HPLC analysis of the amino acid contents of the second- and third-order giant fibres at the giant synapse in the stellate ganglion of the squid
Loligo vulgaris shows that there are significantly ...higher amounts of
l-glutamate and
l-aspartate in the second-order (presynaptic) fibre than in the third-order (postsynaptic) fibre. Immunocytochemical staining of sections of the ganglion with an antibody raised against
l-glutamate produces specific positive staining in the synaptic region of the second-order fibre. In contrast, staining with antibodies raised against glutamate-receptors (mammalian GluR1 with GluR2/3) produces positive staining in the third-order fibre at the postsynaptic region. These data provide further evidence for the hypothesis that
l-glutamate is an excitatory transmitter at the giant synapse.
Infection of avocado seedlings by Phytophthora cinnamomi in infested soil was decreased by 71% by the addition of gypsum soil amendments in replicated greenhouse experiments. Root weights and total ...seedling weights were not significantly increased by gypsum amendments compared with unamended soil; however, the significant reduction in total seedling weight and root weight caused by P. cinnamomi was largely eliminated by the addition of gypsum. Fresh organic matter amendments alone did not significantly affect the total fresh weight or root weight of avocado seedlings. Root fresh weight was decreased in uninfested soil amended with 5% gypsum and organic matter, but in infested soils, the same treatment increased root fresh weight. Root fresh weight of mature avocado trees in an uninfested grove was not significantly affected by gypsum soil amendments. Avocado seedlings grown in gypsum-amended soil and the roots inoculated with suspensions of Phytophthora cinnamomi zoospores were no more resistant than seedlings grown in unamended soil. Permeability of avocado root membranes, as determined by the amount of
Rb exuded from root segments over time, was unaffected by growth in gypsum-amended soil. Infiltration of water into soil amended with fine-grade gypsum was impeded initially; whereas soil amended with coarse drywall gypsum drained faster than unamended soil. Drainage was not correlated with root infection of avocados grown in soil infested with P. cinnamomi and amended with gypsum. It appears that large reductions in infection of avocado seedlings by P. cinnamomi in gypsum-amended soil are not caused by an avocado growth response, increased root resistance, or reduced root membrane permeability. Infection is not markedly affected by poor drainage when the soil is amended with high levels of gypsum.
1. Pharmacological evidence suggests L-glutamate is a strong candidate as a transmitter at the giant synapse of the squid.
Postsynaptic activation at the giant synapse cannot be effected by ...conventional application of putative neurotransmitters
by iontophoresis or perfusion, apparently because the complex structure of the synapse prevents a sufficiently rapid change
in concentration at the postsynaptic membrane. Flash photolytic release of L-glutamate from a pharmacologically inert 'caged'
L-glutamate pre-equilibrated in the stellate ganglion of Alloteuthis or Loligo was used to determine whether L-glutamate can
produce postsynaptic activation when released rapidly in the synaptic clefts. 2. The preparation, reaction mechanism and properties
of the caged L-glutamate, N-1-(2-nitrophenyl)ethoxycarbonyl-L-glutamate, are described. The product quantum yield on photolysis
was 0.65 (+/- 0.05). On flash photolysis glutamate release followed a single exponential time-course in the pH range 5.5-7.8.
The rate constant was proportional to H+ and was 93 s-1 at pH 5.5 and 16 degrees C in artificial sea water (ionic strength,
I = 0.68 M). 3. At pH 7.8 flash photolysis of caged glutamate pre-equilibrated in the synapse caused only a slow depolarization.
A second photolytic release of L-glutamate or transsynaptic activation produced no further depolarization, suggesting desensitization
and inactivation of postsynaptic mechanisms by the initial pulse of L-glutamate. 4. Synaptic transmission in the giant synapse
was normal at pH 5.5. Flash photolysis at pH 5.5 caused rapid production of L-glutamate within the synaptic cleft and a fast
postsynaptic depolarization which generated postsynaptic action potentials.
Rotation (roll or pitch) of a cuttlefish away from its normal orientation produces countershading reflexes (CSRs) that consist of chromatophore expansion on the ventral body surface. When rotation is ...in the roll plane, the CSR has two components on each side of the body. The first (component A) consists of a unilateral expansion of chromatophores on the uppermost latero-ventral edge of the mantle, the underside of the upper fin and the uppermost side of the head; it occurs when the angle of rotation is less than 90°. Further rotation (from approximately 90° to approximately 180°) adds the second component (component B): a unilateral expansion of the chromatophores on the upper half of the ventral surface of the mantle, funnel, head and arms. When rotation is in the pitch plane, chromatophores expand on the posterior part of the ventral mantle and fins when the head is down; when the head is up, chromatophores expand on the ventral surface of the arms, head and funnel and on the anterior part of the ventral mantle and fins. The pitch CSR is always bilateral. Destruction of the gravity or the angular acceleration receptor systems of the statocysts demonstrates that it is the gravity receptor systems that drive the CSRs. Unilateral destruction of the gravity receptor systems shows that the pitch CSR is driven bilaterally, whereas the roll CSR is driven unilaterally. Components A and B of the roll CSR are driven by input from the ipsilateral statocyst, but component A is additionally driven by light. Brain lesions provide evidence that the pathways for the CSRs run through the lateral basal lobes in the supraoesophageal part of the brain.
