Protection from habituation of the crayfish lateral giant fibre escape response

1. Habituation of the lateral giant fibre escape response in the crayfish to repetitive tactile stimuli is believed to result from homosynaptic depression at the first synapse of the reflex, between tactile afferents and interneurones. Normally, habituation of escape responses to repeated innocuous stimuli is presumed to be adaptive. Experiments reported here were undertaken to determine whether habituation would occur under circumstances when it would presumably be maladaptive - in particular, when tactile receptors are stimulated by an animal's own tail-flip movements.2. Experiments were carried out on the crayfish isolated abdominal nerve cord, which contains the lateral giant reflex pathway.3. Compound e.p.s.p.s elicited in the lateral giant by electrical stimulation of tactile afferents decline by from 25 to 36% over a series of eleven trials at 1/5 sec (control series).4. To determine whether such a decline would occur when sensory afferents are stimulated during a ;tail-flip', stimuli were given as in the control series but each stimulus occurred 20 msec after direct electrical stimulation of a medial giant or lateral giant escape-command fibre at which time tail flexion movements of an intact animal would be in progress. Under these conditions% e.p.s.p. decline over 11 trials at 1/5 sec was only 16-45% of that occurring on the control series.5. This protective effect starts at about 10 msec after escape command neurone firing, is maximal at 20 msec, and thereafter declines, remaining weakly detectable at 100 msec. This time course is commensurate with that required for execution of a tail-flip movement. Thus, sensory afferent-to-lateral giant transmission is protected from depression if stimuli occur when a tail-flip movement is or should be occurring.6. Giant fibre spikes do not superimpose facilitation upon a depressed reflex pathway, nor accelerate rate of recovery from depression; rather, protection is attributable to actual prevention of development of the depressed state.7. Protection was also examined at the first synapse of the reflex, where the depression responsible for habituation is believed to occur, by recording intracellularly in the largest of the first-order interneurones (interneurone A) of the pathway. In absence of protection, ten stimuli presented at 1/4 sec caused a mean decline of 32% in the e.p.s.p. in interneurone A. When such stimuli followed directly evoked escape command neurone firing by 20 msec this decline was reduced by 59-100%.8. We suggest that protection serves to prevent crayfish from habituating to stimuli produced by their own tail-flip movements.     

FMRFamide-related peptides potentiate transmission at the squid giant synapse.

The stellate ganglion of the squid Loligo pealli contains the neuropeptides Phe-Met-Arg-Phe-NH2 (FMRFamide), Phe-Leu-Arg-Phe-NH2 (FLRFamide) and at least one N-terminally extended FMRFamide-related peptide that is yet to be fully characterized. Both local application and arterial perfusion of FLRFamide potentiate transmission at the giant synapse. The N-terminally related peptide Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRFamide) produced a similar effect. The threshold for both the tetra- and the hepta-peptides was less than 10 microM. Potentiation could be detected as an increase in rate of rise of the EPSPs, as an increase in amplitude of the EPSP in the absence of spikes, or under voltage clamp as an increase in the EPSC. The effect was most pronounced when the synapse was fatigued by high frequency stimulation. Another molluscan peptide, eledoisin and also leucine enkephalin were without effect. In the absence of any detectable effects of FLRFamide on the resting membrane potential of either pre- or postsynaptic terminals or on the presynaptic spike, it is suggested that the peptide influences transmitter mobilization. However, the peptide could also exert small changes in preterminal calcium currents, which so far we have been unable to detect.     

