THE EFFECT OF SPIKE ACTIVITY VERSUS SYNAPTIC ACTIVATION ON THE METABOLISM OF RIBONUCLEIC ACID IN A MOLLUSCAN GIANT NEURONE

—Previous experiments on a giant neurone (R2) from Aplysia californica have shown that a prolonged electrical stimulation of ganglionic nerves, strong enough to elicit post-synaptic spikes in the giant neurone, caused a marked increase in the uptake of labelled nucleosides into the neuronal RNA. The results described in the present paper very strongly indicate that these effects of synaptic activation were not due to the discharge of spikes in the giant neurone itself. Spikes which were directly elicited in the giant neurone by current pulses injected into the cell through an intracellular microelectrode had no significant effect on RNA labelling. Weak stimulation of ganglionic nerves, eliciting post-synaptic potentials but few spikes in the giant neurone, produced a small but significant increase of RNA labelling.     

SEROTONIN AND FREE AMINO ACID ANALYSIS OF GANGLIA AND ISOLATED NEURONES OF APLYSIA DACTYLOMELA

—The presence of serotonin and different amino acids was investigated in the ganglia and in isolated giant neurones of Aplysia dactylomela. With a few exceptions the pattern of substances was similar in all the ganglia. Of the many identified neurones studied only one giant neurone located in each cerebral ganglion was found to contain serotonin. GABA was detected in most extracts, including those of the serotonin-containing neurone, known cholinergic, and known neurosecretory neurones. Putrescine, recently detected in extracts of nervous tissue and isolated neurones of Helix, was not detected in Aplysia nervous tissue.     

Nicotinic acetylcholine receptors on a cholinergic nerve terminal in the cockroach,Periplaneta americana

Summary Intracellular microelectrode recording and ionophoretic application of carbamylcholine (CCh) were used to compare the cholinergic sensitivity of postsynaptic dendrites of an identified neurone with that of an identified presynaptic cholinergic axon.The axon of the lateral filiform hair sensory neurone (LFHSN) in the first-instar cockroachPeriplaneta americana was found to be as sensitive to CCh as the dendritic regions of giant interneurone 3 (GI 3). The CCh response of both neurones was unaffected by replacing Ca²⁺ with Mg²⁺, confirming that the ACh receptors are present on the neurones under test. The CCh response of both neurones was mimicked by ionophoretic application of nicotine. The responses were blocked by 10^–5 M mecamylamine and 10^–6 M d-tubocurarine and were not affected by muscarinic antagonists, suggesting that the ACh receptors present on GI 3 and LFHSN are predominantly nicotinic.The muscarinic agonist oxotremorine and the antagonists atropine and quinuclidinyl benzilate had no modulatory effect on LFHSN-GI 3 synaptic transmission.The latency of the LFHSN response to CCh was consistent with the hypothesis that ACh receptors are situated on the main axon/terminal within the neuropil of the ganglion. It has previously been shown that this region of the axon does not form output synapses (Blagburn et al. 1985a). This indirect evidence indicates that presynaptic or extrasynaptic ACh receptors are present in the membrane of a cholinergic axon.LFHSN was depolarized by synaptically-released ACh after normal or evoked spike bursts, suggesting that the nicotinic ACh receptors act as autoreceptors. However, it was not possible to obtain direct evidence to support the hypothesis that these receptors modulate ACh release.Abbreviations CCh carbamylcholine - GI giant interneurone - FHSN filiform hair sensory neurone - LFHSN lateral filiform hair sensory neurone - R _in input resistance - V depolarization - V _m resting potential     

Thoracic output of crayfish giant fibres

Summary The thoracic homologue of the abdominal segmental giant neurone of crayfish Pacifastacus leniusculus is identified and described. It has a small cell body located in the anterior ventro-lateral quadrant of the ganglion and a large neuropil arborization, with dendrites aligned along the tracts of the giant fibres. The SG axon exits the ganglion within the major root which innervates the leg, usually in the anterior region of this root. Within 1–2 mm of the ganglion the axon terminates in a mass of fine branches, apparently randomly located within the base of the root.The SG receives suprathreshold input from the ipsilateral MG and LG fibres through rectifying electrical synapses. It makes output to FF motor neurones, also through electrical synapses. The SG also makes output to at least one corollary discharge interneurone. The SG receives depolarizing inhibitory synaptic potentials which can prevent its activation by the GFs. Some but not all of these synaptic potentials are common to similar potentials occurring in a large leg promotor motor neurone.Abbreviations AC anterior connective - GF giant fibre - IPSP inhibitory post-synaptic potential - LG lateral giant fibre - MG medial giant fibre - MoG motor giant neurone - PC posterior connective - PMM promotor motor neurone - r1 first root - r3 third root - rAD anterior distal root - rPD posterior distal root - rPM promotor muscle root - SG segmental giant neurone     

