Ultrastructural correlates of luminescence in Porichthys photophores. II. Effects of metabolic inhibitors.

Prolonged, bright luminescent glows in Porichthys photophores are elicited by administration of 2,4-dinitrophenol (DNP) and potassium cyanide (KCN). Ultrastructural alterations of varicose nerve endings precede photocyte changes during such luminescent activity. Common alterations of nerve profiles include mitochondrial disruptions, flattening and depletion of synaptic vesicles, formation of large vacuolar cisternae, and invaginations in the contour of axolemma. Protracted luminescent activity in response to DNP results in depletion of photocyte vesicle material while vesicle and ER membranes accumulate and coil inside coalesced vesicle pools, and photocyte microvilli disappear completely. Although similar photocyte alterations are initially observed in KCN treated luminescing photophores, the early extinction of the response to KCN is related to deleterious, irreversible effects of this chemical on photocytes. These observations, along with some pharmacological manipulations, indicate that at least DNP acts initially and primarily on neural structures, probably the mitochondria, to induced transmitter release and consequent photocyte activity. Based on this and earlier studies, a chain of subcellular events leading to light emission of Porichthys photophores is proposed and discussed.     

Ultrastructural correlates of luminescence in Porichthys photophores. I. Effects of spinal cord stimulation and exogenous noradrenaline.

Light emission induced by electrical stimulation of the spinal cord is associated with ultrastructural alterations of the varicose endings, photocyte organelles and membranes in Porichthys photophores. Changes in nerve profiles appear first and include alterations of the shape, number and distribution of synaptic vesicles, as well as invaginations of the axolemma and mitochondrial swellings. Protocyte vesicles become markedly coalesced and their membranes are incorporated inside vesicular pools, whereas photocyte microvilli become sharply reduced at the cell periphery. Luminescence obtained by administration of noradrenaline is accompanied by similar, but more acute changes in the ultrastructure of photocytes, whereas no marked alteration of nerve profiles is noted after this treatment. These and other observations, such as the presence of specialized synaptic contacts, are discussed in terms of neural activity and processes within the photocytes which may lead to light production.     

Uptake and release of [3H]-serotonin in photophores of the midshipman fish, Porichthys notatus

A kinetic analysis of [3H]-5-HT uptake in the photocytes of the photophores of Porichthys notatus revealed a high affinity (Km: 1.71 X 10(-7] and low affinity component (Km: 1.10 X 10(-5) M). The high affinity uptake was sodium- and potassium-dependent but largely insensitive to temperatures between 0 and 20 C. Ouabain (5 X 10(-3) M) and dinitrophenol (10(-3) M) reduced uptake significantly. DMI, imipramine and fluoxetine, in that order of potency, greatly inhibited [3H]-5-HT uptake. Noradrenaline and adrenaline reduced uptake in a non-competitive manner, while dopamine, tryptophan, 5-hydroxytryptophan and Cypridina luciferin had little or not effect on uptake. Non-facilitated luminescent responses to electrical stimulation were accompanied by release of [3H]-5-HT accumulated in the photocytes. Facilitatory luminescence excitation consistently failed to induce the release of [3H]-5-HT. Electrical and adrenaline (10(-5) M) stimulation of photophores after [3H]-5-HT release has occurred, failed to elicit any additional luminescent response. The photophores were responsive to KCN (10(-3) M) under these conditions. The results indicate that a specific carrier-mediated transport system is responsible for photocytic [3H]-5-HT uptake, and that release of photocytic [3H]-5-HT is stringently regulated and followed by inhibition of luminescence excitability.     