Mechanisms of depolarizing inhibition at the crayfish giant motor synapse. I. Electrophysiology

1. Mechanisms of depolarizing synaptic inhibition were investigated at the crayfish giant motor synapse with the use of two-electrode current- and voltage-clamp techniques. Depolarizing inhibitory postsynaptic potentials (d-IPSPs) of between 5 and 15 mV in amplitude are produced there in the motor giant motoneuron (MoG) by motor giant inhibitor (MoGI) interneurons. 2. Three mechanisms of inhibition are activated by the d-IPSP: inactivation of a voltage-sensitive inward current (probably sodium), activation of the delayed rectifier, and reverse bias of the electrically rectifying giant motor synapse (GMS). These mechanisms supplement the inhibition produced by a gamma-aminobutyric acid (GABA)-mediated increase in postsynaptic conductance. 3. The d-IPSP is produced by a fast-rising increase in postsynaptic membrane conductance that peaks at 10 microS and lasts nearly 100 ms. 4. An 8-ms, 10-mV depolarizing prepulse inactivated 90% of the inward current evoked by a subsequent step to 33 mV above rest potential, which was -70 mV. d-IPSPs having similar amplitudes should have similar effects on the inward current evoked by an excitatory postsynaptic potential (EPSP). 5. The input resistance of MoG decreased by greater than 60% when the cell was depolarized to 11 mV above rest. This resistance change corresponds to delayed rectification, which should also contribute to the increase in input conductance during a d-IPSP. 6. Depolarization of MoG by 10 mV reduced the excitatory postsynaptic current through the GMS by up to 30%. The reduction in synaptic current occurs because postsynaptic depolarization reduces the transynaptic driving force and increases the reverse bias of the electrically rectifying synapse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depression and recovery of transmission at the squid giant synapse.

1. The process of synaptic depression and recovery were studied in the squid (Loligo pealii) giant synapse with intracellular recording and stimulating electrodes in the prescence of tetrodotoxin (10-minus 7 M). 2. When the synapse was stimulated at 50 Hz, depression occurred rapidly. Recovery after the tetanus was a first-order process with an average recovery time constant of 4-9 sec. The rate of recovery was independent of the amplitude of the post-synaptic potential (p.s.p.) or the degree of depression. 3. For the first five to seven p.s.p.s in the train there was a linear relationship between depression and the total amount of transmitter previously released. This may indicate that depression in this preparation was caused by the depletion of the presynaptic store of transmitter (S). 4. Assuming that this interpretation was correct, we could show that recovery from depression during the tetanus (i.e. 'mobilization') proceeded about 10 times faster than after the end of the tetanus. 5. When the amplitude of the p.s.p. was varied by changing the bathing calcium concentration, [Ca], the degree of depression was correlated to the amplitude of the p.s.p. 6. When the amplitude of the p.s.p. was increased by increasing pre-synaptic depolarization, synaptic depression was found to increase as well. However, synaptic depression increased less than the amplitude of the p.s.p., the relationship between these two measures being non-linear. 7. This finding is interpreted to indicate that the transmitter stores, S, are closely related to the area of the presynaptic membrane which is sufficiently depolarized to release transmitter.     

Mechanisms of depolarizing inhibition at the crayfish giant motor synapse. II. Quantitative reconstruction

1. The relative strengths of four mechanisms of depolarizing synaptic inhibition described in the previous paper were evaluated with an electrical model of the giant motor synapse (GMS) and postsynaptic region of the motor giant motoneuron (MoG). 2. The model consists of one compartment that represents the presynaptic region of the medial giant (MG) interneuron and three compartments that represent the postsynaptic region and proximal axon of the MoG. The presynaptic MG compartment is linked to a postsynaptic MoG compartment by a rectifying conductance that represents the GMS. Each compartment consists of parallel paths to ground for active and/or passive membrane currents. 3. Parameter values of the model were set so the MG compartment would replicate an MG impulse and the MoG compartments would replicate the current-clamp, voltage-clamp, and synaptic responses of a single MoG neuron described in the previous paper. The Hodgkin-Huxley equations described voltage-sensitive sodium and potassium currents. 4. Comparison of the MoG compartment currents that mediate an inhibited excitatory postsynaptic potential (EPSP) [triggered during a depolarizing inhibitory postsynaptic potential (d-IPSP)] with those of an uninhibited EPSP indicate that all four mechanisms have significant inhibitory effects. Reverse bias of the GMS by the d-IPSP reduced the GMS current by 65 nA (12%). The remaining inward current was further reduced by a 243-nA outward current through the inhibitory postsynaptic conductance. The d-IPSP inactivated sodium conductance so the inward sodium current evoked by the EPSP was reduced by 319 nA (-68%). The d-IPSP reduced the latency for potassium activation by the EPSP so that the outward potassium current coincided with the inward sodium current and reduced the net inward current by 100 nA. Together, these mechanisms reduced the EPSP amplitude by 69%. 5. The resting potential of MoG is normally 15 mV more positive than MG rest potential, but in some preparations this difference may be as much as 25 mV or as little as 0 mV. Corresponding differences in the rest potentials of the MoG and MG models have little effect on the amplitude of the model MoG EPSP because changes in the inward synaptic and sodium currents are balanced by corresponding changes in the outward potassium current.     

