Central inhibition of an identified mechanosensory interneuron in the crayfish

Inhibitory input from crayfish mechanoreceptors is mediated polysynaptically to sensory interneurons. An identifiable sensory interneuron, the caudal photoreceptor (CPR), has been used as a model system to characterize inhibitory intermediate cells. A survey of the abdominal connectives, by antidromic stimulation, has identified eleven inhibitory cells, some of which also function as ascending sensory interneurons. These results indicate that lateral interactions within networks of mechanosensory interneurons form an integral part of the information processing mechanisms.     

Cholinergic transmission from mechanosensory afferents to an identified nonspiking interneuron in the crayfish Procambarus clarkii girard

Pharmacological properties of excitatory synaptic transmission from mechanosensory afferents to an identifiable nonspiking interneuron of crayfish were studied by drug perfusion experiments using acetylcholine (ACh) agonists and antagonists. Application of carbachol, a general agonist of ACh, caused sustained depolarization of the interneuron and a decrease in the peak amplitude of its excitatory synaptic response to sensory stimulation on the soma side. Similar depolarization was observed during application of carbachol under the low-Ca²⁺, high-Mg²⁺ condition. The peak amplitude was also reduced by application of nicotine and tetramethylammonium, both of which also caused sustained depolarization of the inter-neuron. By contrast, perfusion of muscarinic agonists, muscarine, oxotremorine and pilocarpine, reduced the peak amplitude without affecting the membrane potential of the interneuron. Perfusion of nicotonic antagonists of ACh, d-tubocurarine and hexamethonium, caused reduction of the peak amplitude without any change in the membrane potential. A muscarinic antagonist atropine was also effective in blocking the synaptic transmission but at higher concentration than d-tubocurarine. The results suggest that the ACh receptors on the nonspiking interneuron belong to a previously characterized class of crustacean cholinergic receptors resembling the nicotinic subtype of vertebrates.     

Silencing normal input permits regenerating foreign afferents to innervate an identified crayfish sensory interneuron

Interneuron A, an identified first-order sensory interneuron that is innervated by mechanoreceptors on one side of the crayfish tailfan, normally resists extra innervation by regenerating contralateral mechanoreceptor axons. However, if its normal innervation is silenced by covering mechanosensory hairs with a surgical glue, it accepts contralateral innervation. This finding on an arthropod provides evidence for the generality and antiquity of the principle, critical in development of the vertebrate nervous system, that activity of one set of afferents can control whether other afferents form synapses on a target.     

Transplantation and functional integration of an identified respiratory interneuron in Lymnaea stagnalis.

The possibility that damaged neural circuitries can be repaired through grafting has raised questions regarding the cellular mechanisms required for functional integration of transplanted neurons. Invertebrate models offer the potential to examine such mechanisms at the resolution of single identified neurons within well-characterized neural networks. Here it is reported that a specific deficit in the respiratory behavior of a pulmonate mollusc, caused by the ablation of a solitary interneuron, can be restored by grafting an identical donor interneuron. The transplanted interneuron not only survives and extends neurites within the host nervous system, but under specific conditions forms synapses with appropriate target neurons and is physiologically integrated into the host's circuitry, thereby restoring normal behavior.     

Cholinergic transmission at mechanosensory afferents in the crayfish terminal abdominal ganglion

Electrical stimulation of mechanosensory afferents innervating hairs on the surface of the exopodite in crayfish Procambarus clarkii (Girard) elicited reciprocal activation of the antagonistic set of uropod motor neurones. The closer motor neurones were excited while the opener motor neurones were inhibited. This reciprocal pattern of activity in the uropod motor neurones was also produced by bath application of acetylcholine (ACh) and the cholinergic agonist, carbamylcholine (carbachol). The closing pattern of activity in the uropod motor neurones produced by sensory stimulation was completely eliminated by bath application of the ACh blocker, d-tubocurarine, though the spontaneous activity of the motor neurones was not affected significantly. Bath application of the acetylcholinesterase inhibitor, neostigmine, increased the amplitude and extended the time course of excitatory postsynaptic potentials (EPSPs) of ascending interneurones elicited by sensory stimulation. These results strongly suggest that synaptic transmission from mechanosensory afferents innervating hairs on the surface of the tailfan is cholinergic.Bath application of the cholinergic antagonists, dtubocurarine (vertebrate nicotinic antagonist) and atropine (muscarinic antagonist) reversibly reduced the amplitude of EPSPs in many identified ascending and spiking local interneurones during sensory stimulation. Bath application of the cholinergic agonists, nicotine (nicotinic agonist) and oxotremorine (muscarinic agonist) also reduced EPSP amplitude. Nicotine caused a rapid depolarization of membrane potential with, in some cases, spikes in the interneurones. In the presence of nicotine, interneurones showed almost no response to the sensory stimulation, probably owing to desensitization of postsynaptic receptors. On the other hand, no remarkable changes in membrane potential of interneurones were observed after oxotremorine application. These results suggest that ACh released from the mechanosensory afferents depolarizes interneurones by acting on receptors similar to vertebrate nicotinic receptors.Abbreviations ACh cetylcholine - mns motor neurones - asc int ascending interneurone     

