of the AMe to the medulla and lamina. Furthermore, one distal-frontoventral local neuron with arborizations in all glomeruli of the AMe colocalized anti-AT- and anti-MIP immunoreactivity. As candidates for contralateral light entrainment pathways, one ventromedian and one ventral neuron colocalized MIP- and ORC immunoreactivity, projecting via posterior and anterior commissures. Both branched in the interglomerular region of the AMe, where arborizations co-labeled with anti-ORC- and anti-MIP antisera. A possible role for MIP in light entrainment is supported also by injections of Rhyparobia maderae-specific MIP-2, which generated an all-advance phase-response curve late at night. Future experiments will challenge our hypothesis that GABA-, MIP- and AT-ir neurons provide ipsilateral light entrainment to all glomeruli, while MIP- and ORC-ir neurons carry contralateral light entrainment to the AMe's interglomerular region, either delaying or advancing AMe neurons light-dependently.

Acetylcholine release and choline uptake by cuttlefish (Sepia officinalis) optic lobe synaptosomes.

Acetylcholine (ACh), which is synthesized from choline (Ch), is believed to hold a central place in signaling mechanisms within the central nervous system (CNS) of cuttlefish (Sepia officinalis) and other coleoid cephalopods. Although the main elements required for cholinergic function have been identified in cephalopods, the transmembrane translocation events promoting the release of ACh and the uptake of Ch remain largely unsolved. The ACh release and Ch uptake were quantitatively studied through the use of in vitro chemiluminescence and isotopic methods on a subcellular fraction enriched in synaptic nerve endings (synaptosomes) isolated from cuttlefish optic lobe. The ACh release evoked by K+ depolarization was found to be very high (0.04 pmol ACh.s(-1).mg(-1) protein). In response to stimulation by veratridine, a secretagogue (a substance that induces secretion) that targets voltage-gated Na+ channels, the release rate and the total amount of ACh released were significantly lower, by 10-fold, than the response induced by KCl. The high-affinity uptake of choline was also very high (31 pmol Ch.min(-1).mg(-1) protein). The observed ACh release and Ch uptake patterns are in good agreement with published data on preparations characterized by high levels of ACh metabolism, adding further evidence that ACh acts as a neurotransmitter in cuttlefish optic lobe.

Comparative effects of aluminum and ouabain on synaptosomal choline uptake, acetylcholine release and (Na+/K+)ATPase.

Closing the gap between adverse health effects of aluminum and its mechanisms of action still represents a huge challenge. Cholinergic dysfunction has been implicated in neuronal injury induced by aluminum. Previously reported data also indicate that in vivo and in vitro exposure to aluminum inhibits the mammalian (Na(+)/K(+))ATPase, an ubiquitous plasma membrane pump. This study was undertaken with the specific aim of determining whether in vitro exposure to AlCl(3) and ouabain, the foremost utilized selective inhibitor of (Na(+)/K(+))ATPase, induce similar functional modifications of cholinergic presynaptic nerve terminals, by comparing their effects on choline uptake, acetylcholine release and (Na(+)/K(+))ATPase activity, on subcellular fractions enriched in synaptic nerve endings isolated from rat brain, cuttlefish optic lobe and torpedo electric organ. Results obtained show that choline uptake by rat synaptosomes was inhibited by submillimolar AlCl(3), whereas the amount of choline taken up by synaptosomes isolated from cuttlefish and torpedo remained unchanged. Conversely, choline uptake was reduced by ouabain to a large extent in all synaptosomal preparations analyzed. In contrast to ouabain, which modified the K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions, AlCl(3) induced reduction of stimulated acetylcholine release was only observed when rat synaptosomes were challenged. Finally, it was observed that the aluminum effect on cuttlefish and torpedo synaptosomal (Na(+)/K(+))ATPase activity was slight when compared to its inhibitory action on mammalian (Na(+)/K(+))ATPase. In conclusion, inhibition of (Na(+)/K(+))ATPase by AlCl(3) and ouabain jeopardized the high-affinity (Na(+)-dependent, hemicholinium-3 sensitive) uptake of choline and the Ca(2+)-dependent, K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions. The effects of submillimolar AlCl(3) on choline uptake and acetylcholine release only resembled those of ouabain when rat synaptosomes were assayed. Therefore, important differences were found between the species regarding the cholinotoxic action of aluminum. The variability of (Na(+)/K(+))ATPase sensitivity to aluminum of cholinergic neurons might contribute to their differential susceptibility to this neurotoxic agent.