Different types of rectification at electrical synapses made by a single crayfish neurone investigated experimentally and by computer simulation

Summary The rectification properties of electrical synapses made by the segmental giant (SG) neurone of crayfish (Pacifastacus leniusculus) were investigated. The SG acts as an interneurone, transmitting information from the giant command fibres (GFs) to the abdominal fast flexor (FF) motoneurones. The GF-SG (input) synapses are inwardly-rectifying electrical synapses, while the SG-FF (output) synapses are outwardly rectifying electrical synapses. This implies that a single neurone can make gap junction hemichannels with different rectification properties.The coupling coefficient of these synapses is dependent upon transjunctional potential. There is a standing gradient in resting potential between the GFs, SG and FFs, with the GFs the most hyperpolarized, and the FFs the most depolarized. The gradient thus biases each synapse into the low-conductance state under resting conditions.There is functional double rectification between the bilateral pairs of SGs within a single segment, such that depolarizing membrane potential changes of either SG pass to the other SG with less attenuation than do hyperpolarizing potential changes. Computer simulation suggests that this may result from coupling through the intermediary FF neurones.Abbreviations l left - r right - FF fast flexor motoneurone - GF giant fibre - LG lateral giant interneurone - MG medial giant interneurone - MoG motor giant motoneurone - R root, e.g. 1R1 is the first root on the left side - SG Segmental giant neurone     

Electrophysiological responses from the peripheral olfactory system of the American eel,Anguilla rostrata

Summary A method is described for recording with microelectrodes from central neurones in locusts,Schistocerca gregaria americana, that are free to perform a large fraction of their behavioural repertoire. This tethered preparation has been used to examine the individual responses of large neurones in the neck connectives to a range of sensory stimuli.From differences in the responses of the units examined and from their positions in the connective, as determined by dye iontophoresis, 31 separate neurones have been identified. The axons of these cells had relatively constant diameters and cord positions in different animals and appeared in both right and left connectives but with their positions mirror reversed. The majority of these 31 cells carried descending information from the head ganglia and under our experimental conditions, 7 were found to have wind stimulation as their strongest sensory input, 17 had visual stimulation, 4 had sound stimulation and 3 had proprioceptive input.Abbreviations DCMD descending contralateral movement detector (neurone) - DIMD descending ipsilateral movement detector (neurone)     

THE SYNTHESIS OF CHOLINE PHOSPHOGLYCERIDES IN THE GIANT FIBRE SYSTEM OF THE SQUID

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion-nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP-choline in the intact ganglion-nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base-exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations. Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.     

Elementary transcallosal connections of the rabbit sensomotor cortex]

Antidromic responses of two callosal neurones to a local electrical stimulation of the rabbit sensorimotor cortex may be recorded simultaneously with one microelectrode in the homotopic cortical area. In such recording conditions the relative amplitude of extracellularly recorded action potentials of the two neurones is determined primarily by the distance between these neurones and the electrode's tip. In response to the stimulation of the symmetrical area transcallosal monosynaptic excitation of the callosal neurone may occur; two callosal neurones may exite monosynaptically one and the same recorded neurone. The results suggest the existence of clusters or columns, formed jointly by the bodies and terminals of callosal neurones; a functional interconnection between symmetrical clusters or columns may exist, in particular a positive feedback.     

Axoplasmic RNA species synthesized in the isolated squid giant axon

Isolated squid stellate nerves and giant fiber lobes were incubated for 8 hr in Millipore filtered sea water containing [³H]uridine. The electrophoretic patterns of radioactive RNA purified from the axoplasm of the giant axon and from the giant fiber lobe (cell bodies of the giant axon) demonstrated the presence of RNA species with mobilities corresponding to tRNA and rRNA. The presence of labeled rRNAs was confirmed by the behavior of the large rRNA component (31S) which, in the squid, readily dissociates into its two constituent moyeties (17S and 20S). Comparable results were obtained with the axonal sheath and the stellate nerve. In all the electrophoretic patterns, additional species of radioactive RNA migrated between the 4S and the 20S markers, i.e. with mobilities corresponding to presumptive mRNAs. Chromatographic analysis of the purified RNAs on oligo(dT)cellulose indicated the presence of labeled poly(A)⁺ RNA in all tissue samples. Radioactive poly(A)⁺ RNA represented approximately 1% of the total labeled RNA in the axoplasm, axonal sheath and stellate nerve, but more than 2% in the giant fiber lobe. The labeled poly(A)⁺ RNAs of the giant fibre lobe showed a prevalence of larger species in comparison to the axonal sheath and stellate nerve. In conclusion, the axoplasmic RNAs synthesized by the isolated squid giant axon appear to include all the major classes of axoplasmic RNAs, that is rRNA, tRNA and mRNA.Special Issue dedicated to Prof. Holger Hydén.     

