Analysis of evoked and spontaneous quantal release at high pressure in crustacean excitatory synapses

The cellular mechanisms underlying the effect of high pressure on synaptic transmission were studied in the opener muscle of the lobster walking leg. Excitatory postsynaptic currents (EPSCs) were recorded using a loose macropatch-clamp technique at normal pressure and 3.5, 6.9 MPa helium pressure. Responses of the single excitatory axon could be grouped into two types: low-yield (L) synapses exhibiting small EPSCs with a considerable number of failures, and high-yield (H) synapses having larger EPSCs with very few failures. High pressure reduced the average EPSC amplitude in all synapses and shifted their amplitude histograms to the left by decreasing the quantal content (m) without changing their quantum current (q). A binomial distribution fit of EPSC amplitudes revealed that high pressure greatly decreased n, the number of available active zones, but the effect on p, the probability of release for each zone, was not consistent. Many of the spontaneous miniature EPSCs (mEPSCs), observed only in L-type synapses, were giant (size=2–5 q). High pressure increased the frequency of the giant mEPSCs but had little effect on their amplitude histogram. High pressure depressed evoked synaptic transmission by modulating the presynaptic quantal release parameters, but concomitantly enhanced spontaneous quantal release by an unknown mechanism.     

Synapsin I injected presynaptically into goldfish mauthner axons reduces quantal synaptic transmission

1. Synapsin I was injected into a vertebrate presynaptic axon to analyze its action on quantal synaptic transmission. Two microelectrodes were used for simultaneous intracellular recording from pairs of identified neurons in the goldfish brain. The postsynaptic electrode was placed in a cranial relay neuron (CRN) within 100 microns of its synapse with the Mauthner neuron. The presynaptic electrode impaled the Mauthner axon (M-axon) 50-200 microns from the first electrode. 2. Spontaneous miniature excitatory postsynaptic potentials (mEPSPs) and evoked postsynaptic potentials (EPSPs) were recorded at steady states before and after synapsin I was microinjected into the presynaptic M-axon. Responses were digitized and subsequently analyzed by computer for quantal parameters. 3. In 12 experiments, injection of synapsin I resulted in a reduction in transmission. The decrease in EPSP amplitude began approximately 30 s after the injection, reached a plateau within 10 min, and appeared to be reversible and dose dependent. 4. Injection of synapsin I decreased quantal content (m), with no effect on postsynaptic receptor sensitivity or on amount of transmitter per quantum. Further analysis based on the simplest binomial model for quantal release revealed that synapsin I consistently reduced the number of quantal units available for release (n) although the probability of release (p) was either unchanged or slightly increased. Injected synapsin I may thus bind to presynaptic vesicles and prevent transmitter quanta from entering a pool subject to evoked release.     

Effects of ethanol and other drugs on excitatory and inhibitory neurotransmission in the crayfish.