Presynaptic inhibition: the mechanism of protection from habituation of the crayfish lateral giant fibre escape response.

1. Mechanism of protection from habituation of the lateral giant escape reflex of the crayfish was studied. Experiments were designed to determine whether presynaptic inhibition of primary afferents for the reflex occurs following escape command neurone firing, and if so, whether it could account for protection of the first synapse from depression. 2. Synaptic transmission between afferents and interneurone A of the escape reflex is strongly inhibited following giant fibre spikes. 3. Giant fibre firing results in post-synaptic inhibition of interneurone A. However, inhibition of afferent input to interneurone A consistently outlasts both i.p.s.p.s and post-synaptic conductance increases in the neurone; the inhibition, therefore, is probably not exclusively post-synaptic. 4. Giant fibre firing results in excitability changes in sensory afferent terminals as measured by the amplitude of antidromic compound action potentials to focal electrical stimuli applied in the region of afferent terminals in the last abdominal ganglion. The time course of this effect parallels those of protection and inhibition of the first synapse. 5. The magnitude and time course of protection and inhibition of transmission to interneurone A parallel each other closely. Both processes considerably outlast measurable signs of post-synaptic inhibition. 6. We conclude that following giant fibre activity the first synapse of the lateral giant reflex is presynaptically inhibited and the presynaptic inhibition is responsible for the protection effect described in the preceding paper.     

Facilitation of transmission at the crayfish neuromuscular synapse by a convulsant phenol

The effects of the convulsant drug 4-Cl phenol on synaptic transmission were studied in the opener muscle of the crayfish walking leg. 4-Cl phenol was found to increase the amplitude of the excitatory postsynaptic potential without affecting the resting potential or input resistance of the muscle fiber. The drug did not change the frequency of spontaneous miniature postsynaptic potentials in K+-depolarized fibers. The postsynaptic voltage response to bath-applied glutamate (the excitatory transmitter compound) was decreased while the Cl(-) -conductance increase related to the action of bath-applied gamma-aminobutyric acid (the inhibitory transmitter) was not affected. In the light of previous results obtained on crayfish axons it is concluded that convulsant phenols induce an increase in the evoked release of transmitter by increasing the duration of the presynaptic depolarization through a block of voltage-dependent potassium channels.     

Extracellular potassium and trasmitter release at the giant synapse of squid.

1. The effects of changes in extracellular K concentration, [K]0, on synaptic transmission were studied at the squid giant synapse with intracellular recording from the presynaptic terminal and post-synaptic axon. 2. The amplitudes of both the presynaptic spike and the e.p.s.p. varied inversely with [K]0. On the average, a 10 mV change in spike height was accompanied by a 3-1 mV change in e.p.s.p. amplitude. 3. The amplitude of the presynaptic spike after-hyperpolarization (AH) varied inversely with [K]0. On the average, increasing [K]0 resulted in a 20% change in e.p.s.p. amplitude per mV change in presynaptic spike AH. 4. Repetitive antidromic stimulation of the post-synaptic giant axon resulted in an exponential decline in the post-synaptic spike AH, a depolarization of the presynaptic membrane potential and a reduction in the AHs of presynaptic spikes. This suggests that the K which accumulates in the extracellular spaces around the post-synaptic axon also affects the presynaptic terminal. 5. Repetitive antidromic stimulation of the post-synaptic axon resulted in a reduction in the amplitude of e.p.s.p.s. elicted by stimulation of the presynaptic axon. The reduction in e.p.s.p. amplitude relative to the change in presynaptic spike AH was quantitatively close to the change produced by increasing [K]0, suggesting that the reduction in e.p.s.p. amplitude is due to the accumulation of extracellular K at the presynaptic terminal. 6. Repetitive stimulation of the presynaptic axon reduced the amplitudes of the e.p.s.p. and the presynaptic spike AH. On the average, a 1 mV change in presynaptic spike AH was accompanied by a 204% change in e.p.s.p. amplitude, suggesting that K accumulation may only contribute to a small extent, under these conditions, to the depression of transmitter release.     