Dopamine modulates synaptic activity in the optic lobes of cuttlefish, Sepia officinalis.

The effects of dopamine on spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs) in three different classes of neurones within the optic lobe of cuttlefish were investigated using whole-cell voltage clamp techniques in a slice preparation. The neuronal types were centrifugal and amacrine neurones, located in the inner granular cell layer, and medullar interneurones, located within the central medulla of the optic lobes. The results demonstrate that bath application of dopamine (50 microM) reversibly reduced both the frequency and amplitude of sEPSCs and of sIPSCs in these optic lobe neurones. The inhibitory effects of DA were dose-dependent and neither D1- nor D2-like receptors appear to be implicated, but probably D4-like receptors are involved in these actions. By pre-applying tetrodotoxin (TTX, 0.5 microM), to block action potential-dependent EPSCs and IPSCs, it is shown that dopamine has no effect on the amplitude, frequency or decay time constant of the mEPSCs or mIPSCs. The results are the first to identify a specific physiological action of dopamine on cephalopod brain activity, they indicate that this effect is probably presynaptic to the specific classes of cells recorded from, and they provide information on the pharmacological profile of the receptors involved. The widespread inhibitory effect of dopamine on the activity of cuttlefish optic lobe neurones is discussed in the context of comparable data from vertebrate preparations and the actions of other neuromodulators in the cuttlefish brain.

Cholinergic and glutamatergic spontaneous and evoked excitatory postsynaptic currents in optic lobe neurons of cuttlefish, Sepia officinalis.

Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from two different classes of neurons in the optic lobes of the cuttlefish brain and their synaptic activities analyzed and compared. The cell types were as follows: efferent centrifugal neurons, with cell bodies in the inner granule layer and axons projecting to the retina, and interneurons local to the medulla. For both neuronal groups, the sEPSCs reversal potentials were around 0 mV and there were no significant differences in their mean amplitude and rise times. However, the sEPSCs from the centrifugal neurons had a significantly higher frequency and faster decay time constant than those recorded from the medulla. Tetrodotoxin (TTX) reduced the mean frequency of the sEPSCs from both the medulla and centrifugal neurons by 69.66 +/- 4.05% and 57.80 +/- 3.87%, respectively, implying that more than half of these excitatory synaptic inputs were due to action potential-mediated release of neurotransmitter. Pharmacological examination revealed that the centrifugal neurons were driven by spontaneous synaptic inputs mediated by glutamatergic and cholinergic receptors, because co-application of the glutamate antagonist kynurenic acid (KYNA) and the nicotinic antagonist mecamylamine hydrochloride (MCM) resulted in complete blockade of these excitatory inputs. For the medulla neurons, the synaptic inputs were driven by glutamate and other transmitters yet to be identified. Evoked EPSCs (eEPSCs) were recorded from both types of neurons by stimulating the appropriate optic nerve bundles; in centrifugal neurons, the eEPSCs were blocked by co-application of KYNA and MCM, whereas in the medulla neurons, KYNA alone either totally or partially blocked the eEPSCs.

Acute hypoxic hypoxia alters GABA(A) receptor modulation by allopregnanolone and pentobarbital in embryonic chick optic lobe.

Using a previously developed model of acute normobaric hypoxic hypoxia on chick embryos, here we studied at embryonic day 12 the in vitro effect of two positive allosteric modulators of GABA binding, the barbiturate sodium pentobarbital and the neurosteroid allopregnanolone. In both cases an increase in E(max) values in membranes obtained from hypoxic embryos was observed. Studies of GABA-gated chloride influx showed that there were no differences in maximal chloride uptake between hypoxic and control membranes. We have already demonstrated that maximal density of GABA binding sites was decreased after hypoxia, suggesting that each of the remaining GABA(A) receptors display a greater chloride flux than controls. To further characterize GABA(A) receptor alterations, GABA-gated chloride influx modulated by the above barbiturate and neurosteroid was determined, finding that E(max) values were increased 60% and 42%, respectively. The increase in Cl(-) influx per receptor subsequent to hypoxic trauma, and the enhancement in the modulatory properties studied, may mediate neuronal damage by potential changes in subunit interaction at the GABA(A) receptor level.