1. Crayfish exposed to 434 mM ethanol (EtOH) showed signs of hyperactivity within 0.5-2 h, at which times crayfish hemolymph EtOH concentration had reached 60-90 mM. 2. A 10-min exposure to 60-90 mM EtOH reduced presynaptic inhibition of excitatory postsynaptic currents (EPSCs) at the crayfish opener neuromuscular junction (NMJ) in vitro but did not significantly alter excitatory neurotransmission. The same concentrations of EtOH did not alter other potentials or currents associated with inhibition at this synapse, such as presynaptic inhibitory potentials (PIPs), inhibitory postsynaptic potentials (IPSPs), and inhibitory postsynaptic currents (IPSCs). 3. Intermediate EtOH concentrations (120-180 mM) applied for 10 min in vitro reduced the amplitude of excitatory postsynaptic potentials (EPSPs) by decreasing the membrane resistance of opener muscle fibers and by reducing the amplitude of EPSCs. 4. High EtOH concentrations (434 mM) applied for 10 min in vitro had yet greater depressive effects on measures of postsynaptic properties described above. The time course of EPSCs was also significantly reduced. In addition, presynaptic properties such as action-potential (AP) amplitude and frequency of spontaneous release of neurotransmitter were reduced by 434 mM EtOH. 5. Presynaptic inhibition, gamma-aminobutyric acid (GABA; 250-500 microM), muscimol (50 microM), and baclofen (75 microM) all reduced the depolarizing afterpotential of APs in the excitor axon and reduced EPSPs in opener muscle fibers. GABA (500 microM) and baclofen (75 microM) significantly reduced presynaptic AP amplitudes, whereas presynaptic inhibition, GABA (250 microM), and muscimol (50 microM) had no effect on AP amplitude. Bicuculline (250-500 microM), a GABAA antagonist, did not entirely eliminate presynaptic inhibition, whereas picrotoxin (50 microM), another GABAA antagonist, completely removed presynaptic inhibition. Thus presynaptic inhibitory mechanisms may involve both GABAA and GABAB receptors on the opener excitor axon. 6. Our data suggest that the behavioral hyperactivity seen at hemolymph EtOH concentrations of 60-90 mM is not accompanied by a change in excitatory synaptic transmission observed at the opener NMJ. Rather, crayfish hyperactivity may be due to depressive effects of EtOH on inhibitory synapses in the CNS similar to the disinhibition evoked by EtOH at the opener NMJ.     

Synaptic depression related to presynaptic axon conduction block.

1. The depression of synaptic transmission, which occurs during prolonged repetitive activation, was examined in the opener muscle of the crayfish walking leg. 2. Excitatory post-synaptic potentials (e.p.s.p.s) initially facilitated but then declined to low amplitudes after about 4000 stimulus pulses had been delivered; this depression is presynaptic in origin; 3. Axon conduction blocks occured at points of bifurcation along the entire length of the presynaptic nerve. This resulted in failure of the nerve impulse to invade some branches of the terminal arborization. 4. Nerve terminal invasion failure caused either intermittent or complete inactiviation of some synaptic release sites; this was associated with depression of the post-synaptic response. 5. The statistics of transmitter release during prolonged repetitive stimulation were examined by focal extracellular recording methods. Transmitter release could be described by binomial statistics, and depression involved a drop in m, n and p. 6. The rate of spontaneous quantal release did not decrease, however, arguing against transmitter depletion. 7. It is concluded that repetitive stimulation eventually leads to depolarization of the axon membrane. This causes impulse propagation failure which reduces the number of synaptic release sites that are activated and mimics a drop in the effective stimulation rate; both effects cause synaptic depression.     

Phosphatidylinositol system's role in serotonin-induced facilitation at the crayfish neuromuscular junction

1. In a crustacean neuromuscular preparation, the walking leg opener muscle of the freshwater crayfish Procambarus clarkii, application of serotonin (1 microM) produces presynaptic depolarization and long-lasting facilitation of excitatory postsynaptic potentials (EPSPs). The frequency of spontaneously released transmitter quanta also increases. Facilitation of evoked EPSPs declines after serotonin application in two phases. 2. Serotonin-induced facilitation was examined using simultaneous pre- and postsynaptic intracellular microelectrode recording. A presynaptic microelectrode recorded action potentials and membrane potential of a presynaptic axonal branch, and one or more postsynaptic microelectrodes recorded EPSPs in muscle fibers innervated by the excitatory motor axon. Components of the phosphatidylinositol second messenger system and pharmacologic agents affecting this system were injected through the presynaptic electrode, and changes in synaptic transmission were measured. 3. Presynaptic injection of inositol 1,4,5-triphosphate (IP3) causes presynaptic depolarization, increases the frequency of spontaneously released transmitter quanta, and promotes a relatively short-lasting facilitation of evoked EPSPs. These actions are consistent with elevation of intracellular Ca2+ and resemble the early phase of serotonin-induced facilitation. 4. Application of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), that activates protein kinase C (C-kinase), produces a long-lasting, low-level facilitation of evoked EPSPs. Application of another phorbol ester, phorbol-12-monoacetate (PTMA), which does not activate C-kinase has no effect. 5. Presynaptic injection of RA 233, a phospholipase C (PLP-C) inhibitor, blocks all aspects of serotonin-induced facilitation. This compound was found to have no general deleterious effects on synaptic transmission and does not block other forms of synaptic facilitation in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crayfish neuromuscular facilitation activated by constant presynaptic action potentials and depolarizing pulses