THE ULTRASTRUCTURE AND HISTOCHEMISTRY OF A NEMATODE-INDUCED GIANT CELL

The development of giant cells induced by the nematode Meloidogyne in tomato roots has been followed under controlled growth conditions and the ultrastructure and histochemistry of these structures have been examined. Entry of the nematode larvae into the roots took place within 24 hours; giant cell formation started on the 4th day and involved breakdown of the cell walls accompanied by thickening of a surrounding giant cell wall and an increase in density and area of the cytoplasm. The nuclei increased in number by simultaneous mitosis throughout a single giant cell. The peak of cytoplasmic density was reached after moulting and during egg production. The rate of protein synthesis in the giant cell is correlated with the rate of growth of the nematode. The giant cell wall is a thick, irregularly surfaced structure which contains all the normal polysaccharide components of a cell wall. The cytoplasm is rich in protein and RNA and contains mitochondria, proplastids, Golgi bodies, and a dense endoplasmic reticulum. The nuclei are large and irregular in shape and contain large nucleoli and a number of Feulgen-positive bodies scattered irregularly along the nuclear envelope. The nucleolus contains RNA and fat as well as Feulgen-positive granules which are revealed after treatment with ribonuclease. It consists of a dense outer cortex surrounding a much lighter central core and is connected at times with the Feulgen-positive bodies in the nucleus. Speculation is provided on the role of these bodies in cytoplasmic protein synthesis.     

Length of human prematurely condensed chromosomes during G0 and G1 phase

Length measurements on C-banded prematurely condensed no. 1 human chromosomes of G0 and G1 lymphocytes, as well as of synchronized G1 HEp cells revealed that (i) no length difference exists between mitotic chromosomes and G0 chromosomes; (ii) 1 h after PHA stimulation a clear increase in length is detectable; (iii) in isolated cases an increase by the factor 5 can be observed during G1; (iv) the increase is significantly less for constitutive heterochromatin than for euchromatin. The possibility is discussed that these conformational changes of chromatin reflect physiological differences, i.e. the rate of RNA synthesis during interphase.     

Identification and pharmacological characteristics of giant neurones situated in the buccal ganglia of an African giant snail (Achatina fulica Férussac)

1. The following four giant neurones were identified on the dorsal surface of the left buccal ganglion of an African giant snail (Achatina fulica Ferussac): d-LBAN (dorsal-left buccal anterior neurone), d-LBMN (dorsal-left buccal medial neurone), d-LBCN (dorsal-left buccal central neurone) and d-LBPN (dorsal-left buccal posterior neurone). The axonal pathways of the neurones were studied by the intracellular injection of Lucifer Yellow; their pharmacological characteristics with respect to common putative neurotransmitters were also investigated.2. The axonal pathways of d-LBAN and d-LBCN were simple, innervating some left lateral buccal nerves or the left accessory connective buccal nerve. On the other hand, those of d-LBMN and d-LBPN were much more widespread, projecting not only to the left buccal nerves, but also to the right buccal nerves through the buccal commissure.3. No direct axonal pathway from any of the four buccal neurones tested to the other ganglioncomplexes through the cerebral buccal connectives was demonstrated.4. The pharmacological characteristics of the four neurones tested were not identical. Only 5-hydroxytryptamine excited all of the neurones, whereas dopamine, l-epinephrine and acetylcholine inhibited all of them. However, the other effective substances, such as dl-octopamine, GABA, l-homocysteic acid, erythro-β-hydroxy-l-glutamic acid and histamine, were either excitatory or inhibitory according to the neurone.     