Serotonin sets the day state in the neurons that control coupling between the optic lobe circadian pacemakers in the cricket Gryllus bimaculatus.

The bilaterally paired optic lobe circadian pacemakers of the cricket Gryllus bimaculatus mutually exchange photic and circadian information to keep their activity synchronized. The information is mediated by a neural pathway, consisting of the so-called medulla bilateral neurons, connecting the medulla areas of the two optic lobes. We investigated the effects of serotonin on the neural activity in this coupling pathway. Spontaneous and light-induced electrical activity of the neurons in the coupling pathway showed daily variations, being more intense during the night than the day. Microinjection of serotonin or a serotonin-receptor agonist, quipazine, into the optic lobe caused a dose- and time-dependent inhibition of spontaneous and light-induced responses, mimicking the day state. The amount of suppression was greater and the recovery from the suppression occurred faster during the night. Application of metergoline, a non-selective serotonin-receptor antagonist, increased spontaneous activity and light-evoked responses during both the day and the night, with higher effect during the day. In addition, metergoline effectively attenuated the effects of serotonin. These facts suggest that in the cricket's optic lobe, serotonin is released during the daytime and sets the day state in the neurons regulating coupling between the bilaterally paired optic lobe circadian pacemakers.

In ovo chronic neurosteroid treatment affects the function and allosteric interactions of GABA(A) receptor modulatory sites.

We investigated the effects of in ovo chronic administration of the endogenous neurosteroid epipregnanolone (5beta-pregnan-3beta-ol-20-one) on the GABA(A) receptor complex present in chick optic lobe synaptic membranes. Chronic epipregnanolone treatment failed to exert any effect on the chick optic lobe total protein content and wet weight at the different doses tested. [3H]Flunitrazepam control binding remained unaltered after neurosteroid exposure, however, the positive allosteric modulation of this ligand by 4 microM allopregnanolone was reduced in a dose-dependent manner by neurosteroid treatment. Embryo exposure to 30 microM epipregnanolone decreased allopregnanolone EC(50) and E(max) values. Analyses of saturation binding isotherms disclosed that such administration had no effect on K(d) and B(max) values for [3H]flunitrazepam and [3H]GABA binding. [3H]GABA binding modulation disclosed an increase in allopregnanolone EC(50) value with a decrease in its E(max) value. Diazepam EC(50) and E(max) values were enhanced, while low affinity sodium pentobarbital EC(50) value was reduced by epipregnanolone treatment. The investigation of the GABA(A) receptor function revealed that administration of this neurosteroid reduces the efficacy of GABA to induce 36Cl(-) influx into microsacs prepared from chick optic lobe. These results indicate that endogenous neurosteroid epipregnanolone chronically administered in ovo produces homologous uncoupling between steroid modulatory sites, and those corresponding to benzodiazepine and GABA receptors. Thus epipregnanolone is able to induce heterologous changes in the allosteric linkage between benzodiazepine and barbiturate modulatory sites, and the GABA receptor site. Taken jointly with results on epipregnanolone enhancing effects on [3H]flunitrazepam and [3H]GABA binding, in the context of its endogenous synthesis, our present findings support this neurosteroid as the endogenous modulator of GABA(A) receptor sites and function during chick optic lobe development.

Ca(2+) dynamics in synaptosomes isolated from the squid optic lobe.