Cyclic AMP mediates serotonin-induced synaptic enhancement of lateral giant interneuron of the crayfish

The lateral giant (LG)-mediated escape behavior of the crayfish habituates readily on repetitive sensory stimulation. Recent studies suggested that the biogenic amines serotonin and octopamine modulate the time course of recovery and/or re-depression of the LG response after habituation. However, little is known of how serotonin and octopamine effect LG habituation and what second-messenger cascades they may activate. To investigate the effect of biogenic amines on LG habituation, serotonin and octopamine were superfused before presenting repetitive sensory stimulation. Serotonin and octopamine increased the number of stimuli needed to habituate the LG response. Their effects were mimicked by mixed application of a cAMP analogue [8-(4-chlorophenylthio)-cAMP (CPT-cAMP)] and a phosphodiesterase inhibitor [3-isobutyl-1-methylxanthine (IBMX)] but not by a cGMP analogue (8-bromoguanosine 3',5'-cyclic monophosphate). Perfusion of the adenylate cyclase inhibitor (SQ22536) abolished the effect of serotonin but not that of octopamine. To investigate the site of action of each biogenic amines in the neural circuit meditating LG escape, the effect of drugs on directly and indirectly elicited postsynaptic potentials in LG was investigated. Serotonin, octopamine, and a mixture of CPT-cAMP and IBMX increased both the direct and indirect synaptic inputs. Simultaneous application of SQ22536 abolished the effect of serotonin on both inputs but did not block the effect of octopamine. Direct injection of the cAMP analogue (Sp-isomer of adenosine-3',5'-cyclic monophosphorothioate) into LG increased both the direct and indirect inputs to LG. These results indicate that serotonin mediates an increase in cAMP levels in LG, but octopamine acts independently of cAMP and cGMP.     

Effect of glycine on synaptic transmission at the third order giant synapse of the squids Alloteuthis subulata and Loligo vulgaris

An in vitro slice preparation was used for intracellular recording from rat central nucleus of inferior colliculus neurons (CNIC). Stable intracellular recordings were made on 184 neurons, 27 of which were successfully stained with iontophoresed biocytin. Twenty one of those were classified as flat (F), four as less flat (LF) and the remaining two neurons as unclassified neurons. Recording in response to depolarizing current steps revealed five firing patterns: onset, adapting (regularly and irregularly), regular and bursting. The onset neurons showed non-linear current-voltage (I-V) relationship, whereas the rest had linear current-voltage relationship. Preconditioning hyperpolarizations changed the response pattern of CNIC neurons. It is concluded that there is no anatomy and physiology correlation and the levels of membrane potential may be important in the production of some firing patterns.     