THE ORGANIZATION AND INNERVATION OF THE LUMINESCENT ORGAN IN A FIREFLY, PHOTURIS PENNSYLVANICA (COLEOPTERA)

The organization of the luminescent organ of an adult firefly has been studied with the electron microscope, and particular attention has been given to the disposition of nerve terminals within the organ. The cytological structure of the cells of the tracheal system, the peripheral and terminal axons, the photocytes and the cells of the dorsal ("reflecting") layer is described. Previous observations on the peripheral course of nerve branches alongside the tracheal trunks at the level of the dorsal layer and photocyte epithelium have been confirmed, and specialised nerve endings containing axoplasmic components structurally identical with "synaptic vesicles" and "neurosecretory droplets" have been identified, not in association with the surface of the photocytes, but lying between the apposed surfaces of two components of the tracheal epithelium: the tracheal end-cell and the tracheolar cell. These cytological findings are discussed in terms of available biochemical and physiological evidence concerning the mechanism of light emission in the firefly, especially with respect to the possible role of chemical "transmitter" action in triggering a response in a luminescent effector system.     

Postnatal development of chronotropic responses to chemical and electrical stimulation of adrenergic nerve endings in the sinoatrial node of the heart of the albino rat

The positive chronotropic response to stimulation of adrenergic nerve endings in the sinoatrial node was studied in isolated atria from the hearts of rats of different ages. Dimethylphenylpiperazinium (DMPP) was used for chemical stimulation and transmural stimulation of the sinoatrial node region as electrical stimulation; in both cases noradrenaline is released from the nerve endings. With both stimulation methods, postnatal development was recorded in two phases. In the first phase, positive chronotropic responses are markedly increased and attained the maximum at the age of 14 days on using DMPP and of 24 days on using electrical stimulation. In the second phase, positive chronotropic responses diminish and at the age of about 45 days, with both stimulation methods, they become reduced to adult level. The first developmental phase can be attributed to an increase in the noradrenaline content of the nerve endings and the release of a larger amount of the transmitter during stimulation, together with an increase in the noradrenaline sensitivity of the cells of the sinoatrial node. It is not clear why positive chronotropic responses decrease in the second phase, when the noradrenaline content of the myocardial tissue continues to rise and pacemaker sensitivity to noradrenaline is not reduced.     

Evidence for a widespread involvement of NO in control of photogenesis in bioluminescent fish

Krönström, J. and Mallefet, J. 2009. Evidence for a widespread involvement of NO in control of photogenesis in bioluminescent fish. —Acta Zoologica (Stockholm) 91 : 474–483. The presence of nitric oxide synthase (NOS) and nerve fibres in the photophores of seven bioluminescent fish species (Hygophum benoiti, Myctophum punctatum, Electrona risso, Cyclothone braueri, Vinciguerria attenuata, Maurolicus muelleri and Porichthys notatus) with endogenous photocytes, were investigated. Antibodies directed against neuronal and inducible NOS (n and iNOS respectively) and NADPH‐diaphorase activity were used to reveal the locations of NOS, while antibodies directed against acetylated tubulin were used to visualize nerve fibres. The nNOS antibody labelled structures in all investigated photophores except in the organs from P. notatus. The photocytes of P. notatus showed NADPH‐diaphorase activity. In the myctophid species, NOS‐like immunoreactivity was found in small intracellular structures of the photocytes and in nerve fibres reaching the photocytes. nNOS‐positive fibres were also found among lens/filter cells in V. attenuata, and in M. muelleri the cytoplasm of lens/filter cells contained NOS‐like material. In C. braueri, a cell type located at a collecting chamber for luminous products in the photophore contained NOS‐like material. All photophores received an innervation reaching the photocytes, as well as other components including lens/filter areas. The results of this study comply with an involvement of nitric oxide in the control of bioluminescence in several fish species.     

Effect of prostaglandin E2 on the pressor response to periarterial stimulation and norepinephrine of the isolated perfused rabbit kidney.