The effect of synaptic stimulation on RNA and protein metabolism in the R2 soma of Aplysia

Excitatory synaptic stimulation of the R2 neuron in the abdominal ganglion of Aplysia californica causes an increased incorporation of ³Huridine into RNA. However, this could be the result of a change in precursor specific activity rather than an increase in RNA synthesis. We find that at low external uridine concentrations (1.5 μM) there is no increase in ³H-uridine incorporation correlated with synaptic stimulation. In addition, no change in incorporation of ³H-leucine into total protein or in the pattern of newly-synthesized proteins, resolved by electrophoresis on SDS-polyacrylamide gels, was detected with stimulation. Since the R2 neuron can be stimulated without a detectable change in RNA or protein synthesis, we conclude that the increase in incorporation observed at high external uridine concentrations (100 μM) could be caused by increased specific activity in a precursor pool rather than by an RNA synthesis change.     

Response properties of the prothoracic AN2 auditory interneurone to model calling songs in the cricket Gryllus bimaculatus

Sound processing properties for calling song (CS) models, as described for the prothoracic L3 auditory neurone in Acheta domesticus, are investigated for the homologous auditory neurone 2 (AN2) in female Gryllus bimaculatus De Geer. AN2 of G. bimaculatus responds selectively to the syllable period (SP) of models of a male CS. The selectiveness of this response parallels the selectivity of phonotaxis females perform in response to the same SPs. Both, the responses of AN2 and female behaviour show clear interindividual variability. The SP‐selective responses of AN2 result from an SP‐dependent reduction in the spiking to subsequent syllables of the model CSs, measured as the percentage decrement. This SP‐dependent response does not primarily result from inbuilt properties of the AN2 membrane. Rather, it is dependent on inhibitory input to the AN2. However, clear inhibitory postsynaptic potentials in dendritic recordings of the AN2 are not encountered. This immediate response of AN2 to CSs is followed by an increased rate of tonic firing between stimulus CSs, which is termed the prolonged response, and is dependent on the carrier frequencies that make up the male CSs. With stimulation on the contralateral side of the soma of AN2s, more than 50% of AN2s exhibit a prolonged response. However, with stimulation from the ipsilateral side of the soma, most AN2s exhibit a prolonged response. The prolonged response of AN2 at 5 kHz may be even more sensitive than the immediate response. Thus, the AN2 neurone could provide a basis for phonotaxis that is selective for both the SPs and the carrier frequencies of potentially attractive calling songs.     

Electrophysiological properties ofDictyostelium derived from membrane potential measurements with microelectrodes

Summary Electrical membrane properties of the cellular slime moldDictyostelium discoideum were investigated with the use of intracellular microelectrodes. The rapid potential transients (1 msec) upon microelectrode penetration of normal cells had a negative-going peak-shaped time course. This indicates that penetration of a cell with a microelectrode causes a rapid depolarization, which can just be recorded by the microelectrode itself. Therefore, the initial (negative) peak potential transient valueE _p (–19 mV) should be used as an indicator of the resting membrane potentialE _m ofD. discoideum before impalement, rather than the subsequent semistationary depolarized valueE _n (–5 mV). Using enlarged cells such as giant mutant cells (E _p=–39 mV) and electrofused normal cells (E _p=–30 mV) improved the reliability ofE _p as an indicator ofE _m. From the data we concluded thatE _m ofD. discoideum cells bathed in (mm) 40 NaCl, 5 KCl and 1 CaCl₂ is at least –50 mV. This potential was shown to be dependent on extracellular potassium. The average input resistanceR _i of the impaled cells was 56 M for normalD. discoideum. However, our analysis indicates that the membrane resistance of these cells before impalement is >1 G. Specific membrane capacitance was 1–3 pF/cm². Long-term recording of the membrane potential showed the existence of a transient hyperpolarization following the rapid impalement transient. This hyperpolarization was associated with an increase inR _i of the impaled cell. It was followed by a depolarization, which was associated with a decrease inR _i. The depolarization time was dependent on the filling of the microelectrode. The present characterization of the electrical membrane properties ofDictyostelium cells is a first step in a membrane electrophysiological analysis of signal transduction in cellular slime molds.     

Intersegmental variation of afferent pathways to giant interneurons of the earthworm,Lumbricus terrestris L.