Colour changes in cephalopods are controlled by complex organs termed chromatophores whose radial muscles are directly innervated from the brain. In the squids Alloteuthis subulata and Loligo ...vulgaris, light microscopy of silver- or Methylene-Blue-stained preparations shows that each muscle is innervated by 2-6 nerves running along its length. An electron microscope (EM) study shows that most of these nerves contain 50 nm diameter electron-lucent vesicles organised into numerous synapses along the muscle. Their size and appearance is consistent with their containing l-glutamate (l-Glu). Usually there is one nerve on each muscle containing 95 nm diameter electron-dense vesicles that are not organised into synapses. Such vesicles, whose appearance is consistent with their containing serotonin (5-HT), are never found co-localised with the small, clear vesicles. Topically applied l-Glu causes the radial muscles to contract (and the chromatophore to expand), even after chronic denervation; this effect is blocked by the glutamate antagonists CNQX and DNQX. In contrast, topically applied 5-HT (or its agonists 8-OH-DOPAT and -methyl 5-HT) induces relaxation of precontracted muscle. Incubation with antibodies to l-Glu (Lg-A), using peroxidase anti-peroxidase/diaminobenzidine visualisation, produces specific staining along the radial muscles like that seen with silver. Antibodies to 5-HT produce similar specific staining. When sections of skin that had stained positively with Lg-A in the light microscope are examined at the EM level, it is seen that such staining is confined to nerve axons. These results, showing that l-Glu and 5-HT are endogenous in the nerves innervating squid chromatophores and that the radial muscles contain receptors for both substances, suggest that l-Glu is an excitatory transmitter at squid chromatophore muscles. The way in which 5-HT acts to relax the muscles, however, remains to be established.
From January 2013 scientific projects involving cephalopods became regulated by Directive 2010/63/EU, but at present there is little guidance specifically for cephalopods on a number of key ...requirements of the Directive, including: recognition of pain, suffering and distress and implementation of humane end-points; anaesthesia and analgesia, and humane killing. This paper critically reviews these key areas prior to the development of guidelines and makes recommendations, including identifying topics for further research. In particular: a) Evidence on how cephalopods might experience pain is reviewed; and a draft scheme of behavioural and physiological criteria for recognising and assessing pain, suffering and distress in cephalopods used in scientific procedures is presented and discussed. b) Agents and protocols currently used for general anaesthesia and analgesia are evaluated. Magnesium chloride, ethanol and clove oil are the most frequently used agents, but their efficacy and potential for induction of aversion need to be systematically investigated, according to the species of cephalopod and factors such as body weight, sex and water temperature. Means of sedating animals prior to anaesthesia should be investigated. Criteria for assessing depth of anaesthesia, including depression of ventilation, decrease in chromatophore tone (paling), reduced arm activity, tone and sucker adhesiveness, loss of normal posture and righting reflex, and loss of response to a noxious stimulus, are discussed. c) Analgesia should be provided for cephalopods used in scientific procedures, whenever this would be the case for vertebrates. However, research is needed to evaluate effective agents and administration routes for cephalopods. d) Techniques for local anaesthesia need to be defined and evaluated. e) Currently used methods of killing and criteria for confirmation of death in cephalopods are evaluated. Based on present knowledge, a protocol for humane killing of cephalopods is proposed. However, further evaluation is needed, along with development of humane methods of killing that will not compromise study of the brain. On humane grounds: i. mechanical (as opposed to chemical) methods of killing should not be used on conscious cephalopods (unless specifically authorised by the national competent authority); and ii. hatchlings and larvae should be killed by overdose of anaesthetic and not by immersion in tissue fixative.
Key gaps in current knowledge are also highlighted, so as to encourage research that will contribute to the evidence base needed to develop guidelines to the Directive.
This paper presents the first evidence that some neurons in the octopus CNS contain delta-amino butyric acid (GABA). Using conventional immunohistochemical methods with appropriate controls, we ...obtained positive staining with an antibody to GABA in fibres in the neuropil of many lobes of the brain of the northern octopus Eledone cirrhosa. In several lobes cell bodies were also stained. Staining was not uniformly distributed in the brain nor within a particular lobe: some regions stained strongly, others not at all. These findings suggest that GABA should be added to the already long list of putative neurotransmitters in the cephalopod CNS.
Magnesium chloride as an anaesthetic for cephalopods Messenger, J B; Nixon, M; Ryan, K P
Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology,
1985, Letnik:
82, Številka:
1
Journal Article
Recenzirano
Magnesium chloride is an effective anaesthetic and narcotizing agent for cephalopod molluscs. Individuals belonging to five genera (decapod and octopod) were anaesthetized and subsequently revived at ...temperatures ranging from 13 to 22 degrees C. At no stage of anaesthesia does MgCl2 appear to cause any trauma. There is evidence that MgCl2 acts centrally on the nervous system to induce anaesthesia. This salt, cheap, readily available and convenient to use, is recommended over urethane or ethanol as a suitable anaesthetic or narcotic for use with cephalopods.
A Countershading Reflex in Cephalopods Ferguson, Graham P.; Messenger, John B.
Proceedings of the Royal Society. B, Biological sciences,
01/1991, Letnik:
243, Številka:
1306
Journal Article
Recenzirano
Most cephalopods have more chromatophores on the dorsal body surface than on the ventral and these tend to be kept tonically expanded. As a result the dorsal surface is usually darker than the ...ventral, an effect shown by many animals and known as countershading. We report here that when Sepia officinalis, Loligo vulgaris and Octopus vulgaris are rotated 180° around the longitudinal body axis the ventral chromatophores expand, causing darkening, while the dorsal chromatophores retract, causing paling. When animals are rotated through only 90° the chromatophores on the uppermost half of the ventral and dorsal surfaces expand, while those on the lower half retract. This response, which we term the countershading reflex, can be abolished by ablating the statocysts; and experiments in which the direction of incident light is reversed show that the reflex is not driven by sensory input from the eyes. The function of the reflex is presumably to maintain countershading while the animal is momentarily disoriented; this idea is supported by the fact that it lasts only a few seconds.
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