The interaction of prostaglandin E2 (PGE2) and aspirin with the responses to peri-arterial stimulation (PS) and norepinephrine (NE) was studied in the isolated kidney of rabbit perfused through the renal artery at constant flow with Krebs' solution. NE and PS increased vascular perfusion pressure of kidney and caused a contraction on the isolated rabbit aortic strip superfused with the effluent from kidney. Addition of PGE2 to the perfusion medium decreased the PS-induced rise in perfusion pressure without changing the effect of exogenous NE. In contrast, addition of aspirin to the perfusion medium induced a potentiation of the response to PS but not to NE. These results suggest that PGE2 modulates the effect of PS probably by inhibiting the releases of NE from sympathetic nerve endings.     

Adaptive mechanisms of the noradrenergic cyclic AMP generating system in the limbic forebrain of the rat: adaptation to persistent changes in the availability of norepinephrine (NE).

Abstract— The noradrenergic cyclic AMP generating system in slices of the limbic forebrain of rats displays characteristics which are compatible with those of a central NE receptor. The cyclic AMP response to a K_max concentration of NE (concentration of NE which elicits maximal increase in the level of cyclic AMP) is significantly enhanced in slices from reserpinized animals, although the K_a value of NE (concentration of NE eliciting half-maximum response) was not significantly changed. Chemosympathectomy with 6-hydroxydopamine (6-OHDA) significantly enhanced the activity of the system to NE and isoproterenol but not to adenosine and reduced the K_a value for NE. The changes in the reactivity of the cyclic AMP generating system following 6-OHDA administration appear to be related to a decrease in the availability of NE and not to that of other neurotransmitters as protection by desipramine (DMI) of noradrenergic neurons against the neurotoxic action of 6-OHDA prevented the development of supersensitivity to NE. Conversely, and independent of the actual concentration of NE in brain tissue, a persistent increase in the availability of NE caused by prolonged MAO inhibition lead to a marked decrease in the reactivity of the cyclic AMP generating system. The results provide further evidence for a regulatory mechanism in the CNS involving the noradrenergic receptor that adapts its sensitivity to NE in a manner inversely related to the degree of its stimulation by the catecholamine.     

Reflector of the body photophore in lanternfish is mechanistically tuned to project the biochemical emission in photocytes for counterillumination

Lanternfish, a family Myctophidae, use ventro-lateral body photophores for camouflage of the ventral silhouette, a strategy called counterillumination. While other deep-sea fishes possess pigmented filters and silver reflectors to match sunlight filtering down through the depths, myctophids developed a blue-green reflector for this purpose. In this study, we showed in a lanternfish Diaphus watasei that the reflector comprised monolayered iridophores containing multilayered guanine crystals which enable high reflection with light interference colouration. Platelets shape in body photophores is an unique near-regular hexagonal, probably to allow the homogeneity of reflection angle of the luminescence from photocytes. Focus point of the parabola-like reflector is positioned on the photocytes that ensures the light produced from the photocytes is redirected to the ventral direction. In vitro luminescence reaction using purified luciferase and the substrate coelenterazine showed the light emission at λ_max 454 nm, while reflection spectra of the iridophores exhibit peaks at longer wavelength, which accomplish to alter the luminescence emitted from photocytes to longer wavelength to fit the mesopelagic light environment. Taken together, we revealed multiple mechanistic elaborations in myctophid body photophores to achieve effective control of biochemical luminescence for counterillumination.     

神经生长因子在6－OHDA化学性去交感神经中的保护作用

本实验用６－ＯＨＤＡ造成成年小白鼠领下腺化学性去交感神经,观察了神经生长因子对该神经的保护作用。６－ＯＨＤＡ（１５ｍｇ／ｋｇｉｐ）处理后２４ｈ腺体内去甲肾上腺素（ＮＥ）含量降至正常水平的２％以下。若在６－ＯＨＤＡ处理同时开始多次给予神经生长因子（ＮＧＦ）,则ＮＥ残留量明显提高。减小６－ＯＨＤＡ剂量至１０ｍｇ／ｋｇ,ＮＥ残留量增加,同时ＮＧＦ的作用亦较用６－ＯＨＤＡ１５ｍｇ／ｋｇ时更为显著。若提前２４ｈ给予ＮＧＦ,尽管仍显著提高ＮＥ残留量,但程度却显然低于与６－ＯＨＤＡ同时给予者。以上结果表明外源性ＮＧＦ对６－ＯＨＤＡ造成交感神经化学性损毁有保护作用,此作用与神经受损的严重程度以及ＮＧＦ处理时间有关。     