Cholinergic mechanisms in the reticular control of transmission in the cat lateral geniculate nucleus

1. We examined the hypothesis that the ascending reticular arousal system influences thalamic transmission through a cholinergic mechanism. Extra- and intracellular recordings were obtained from neurons of the dorsal lateral geniculate nucleus (LGNd) and the perigeniculate nucleus (PGN) of cats anesthetized either with N2O and pentobarbital or with N2O and halothane. We compared the effects that electrical stimulation of the mesencephalic reticular formation (MRF) and ionophoretically applied acetylcholine (ACh) have on spontaneous and evoked activity of individual neurons and tested whether these effects could be antagonized by ionophoretic administration of the muscarinic receptor blocker scopolamine. The effects of ionophoretically applied glutamate (GLU), N-methyl-D-aspartate, and bicuculline were examined in addition. 2. The prominent effects in LGNd relay cells of both ACh application and of MRF stimulation were an enhancement of the resting discharge, a facilitation of the excitatory responses to light, a reduction of the amplitude and duration of evoked inhibitory episodes, and a blockade of postinhibitory rebound burst. These latter effects resembled those induced with bicuculline. Under barbiturate anesthesia neither ACh application nor MRF stimulation elicited discharges when the excitatory input from the retina was blocked. Ionophoretic application of hte muscarinic antagonist scopolamine abolished the effects of ACh ionophoresis in all relay cells tested (n = 20), and in 10 cells it also antagonized completely the effects of MRF stimulation. In the remaining cells scopolamine reduced the effects of MRF stimulation. 3. Increasing the depth of anesthesia reduced or abolished the effects of ACh application and MRF stimulation on the cells' resting activity but did not interfere with the facilitation of evoked responses. 4. The effects of the excitatory amino acids GLU and NMDA differed from those of MRF stimulation and ACh application, since the former always enhanced both spontaneous and evoked discharges but neither shortened phases of evoked inhibition nor abolished postinhibitory rebound bursts. 5. There was a high correlation between the effectiveness of MRF stimulation and ACh application in individual neurons. On the average, the facilitation of evoked responses was more pronounced in X- than in Y-cells, and the fraction of cells responding with an increase of resting activity to both procedures was considerably higher among X- than among Y-cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The development of transmission at an identified molluscan synapse. II. A quantal analysis of transmission

1. A quantal analysis of transmission at the identified molluscan synapse, V2-RPr1, was performed during development. The study was intended to determine the pre- and postsynaptic contributions to the marked changes in transmitter release described in the previous report. 2. The success of the quantal analysis was predicated on overcoming the problems associated with extending the quantal analysis technique to central synapses. This involved adopting the following strategies: 1) using a low-noise recording system coupled with electrical filtering; 2) establishing objective criteria for failures recognition; and 3) using three methods to determine the quantal content: amplitude histograms, failures analysis, and the coefficient of variation. 3. The correlation of the results obtained from an analysis of amplitude histograms and from failures analysis were highly significant (P less than 0.01) at all times studied. A similar significant correlation was observed between the failures method and the coefficient of variation methods. 4. The amplitude of the quantal unit declined progressively during development (range: 131-25 microV), in parallel with the decrease in the postsynaptic input resistance (range: 103-5 M omega). 5. At both frequencies of stimulation (0.02 and 0.2 Hz), there is an approximately 20-fold increase in quantal content over the period of the study. Frequency facilitation at the synapse is due to an increase in quantal content. 6. Possible structural correlates for the developmental increase in quantal content were discussed.     

Postsynaptic factors controlling the shape of potentials at the squid giant synapse

The roles of rectification and cable properties of the squid giant axon in determining the shape of synaptic potentials generated at the giant synapse were investigated. Excitatory postsynaptic potentials were recorded in response to selective stimulation of the main presynaptic axon at various temperatures. Excitatory postsynaptic potentials elicited at low temperatures (less than 18 degrees C) exhibited a marked after-hyperpolarization or undershoot, while those recorded at higher temperatures did not. The postsynaptic current, recorded under voltage clamp conditions, did not show an undershoot. Furthermore, intracellular injection of tetraethylammonium chloride, to block the voltage-dependent rise in potassium conductance, also eliminated the undershoot of the excitatory postsynaptic potential. These results indicate that the duration of synaptic potentials at the squid giant synapse is reduced by rectification due to a delayed rise in potassium conductance. Computer simulations of these synaptic potentials suggested that the effects of rectification will be more prominent in spherical (isopotential) cells than in cells with more complicated geometries.