Synaptosomes from the optic lobes of squid (Loligo forbesi) were prepared by homogenization and allowed to settle onto glass coverslips. Synaptosomes were loaded with Ca(2+) sensitive dyes (Fura-2 AM, Calcium Green-1 AM and Calcium Green-5N AM), visualized by light microscopy and Ca(2+) sensitive fluorescence signals recorded and analyzed. With Fura-2, resting Ca(2+) was found to be 80 nM (n = 10, SEM 5.7). Addition of K(+) (30 mM), caffeine (3 mM) and thapsigargin (10 microM) evoked transient increases in cytoplasmic Ca(2+). Addition of BAPTA-AM (20 microM) decreased intrasynaptosomal free Ca(2+). Similar results were obtained with Calcium Green-1 AM but not with Calcium Green-5N AM. We conclude that synaptosomes from the squid optic lobe posses intact membranes and mechanisms to regulate intrasynaptosomal free [Ca(2+)], as well as caffeine sensitive Ca(2+) stores. The results of this study are discussed with respect to the role of Ca(2+) in presynaptic protein synthesis.

Treatment with 6-hydroxydopamine in planaria (Dugesia gonocephala s.l.): morphological and behavioral study.

Morpho-functional and behavioral effects of exposure to 6-hydroxydopamine (OHDA)-HCI (24 microg/ml per day for 24 h and 7 days) were studied in planarias (Dugesia gonocephala s.l.). Exposure to 6-OHDA-HC1 for 24 h produced hypokinesia of the specimens. These behavioral changes were more pronounced, leading to complete immobility, after 7 days of exposure to the neurotoxin. Moreover, specimens exposed to 6-OHDA-HCI for 24 h and 7 days failed to show any behavioral response to nomifensine, thus furnishing evidence of the damage of presynaptic dopamine terminals. Exposure to 6-OHDA-HCl for 24 h significantly reduced cathecolamine content in neuropil region, as demonstrated by histochemistry, and electron-dense presynaptic vesicles, as observed on electron microscopy examination. All these alterations were significantly more pronounced and were accompanied by swelling and strong increase of electron-density in cytoplasm of numerous neurons after exposure to the neurotoxin for 7 days. This appears to be the first demonstration of the neurotoxic effects of 6-OHDA-HCI in flatworms.

Modulatory action of zinc on GABA(A) receptor complex during avian CNS development.

In the present work, we studied the effect of zinc on GABA(A) receptor complex at three developmental stages of chick optic lobe (embryonic day 14, post-hatching day 1, and adulthood), in order to investigate the role of this cation in central nervous system (CNS) functional maturation. It was demonstrated that zinc exerts an inhibitory modulation of both GABA binding and GABA-gated chloride flux in a concentration-dependent manner with maximal effects at 100 microM zinc concentration. Maximal inhibition was higher at the embryonic stage and declined thereafter, disclosing minimal values at the adult stage. The effect of zinc on saturation GABA binding experiments performed at embryonic day 14 demonstrated that the cation decreased the maximal number of binding sites (B(max)) from 7. 53 +/- 1.06 pmol/mg protein to 4.63 +/- 0.53 pmol/mg protein, in the absence and presence of 100 microM zinc, respectively, while the dissociation constant (K(d)) remained unchanged. Analysis of the GABA concentration-effect curve at the embryonic stage revealed that the addition of 100 microM zinc decreased E(max) values for GABA stimulation of chloride uptake from 26.46 +/- 2.64% to 16.40 +/- 1. 96%, while EC(50) values were unaffected. In conclusion, our results suggest that zinc acts as a non-competitive inhibitor of both GABA binding and GABA responses during avian CNS development, with its effect inversely related to age.

Biosynthesis of progesterone derived neurosteroids by developing avian CNS: in vitro effects on the GABAA receptor complex.