Directional sensitivity of an identified wind-sensitive interneuron during the postembryonic development of the locust

Simultaneous extracellular recordings from both locust abdominal connectives show a differential activation of both bilateral homologues of an identified long projection interneuron (A4I1) in response to wind stimuli from different directions. Despite the previously shown extensive structural dynamics of sensory afferents and synaptic rearrangement of the direct afferent-to-interneuron connections during postembryonic development, a directional sensitivity is already present in first instar nymphs. Only quantitative changes in the strength of the directional response can be detected. Intracellular stainings of the A4I1 interneuron in first instar nymphs and adults show that general morphological features do not change during postembryonic development, in contrast to the presynaptic sensory afferents. This also holds for general morphological features of pleuroaxillary flight motoneurons. The output connections of A4I1 to these motoneurons and an unidentified intersegmental interneuron are already present in flightless nymphs.     

Ionic dependence of the axotomy-induced long-lasting firing in an identified crayfish motoneuron

I have reported previously that axotomy of an identifiable anal motoneuron of crayfish Procambarus clarkii induces a long-lasting firing and that a prolonged depolarizing pulse to its cut end can induce a similar response. In this study, I confirmed that this stimulus is comparable to axotomy; the frequency of stimulus-induced firing increases linearly with the stimulus intensity and its firing pattern is the same as that following axotomy. Then, when the cut end was bathed for more than 1 hr in test solutions, it was examined whether the stimulus to the cut end induces or blocks the response. Na(+)-free saline (Tris(+) replaced Na(+)) or TTX (3 x 10(-7) M) reversibly blocked the response within 30 min. By contrast, Mn(2+) saline (40 mM Mn(2+) replaced Ca(2+)) or Ca(2+)-free salines (Mg(2+) or 1 mM EDTA replaced Ca(2+)) cannot block the response, but instead increased the firing frequency. These results obtained with stimulus were confirmed also by those with axotomy. I concluded that axotomy-induced firing, which occurs locally at its cut region, is primarily responsible for voltage-dependent Na(+) conductances, but not for Ca(2+) ones.     

Spontaneous firing in the isolated anucleate axonal segment of an identified crayfish motoneuron

The aim of this study was to present evidence for prolonged spontaneous firing in an anucleate axonal segment of an identifiable crayfish anal motoneuron L (AML) and to determine the initiation site of this firing. AML has its soma in the 6th abdominal ganglion (A6). By separating a nerve with the AML axon from A6 and the target muscle, various lengths of an anucleate AML axonal segment were procured. Then, AML activity was recorded extracellularly for 14-26 hr from the distal end of this axonal segment. This segment (n=19) exhibited spontaneous firing, which occurred without any stimulation 0.03-5.13 hr after the A6-cut and persisted tonically for 0.20-19.98 hr. During firing, the frequency augmented gradually, whereas the amplitude decreased gradually. There was no significant correlation between latency and duration of the firings. No correlation was noted between latency and length of the axonal segment or its size, or between duration and length or size. These results revealed that the anucleate AML axon itself can inherently generate prolonged firing. The delay in the appearance of AML impulses between the proximal and distal regions at the same axonal segment proved that the firing occurred proximally. There was no significant difference in delays between firing following the A6-cut and the spontaneous firing observed after the A6-cut. This suggests that the initiation site of the spontaneous firing is at the proximal end of the AML axonal segment, since the AML firing following the A6-cut occurs at its cut end.     