Effects of temperature on alpha adrenoceptors in limb veins: role of receptor reserve

In cutaneous veins of the dog, cooling augments the response to sympathetic nerve stimulation and exogenous norepinephrine (NE). The postjunctional alpha adrenoceptors in this blood vessel belong to both the alpha 1 and alpha 2 subtypes. Cooling augments alpha 2-adrenergic responses (presumably because of an increased receptor affinity), but depresses alpha 1-adrenergic responses (presumably because of a direct inhibitory effect on the contractile process). When agonists of high efficacy such as NE or phenylephrine are used, an alpha 1-adrenoceptor reserve is present that buffers the response from the inhibitory effect of cooling. This allows the potentiating effect of cold on the alpha 2-adrenergic component of the response to catecholamines to predominate, and the contractile response to exogenous NE and sympathetic nerve stimulation is augmented. By contrast, in deep veins of the limb, cold reduces the contractions evoked by alpha 1- and alpha 2-adrenergic activation. This can be explained best by the absence of a receptor reserve for alpha 1-adrenergic agonists of high efficacy, combined with a reduced density of postjunctional alpha 2 adrenoceptors.     

Effect of prostaglandin E2on the pressor response to peri-arterial stimulation and norepinephrine of the isolated perfused rabbit kidney

The interaction of prostaglandin E₂ (PGE₂) and aspirin with the responses to peri-arterial stimulation (PS) and norepinephrine (NE) was studied in the isolated kidney of rabbit perfused through the renal artery at constant flow with Krebs' solution. NE and PS increased vascular perfusion pressure of kidney and caused a contraction on the isolated rabbit aortic strip superfused with the effluent from kidney. Addition of PGE₂ to the perfusion medium decreased the PS-induced rise in perfusion pressure without changing the effect of exogenous NE. In contrast, addition of aspirin to the perfusion medium induced a potentiation of the response to PS but not to NE. These results suggest that PGE₂ modulates the effect of PS probably by inhibiting the release of NE from sympathetic nerve endings.     

Insulin inhibits norepinephrine overflow from peripheral sympathetic nerve ending.

The effects of insulin on peripheral nervous system are unknown. We therefore studied the effects of insulin on sympathetic nerve activity in isolated mesenteric arteries of Sprague-Dawley rats. The overflow of norepinephrine (NE) by electrical stimulation was used as the index of sympathetic nervous system activity. Insulin (0.5 to 1U/l) decreased the NE release in a dose-dependent fashion. This inhibitory effect was, however, reversed by either 5 x 10(-5)M cocaine or 5 x 10(-4)M ouabain treatment. Thus, we postulate that insulin attenuates NE overflow from peripheral sympathetic nerve endings, probably due to enhanced NE reuptake.     

The photophore morphology of Selenoteuthis scintillans Voss and other lycoteuthids (Cephalopoda: Lycoteuthidae)