Summary We have investigated the connectivity of four classes of mechanosensory afferents to giant interneurons in the earthwormLumbricus. Three of these classes of afferents change their specification for connection to medial giant (MGF) and lateral giant (LGF) fibers along the length of the animal. Near the caudal end, stimulation of touch, pressure and small tactile fibers generates excitatory post-synaptic potentials, epsp's, in the two LGF's but not in the MGF. Near the rostral end these afferents produce much smaller epsp's in the LGFs but produce large epsp's in the MGF. In the middle region of the animal an overlap region exists where both giant fibers receive approximately equal inputs from these afferents. The amplitude of these inputs are reduced compared to the maxima seen at either end. The fourth class of sensory afferents investigated, the stretch neurons, have no synaptic effect on the giant fibers anywhere in the nerve cord.These results explain at least part of the basis, in neuronal connectivity, for the differences in response to tactile stimulation of the head and tail segments previously characterized in terms of behavior and giant fiber impulse activity. In this system developmental mechanisms generating synaptic connectivity patterns have coded certain classes of homologous afferent neurons and interneurons to make different connections in different segments.Abbreviations MGF medial giant fiber - LGF lateral giant fiber - SN1 first segmental root - SN2 second segmental root - SN3 third segmental root - RIN giant interneuron     

Deterrence coding by a larval Manduca chemosensory neurone mediating rejection of a non-host plant, Canna generalis L.

Abstract. The physiological basis of phagodeterrence was studied electrophy-siologically and behaviourally in the phytophagous caterpillars Manduca sexta and Manduca quinquemaculata. The model unacceptable non-host plant was the canna lily, Canna generalis. A strongly deterrent extract was obtained from fresh leaves of canna by extraction with hot ethanol or ethyl acetate in a blender. Behavioural rejection of these extracts was similar to that of fresh leaves, although less intense. In contrast, blender extracts using other solvents, as well as leaf surface rinses, were phagostimulant or neutral. Chromatographic fractionation of the deterrent ethanolic extract showed the active principles to be moderately polar and separable into two fractions. Previous ablation experiments had shown that the medial maxillary styloconica and epipharyngeal sensilla are the two most important chemosensory organs in mediating behavioural rejection of canna leaves; if only one of these organs is spared, the animal completely rejects canna. We investigated the neural responses of the medial styloconica and their contribution to the sensory coding responsible for this phagodeterrence. The active fractions of the deterrent ethanolic extract elicited a vigorous response from one chemosensory neurone in the medial styloconica. This neurone is distinguishable from others in the medial styloconica by its unique temporal response parameters and the characteristic shape changes of its action potentials. The response frequency of this neurone correlates with the degree of phagodeterrence in a dose-dependent manner. Threshold deterrence occurs at a concentration of extract (1%) that elicits firing in this neurone at a rate of c. 50 spikes/s peak instantaneous frequency and 30 total spikes in the first Is. We conclude that this is a ’deterrent neurone’ in the sense that vigorous response from this neurone is a sufficient sensory code for behavioural rejection of canna. Thus input from a single sensory neurone is capable of blocking feeding, since only one (unilateral) medial styloconicum is needed to mediate this rejection.     

Further study of effects of synthetic peptides on identifiable giant neurones of an african giant snail (Achatina fulica Férussac)

• 1.1. Effects of the following peptides at 10⁻⁴ M on identifiable giant neurones of Achatina fulica Férussac were examined: physalaemin, eledoisin, bradykinin, neurokinin A, neurokinin B, neuromedin B, gastrin releasing peptide decapeptide (neuromedin C), gastrin releasing peptide (14–27), cholecystokinin tetrapeptide, cholecystokinin octapeptide, thyrotropin releasing hormone, Arg-vasotocin, γ-melanocyte stimulating hormone.
• 2.2. The six neurones tested were as follows: PON (periodically oscillating neurone), TAN (tonically autoactive neurone), RAPN (right anterior pallial neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone) and d-VLN (dorsal-visceral large neurone).
• 3.3. Of the peptides examined, only Arg-vasotocin at 10⁻⁴ M produced the excitatory effects on PON, VIN and d-VLN. Physalaemin showed slight inhibitory effects on TAN; this substance was sometimes almost ineffective on the neurone.
• 4.4. The other peptides examined were completely ineffective on all of the neurones tested.

     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

Different effects of the replacement of extracellular sodium with lithium on the abnormal spike discharges caused by 2 convulsants, Metrazol and strychnine, on an identifiable giant neuron of the mollusc Achatina fulica Ferussac

The influences of the replacement of sodium with lithium in the extracellular medium on the abnormal spike discharges, caused by two convulsants, metrazol and strychnine, of a giant neurone (TAN, tonically autoactive neurone) identified in the suboesophageal ganglia of the African giant snail (Achatina fulica Férussac) were examined. The slow oscillations of potential caused by metrazol disappeared after this replacement. On the other hand, the abnormal action potentials caused by strychnine, as well as the normal action potentials, still remained after the removal of sodium in the medium.