1. Experiments were conducted to test the hypothesis that facilitation of transmitter release in response to repetitive stimulation of the exciter motor axon to the crayfish claw opener muscle is due to an increase in the amplitude or duration of the action potential in presynaptic terminals. No consistent changes were found in the nerve terminal potential (n.t.p.) recorded extracellularly at synaptic sites on the surface of muscle fibres.2. Apparent changes in n.t.p. are attributed to three causes.(i) Some recordings are shown to be contaminated by non-specific muscle responses which grow during facilitation.(ii) Some averaged n.t.p.s exhibit opposite changes in amplitude and duration which suggest a change in the synchrony of presynaptic nerve impulses at different frequencies.(iii) Some changes in n.t.p. are blocked by gamma-methyl glutamate, an antagonist of the post-synaptic receptor, which suggests that these changes are caused by small muscle movements.3. The only change in n.t.p. believed to represent an actual change in the intracellular signal is a reduction in n.t.p. amplitude to the second of two stimuli separated by a brief interval.4. Tetra-ethyl ammonium ions increase synaptic transmission about 20% and prolong the n.t.p. about 15%. This result suggests that an increase in n.t.p. large enough to increase transmission by the several hundred per cent occurring during facilitation would be detected.5. The nerve terminals are electrically excitable, and most synaptic sites have a diphasic or triphasic n.t.p., which suggests that the motor neurone terminals are actively invaded by nerve impulses.6. When nerve impulses are blocked in tetrodotoxin, depolarization of nerve terminals increases the frequency of miniature excitatory junctional potentials (e.j.p.s), and a phasic e.j.p. can be evoked by large, brief depolarizing pulses. Responses to repetitive or paired depolarizations of constant amplitude and duration exhibit a facilitation similar to that of e.j.p.s evoked by nerve impulses.7. It is concluded that facilitation in the crayfish claw opener is not due to a change in the presynaptic action potential, but is due to some change at a later step in the depolarization-secretion process.     

Reciprocal axo-axonal synapses between the common inhibitor and excitor motoneurons in crustacean limb muscles

Summary Nerve terminals of the common inhibitor motoneuron in a crab (Eriphia spinifrons) limb closer muscle and in a crayfish (Procambarus clarkii) limb accessory flexor muscle make neuromuscular synapses with the muscle membrane (postsynaptic inhibition) as well as axo-axonal synapses with the terminals of the excitatory axon (presynaptic inhibition). That transmission is from the inhibitor to the excitor terminals at these axo-axonal synapses is indicated by the occurrence on the inhibitor membrane of presynaptic dense bars denoting sites of transmitter release. Axo-axonal synapses with the opposite polarity, in which transmission is from an excitatory onto an inhibitory terminal, were occasionally seen either adjacent to or separate from the inhibitory axo-axonal synapse. Nerve terminals of the specific inhibitor in the crayfish opener muscle were seen to make numerous axo-axonal output synapses upon excitatory nerve terminals but excitor nerve terminals were not seen to make output synapses onto inhibitor terminals. Thus reciprocal axo-axonal synapses appear to be a feature of the common inhibitor but not of the specific inhibitor. The excitor-to-inhibitor component of these reciprocal synapses may serve to limit transmitter output in the common inhibitor axon by activating glutamate_B receptors which facilitate efflux of K⁺ and hyperpolarization of the membrane.     