It has been demonstrated in different vertebrate species that the GABAA receptor complex is modulated by certain steroids. Theses results prompted work on the synthesis of these neurosteroids in the Central Nervous System. However, there are scarcely any studies analyzing their production or their modulatory effects on this receptor during development. In this work, the biosynthesis of [14C]progesterone metabolites as well as the characterization of their in vitro effects on the GABAA receptor complex in developing chick optic lobe were investigated. Studies on progesterone metabolism indicated that this steroid was converted to 5 beta-pregnanedione, 5 beta-pregan-3 beta-ol-20-one, and a 20-hydroxy derivative. Radioactive progesterone was completely metabolized at early embryonic stages, and a great proportion of 5 beta-pregnanedione was converted to 5 beta-pregnan-3 beta-ol-20-one. Thus, it seems that some of the steroidogenic activities present in chick optic lobe are age-dependent, though greater at embryonic stages. Results from in vitro modulation of [3H]flunitrazepam binding by 5 beta-pregnan-3 beta-ol-20-one indicated that this steroid produces a one-component-concentration dependent enhancement above control binding. 5 beta-pregnan-3 beta-ol-20-one EC50 values were 0.195 +/- 0.049, 0.101 +/- 0.017, 0.147 +/- 0.009, and 0.569 +/- 0.114 microM, and Emax were 22.37 +/- 1.57, 23.67 +/- 4.02, 29.01 +/- 1.08, and 15.11 +/- 2.67% at embryonic days 11, 14, hatching, and postnatal day 21, respectively. In conclusion, the biosynthesis of 5 beta-pregnan-3 beta-ol-20-one from progesterone in developing chick optic lobe, together with its ability to modulate the GABAA receptor present in such tissues, suggests a physiological role of this neurosteroid in developing avian Central Nervous System.

Directionality and inhibition in crayfish tangential cells.

The purpose of this study was to characterize the inhibitory mechanism(s) associated with directionally selective motion detection (DS) in nonspiking tangential cells of crayfish optic lobe. The experiments employed intracellular recording of synaptic potentials elicited with sinewave gratings and pharmacological techniques. Previous studies established that tangential cells are subject to bicuculline-sensitive GABA-mediated inhibition. In this study DS was reduced by 90% by bicuculline. The reduction in DS was accompanied by a substantial increase in the response to null-direction motion. Bicuculline also altered the response to pulses of illumination. The magnitude and time course of inhibition were derived from the time varying difference between the control light response and that elicited during bicuculline perfusion. Both the inhibitory delay (relative to excitation) and the inhibitory amplitude are close to the expectations of a linear model of DS. The inhibition is not prolonged with respect to excitation but its risetime is approximately 2.5 times longer. The result implies a longer time constant in the inhibitory pathway relative to that in the excitatory pathway and places limits on the frequency response of inhibition and DS. The velocity-dependence of DS is related to the time course of inhibition. The stimulus drift velocity eliciting maximum directionality is inversely proportional to the inhibitory delay. Bicuculline did not influence orientation selectivity. It is concluded that the quantitative features of bicuculline-sensitive, GABA-mediated inhibition are consistent with a linear model of DS.

Comparative modulation by 3 alpha,5 alpha and 3 beta,5 beta neurosteroids of GABA binding sites during avian central nervous system development.

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABAA receptor complex. The presence of a 3 alpha-hydroxyl group and a 5 alpha-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [3H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3 alpha-hydroxy,5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 beta-hydroxy,5 beta-pregnan-20-one (3 beta,5 beta-P). Both steroids were found able to enhance [3H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3 alpha,5 alpha-P had greater potency (EC50 0.22 microM) and enhancing effect (Emax: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABAA receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3 alpha,5 alpha-P by its 3 beta,5 beta isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5 alpha-reduced progesterone metabolites present since early development, while 3 alpha,5 alpha-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5 beta-steroids in early avian development, we propose that 3 beta,5 beta-P, instead of the classical potent 3 alpha,5 alpha-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.

T3 treatment increases mitosis, then bax expression and apoptosis in the optic lobe of the chick embryo.

We investigated T3 effects on cell proliferation and apoptosis in the optic lobe of the chick embryo between embryonic days (E) 6 and 11. Injection of T3 into the yolk increased [3H]thymidine incorporation between E7 and E9. This increased mitosis was followed by altered timing and degree of apoptosis during E9-11. In T3-treated embryos a marked increase in apoptosis occurred on E10, coincident with increased levels of mRNA encoding Bax, a pro-apoptotic protein. By E11, the overall morphology and total number of cells in each layer of the optic lobe were not different in control and treated embryos. Thus, although T3 transiently increases cell number, a homeostatic mechanism enters into play re-adjusting the balance between cellular proliferation and cell death.

Ethanol alters brain phospholipid levels which correlate with altered brain morphology.