Effects of active conductance distribution over dendrites on the synaptic integration in an identified nonspiking interneuron

The synaptic integration in individual central neuron is critically affected by how active conductances are distributed over dendrites. It has been well known that the dendrites of central neurons are richly endowed with voltage- and ligand-regulated ion conductances. Nonspiking interneurons (NSIs), almost exclusively characteristic to arthropod central nervous systems, do not generate action potentials and hence lack voltage-regulated sodium channels, yet having a variety of voltage-regulated potassium conductances on their dendritic membrane including the one similar to the delayed-rectifier type potassium conductance. It remains unknown, however, how the active conductances are distributed over dendrites and how the synaptic integration is affected by those conductances in NSIs and other invertebrate neurons where the cell body is not included in the signal pathway from input synapses to output sites. In the present study, we quantitatively investigated the functional significance of active conductance distribution pattern in the spatio-temporal spread of synaptic potentials over dendrites of an identified NSI in the crayfish central nervous system by computer simulation. We systematically changed the distribution pattern of active conductances in the neuron's multicompartment model and examined how the synaptic potential waveform was affected by each distribution pattern. It was revealed that specific patterns of nonuniform distribution of potassium conductances were consistent, while other patterns were not, with the waveform of compound synaptic potentials recorded physiologically in the major input-output pathway of the cell, suggesting that the possibility of nonuniform distribution of potassium conductances over the dendrite cannot be excluded as well as the possibility of uniform distribution. Local synaptic circuits involving input and output synapses on the same branch or on the same side were found to be potentially affected under the condition of nonuniform distribution while operation of the major input-output pathway from the soma side to the one on the opposite side remained the same under both conditions of uniform and nonuniform distribution of potassium conductances over the NSI dendrite.     

Proportional inhibition in the cricket medial giant interneuron

Inhibitory effects on the number of wind-evoked impulses were studied in the medial giant interneuron of the cricket, Gryllus bimaculatus. The interneuron receives an inhibitory input from wind receptors on cercus ipsilateral to its soma. Using a dual channel wind stimulator, the intensity of inhibitory input was changed over 1,000-fold and effects on the number of spikes were observed. The ipsilateral inhibition reduced the number of outgoing spikes from a level elicited by excitation alone and it did so in proportion to the level of wind responsiveness displayed by each cell. A proportional coefficient of inhibition was derived and its value depended on the level of total excitation of the medial giant interneuron. The medial giant interneurons with high excitation showed a smaller value of the coefficient than those with low excitation. The proportional inhibition of the medial giant interneuron by the ipsilateral cercus suppresses the number of its spikes to a reasonable level for a wide range of stimulus intensities under natural conditions.     

Antibody block of a neural-tissue-specific glycoconjugate perturbs growth cone guidance of an identified interneuron in the grasshopper

Antibodies against horseradish peroxidase (HRP) recognize a neural-tissue-specific carbohydrate moiety that is expressed on a complex set of developmentally regulated antigens in grasshopper, Drosophila and other insects. The functional role of the neural-specific carbohydrate has been investigated by mutant analysis in Drosophila where subtle defects in wing sensory axon projections have been reported. Here we extend the analysis of this neural-specific carbohydrate to the single cell level by focusing on identified brain interneurons in the grasshopper embryo. Immunological blocking experiments carried out in embryo culture show that the neural-specific carbohydrate is essential for correct axonal guidance of the identified interneurons. Functional block of the carbohydrate epitope causes major aberrations in growth cone guidance and axonal outgrowth in approximately 40% of the cases studied. This analysis reveals an important role of neural-specific glycoconjugate for correct axonal guidance of individual identified neurons.     

Neuromodulation of central inhibition on peripheral mechanosensory afferents

100 g reserpine, suspended in sunflower oil, was injected into the crop of six leeches (Hirudo medicinalis). Five leeches were injected with oil alone. The efficacy of delivering reserpine in this manner was assessed by measuring swimming bout duration. Four days after injection, swimming bouts were significantly longer in reserpine-treated leeches than in oil-treated or normal leeches. The amplitude of excitatory potentials elicited in velocity sensitive skin mechanoreceptors (T cells) by action potentials in mechanosensory cells which respond to sustained deformation of the skin (P cells) was no different in reserpine-treated leeches than in untreated leeches. The amplitude of inhibitory potentials in these T cells, produced by interneurons excited by P cells, was significantly larger in reserpine-treated leeches. Depletion of monoamines enhances inhibition in T cells. This is consistent with the established roles played by monoamines in promoting leech swimming and feeding behaviour.