Females and juveniles of Selenoteuthis scintillans have photophores of several structural types, distributed on the tentacles and eyeballs, and within the mantle cavity and tail. Three distinct photophore types can be recognized on the basis of their accessory structures, though their photocytes are identical. The tail and some tentacular photophores (Type 1) lack any accessory optical structures; other tentacular and abdominal photophores (Type 2) have collagenous diffusing fibres; the anal and ocular photophores (Type 3) have a variety of iridosomes but no collagen. The distal tentacular organ is a double structure composed of a unit each of Type 1 and Type 2. Ocular photophores 1 and 5 are also double structures, composed of two Type 3 units. The photophores closely resemble in structure those of Lycoteuthis diadema. The photocytes have a marked fluorescence and luminesce on treatment with dilute hydrogen peroxide. The bio-luminescence intensity of the tail organ may be modified by chromatophore movements and has a blue-green spectral emission.
The photophores of juvenile Lampadioteuthis megaleia are similar in structure to those of Selenoteuthis but somewhat less complex. A comparison between the morphology of the photophores of lycoteuthid and enoploteuthid squids emphasizes the close similarity between the two families. At the ultrastructural level, certain photophores of both families have very characteristic microvillous blood vessels associated with the photocytes.     

Characteristics of electrical activity in different sections of nerve endings in the frog

In experiments on frog sartorius neuromuscular preparations, the evoked electrical responses of nerve endings were recorded by extracellular microelectrodes. It was shown that in proximal parts of the nerve ending, the three-phase response (+ - +) with a high amplitude negative phase occurred due to motor nerve stimulation. With movement of the extracellular electrode in distal direction a certain increase of the initial positive phase and a significant decrease of the negative one were observed. At the end of the terminal that response transformed to the monophasic one (+). On local iontophoretic application of tetrodotoxin (TTX) to the recording site two components of the nerve ending response were revealed: TTX-insensitive and TTX-sensitive. A significant decrease of the TTX-sensitive component occurred along the course of the nerve ending. That component was absent from the distal synaptic areas. It is concluded that in frog nerve ending, the action potential propogates with decrement while depolarization of the end parts of the terminal is passive in nature.     

Simultaneous monitoring of acetylcholine and catecholamine release in the in vivo rat adrenal medulla

To simultaneously monitor acetylcholine release from pre-ganglionic adrenal sympathetic nerve endings and catecholamine release from post-ganglionic adrenal chromaffin cells in the in vivo state, we applied microdialysis technique to anesthetized rats. Dialysis probe was implanted in the left adrenal medulla and perfused with Ringer's solution containing neostigmine (a cholinesterase inhibitor). After transection of splanchnic nerves, we electrically stimulated splanchnic nerves or locally administered acetylcholine through dialysis probes for 2 min and investigated dialysate acetylcholine, choline, norepinephrine and epinephrine responses. Acetylcholine was not detected in dialysate before nerve stimulation, but substantial acetylcholine was detected by nerve stimulation. In contrast, choline was detected in dialysate before stimulation, and dialysate choline concentration did not change with repetitive nerve stimulation. The estimated interstitial acetylcholine levels and dialysate catecholamine responses were almost identical between exogenous acetylcholine (10 microM) and nerve stimulation (2 Hz). Dialysate acetylcholine, norepinephrine and epinephrine responses were correlated with the frequencies of electrical nerve stimulation, and dialysate norepinephrine and epinephrine responses were quantitatively correlated with dialysate acetylcholine responses. Neither hexamethonium (a nicotinic receptor antagonist) nor atropine (a muscarinic receptor antagonist) affected the dialysate acetylcholine response to nerve stimulation. Microdialysis technique made it possible to simultaneously assess activities of pre-ganglionic adrenal sympathetic nerves and post-ganglionic adrenal chromaffin cells in the in vivo state and provided quantitative information about input-output relationship in the adrenal medulla.     

The comparative morphology of the photophores of the squid Pyroteuthis margaritifera (Cephalopoda: Enoploteuthidae)