The effects of embryonic exposure on brain phospholipid levels were studied by injecting various concentrations of ethanol into fertile chicken eggs at 0 days of development. At 18 days of development, the levels of total phospholipids and various phospholipid classes were assayed in brain tissue and correlated to neuron densities within the cerebral hemispheres and the optic lobes. Although ethanol concentrations ranging from 0 to 3700 microns/Kg egg wt. failed to influence either total brain weight or total brain phospholipid levels, ethanol-induced changes in the levels of individual phospholipid classes were observed. When injected with 7 microns of ethanol/Kg egg wt., a 2- to 3-fold increase in brain phosphatidylethanolamine (PE) levels were observed with reduced levels of brain phosphatidylcholine (PC) and brain sphingomyelin (SP). When injected with 74 microns of ethanol/Kg egg wt., ethanol-induced increases in brain phosphatidylserine (PS) and PE were observed with ethanol-induced decreases in brain PC and SP. Cell fractionation studies demonstrated ethanol-induced increases in brain PE and PS and ethanol-induced decreases in brain PC and SP in nuclear, mitochondrial, and microsomal membranes. These ethanol-induced alterations in brain phospholipid profiles correlated with ethanol-induced reductions in neuron densities within the cerebral hemispheres and optic lobes.

The intrinsic electrophysiological characteristics of fly lobula plate tangential cells: II. Active membrane properties.

The voltage-gated currents in the fly lobula plate tangential cells were examined using the switched electrode voltage clamp technique. In CH cells, two currents were identified (Figs. 1, 2): a slow calcium inward current and a delayed rectifying, noninactivating potassium outward current. HS and VS cells appear to possess similar currents to CH cells, but in addition, exhibit a fast-activating sodium inward current and a sodium-activated potassium outward current (Figs. 3, 4). While the delayed rectifying potassium current in all three cell classes is responsible for the observed outward rectification described previously (Borst and Haag, 1996), the sodium inward current produced the fast and irregular spikelike depolarizations found in HS and VS cells but not in CH cells: When the sodium current is blocked by either. TTX or intracellular QX314, no more action potentials can be elicited in HS cells under current-clamp conditions (Fig. 5). As is demonstrated in HS cells, space clamp conditions are sufficient to suppress synaptically induced action potentials (Fig. 6). The currents described above were incorporated with the appropriate characteristics into compartmental models of the cells (Fig. 7, 8). The anatomical and electrically passive membrane parameters of these cells were determined in a preceding paper (Borst and Haag, 1996). After fitting the current parameters to the voltage-clamp data (Fig. 9), the model cells qualitatively mimicked the fly tangential cells under current clamp conditions in response to current injection (Fig. 10). The simulations demonstrated that the electrical compactness seen in the HS and VS cells, either in passive models or in active models during continuous hyperpolarization, decreased significantly in the active models during continuous depolarization (Fig. 11). Active HS models reproduce the frequency-dependent amplification of current injected into their axon (Fig. 12).

Neurotransmitters regulate rhythmic size changes amongst cells in the fly's optic lobe.

Axon calibre in monopolar cells L1 and L2 of the fly's lamina can change dynamically. Swelling by day, L2 exhibits a daily rhythm of changing size apparently mediated by wide-field LBO5HT and PDH cells. L1/L2 axon profiles were measured planimetrically in the housefly, Musca domestica, from 1 microns cross sections. Four hours after injecting 80-100 nl of 1.25 x 10(-4) M 5-HT into the optic lobe, L1's axon swelled but L2's did not, whereas 2.2 x 10(-5) M of PDH enlarged both axons. Similar to 5-HT, 1.63 x 10(-4) M histamine (the photoreceptor transmitter) enlarged L1 but not L2, mimicking light exposure, while 1.7 x 10(-4) M glutamate and 1.94 x 10(-4) M GABA both decreased L1 and L2. 2.5 x 10(-4) M of 5,7-dihydroxytryptamine decreased L2 and, somewhat, L1, an effect attributable to the loss of LBO5HT neurites. Twenty four hours after cutting LBO5HT and PDH commissural pathways, L1 and L2 both shrank. Apparently, L2's size depends on either LBO5HT or sufficient 5-HT, and L1 and L2 have different response ranges to 5-HT. Responses to PDH imply that daytime PDH release drives a circadian rhythm, enlarging L1 and L2.