Pyroteuthis margaritifera has morphologically distinctive photophores on the tentacles, eyeball and in the mantle cavity. The photogenic tissue in each photophore is identical, has a blue-green fluorescence and luminesces on treatment with dilute hydrogen peroxide. The photocytes frequently contain organized fibrillar material akin to that in the photocytes of certain other cephalopods. Several different types of blood vessel are present among the photocytes, including some, apparently restricted to the photophores, with a microvillous endothelium. Haemocyanin is present not only within identifiable blood vessels but also in some intercellular spaces.
On the basis of their characteristic optical systems the photophores can be separated into three types: (1) tentacular; (2) ocular and anal; (3) branchial and median abdominal. The tentacular photophores have collagenous reflector and light guide systems and the median ones are double organs. The ocular and anal organs do not have collagenous optical structures but an elaborate variety of reflective iridosomes. Those in the aperture of the photophores appear to act as interference filters. The branchial and abdominal organs have iridosomes as the major reflective tissue but collagenous fibrils function as light guides in the aperture of these organs and their emission is diffuse rather than collimated.     

Adrenergic control of luminescence and cellular metabolism in Porichthys isolated luminous organs

1. Isolated photophores from the luminous fish Porichthys produce light in response to adrenaline and the metabolic inhibitors iodoacetic acid (IAA) or potassium cyanide (KCN).2. We attempted to analyse the interactions of cellular metabolism and adrenergic stimulation of the photogenic cells.3. Photophores were treated with IAA in the presence of pyruvate. In these conditions, IAA does inhibit glycolysis without inducing any luminescent activity of the cells.4. Similarly, other photophores were incubated with KCN in the presence of glucose, in order to inhibit cellular respiration while keeping the luminous system inactive.5. We observed that adrenergic stimulation of these photophores remained effective and induced a light emission, demonstrating that glycolytic and oxidative metabolism are not absolutely essential to the mechanism underlying adrenergic activation of the luminous system.6. The comparison of these luminescences with adrenergic responses of control photophores showed that the light emission to adrenaline was markedly inhibited by glycolysis blockade but potentiated by an inhibition of cellular respiration.7. As the inhibitory effect of IAA does not result from a direct action of IAA on the luminous system, these results suggest that adrenaline activation of adrenergic receptors might interact with glycolysis in photogenic cells.8. Glyceraldehyde 3-phosphate, or some derivatives, could be implicated in the glycolytic control of luminescence in the photophores.     

动脉壁脑啡肽的含量,存在部位及作用途径

用放射免疫法测得兔动脉的亮氨酸脑啡肽(L-ENK)和甲硫氨酸脑啡肽(M-ENK)样免疫活性物的含量(pg/mg组织湿重)分别为耳动脉38.99±17.29,134.67±8.11;肾动脉31.10±7.76,93.60±18.22,肠系膜动脉25.70 13.60,88.43±18.16。动物经注射交感神经末梢化学切割剂6-OHDA后,这三种动脉中L-ENK样免疫活性物的含量均明显下降(P<0.001);切除颈上神经节使支配耳动脉的交感神经溃变后,或离体耳动脉条受电场刺激后,脑啡肽(ENK)样免疫活性物的含量也都显著下降(P<0.001);但注射利血平则无明显影响,用荧光法测定培育动脉条的浴槽液中NE的含量,观察到ENK可抑制电场刺激所引起的NE的释放。结果提示,动脉壁的L-ENK和M-ENK存在于交感神经末梢中,它可因受电场刺激而释放,可能通过激活突触前膜阿片受体,减少交感神经末梢释放NE,从而抑制动脉的收缩活动。     

Neuro-pharmacological studies on firefly light organs during metamorphosis

Luminescences in response to electrical stimulation and pharmacological treatment were characterized during progressive degeneration of the larval light organ and differentiation of the adult light organ during metamorphosis. It was found that: (1) neural control of the larval light organ persists during pupation, (2) the differentiating adult light organ, while frequently luminescing, is not directly or neurally excitable until eclosion (3), a gradual loss of postsynaptic receptor chemical responsiveness occurs in parallel with declining neural excitability of the larval light organ, (4) postsynaptic chemical sensitivity of the adult light organ appears prior to establishment of neural control, (5) both larval and adult light organs spontaneously luminesce in the absence of neural control, and (6) synapses between nerve terminals and photocytes of the larval light organ are present throughout pupation.