The binomial nature of transmitter release at the crayfish neuromuscular junction

1. Transmitter release at excitatory junctions on the opener muscle of the crayfish dactyl was studied by recording junctional potentials with extracellular micro-electrodes.2. At low temperatures, evoked release was dispersed sufficiently in time for potentials produced by individual quanta to be counted, and the mean (m) and variance (sigma(2)) of the quantum content distribution for a series of trials measured directly. These values were used to calculate the average probability of quantal release (p), assuming a binomial distribution.3. For all values of m and p, the observed release pattern (number of 0, 1, 2, 3,... quantal releases during a series of trials) was approximated closely by the corresponding binomial distribution. However, Poisson predictions differed significantly from the observed quantal distribution for values of p > 0.2.     

Excitatory potentials induced by stimulation of the inhibitory axon at the crustacean neuromuscular junction.

Synaptic events in a chloride-deficient condition were studied to elucidate functional aspects of presynaptic inhibitory synapses. The extracellular junctional potentials and nerve terminal potentials were concurrently recorded from a synaptic region. Inhibitory stimulation produced repetitive spikes on the inhibitory nerve terminal and then the excitatory nerve terminal, which resulted in the extracellular excitatory junctional potentials. Excitatory stimulation did not produce repetitive spikes on the inhibitory nerve terminal, indicating one-way signal transmission in this axo-axonal synapse from inhibitory to excitatory axon. The interval required for an inhibitory stimulation to produce the first response in the postsynaptic muscle membrane ranged widely from 10 to 800 msec. When gamma-aminobutyric acid (GABA, 1 times 10-minus 4 M) was added in these experimental conditions, the muscle membrane was transiently depolarized by about 10 mV. The action of GABA mimics that of the neurotransmitter at presynaptic inhibitory synapses. The experimental observations may be well explained by the concept of synapses on synapses, i.e., presynaptic inhibition, where the neurotransmitter may be GABA and chloride ions may be playing essential roles as in the case of postsynaptic inhibition.     

High susceptibility to hypoxia of afferent synaptic transmission in the goldfish sacculus

1. The effect of hypoxia on synaptic transmission between hair cells and afferent fibers was examined in the sacculus of goldfish. For this, we recorded potentials, intracellularly, from large afferent fibers. Anoxia was introduced by perfusing the gill with water deprived of oxygen or by halting the water flow to the gill. 2. The ear of the goldfish is most sensitive to hypoxia. Sound-evoked afferent activities were profoundly depressed within several minutes after the introduction of hypoxia. 3. The depressed afferent activity was attributed to a reduction in the amplitude of sound-evoked excitatory postsynaptic potentials (EPSPs) generated at afferent fiber terminals, since no significant change was detected in the resting and action potentials of afferent fibers or in intensity of the threshold current required to set up an action potential. Also, there was no marked change in the electrical activity of hair cells, determined by the finding that the amplitude of intramacularly recorded microphonic potentials and that of the coupling potentials was not altered. 4. A statistical analysis of the amplitude of sound-evoked EPSPs revealed that the binomial parameter n decreased during hypoxia, in parallel with a reduction in the amplitude of EPSPs, while the binomial parameter p either remained unaltered or was augmented. No change was found in the quantal size, thereby indicating that the sensitivity of the postsynaptic membrane remained unchanged. These results indicate that presynaptic mechanisms within hair cells, especially those playing a role in transmitter release or in replenishment of the latter, are suppressed during hypoxia.     

Estimation of single channel conductance underlying synaptic transmission between pyramidal cells in the visual cortex

Axon collaterals originating from pyramidal cells are one of the most abundant presynaptic elements in the neocortical circuits. To understand a quantitative aspect of synaptic transmission between pyramidal cells, we attempted to estimate single channel conductance by applying non-stationary noise analysis to unitary excitatory postsynaptic currents. Simultaneous recordings were carried out in two pyramidal cells of superficial layers in visual cortical slices. Unitary postsynaptic currents, which were evoked by action potentials of presynaptic cells impaled with conventional sharp electrodes, were recorded from postsynaptic cells with whole-cell patch clamp techniques. Estimated single channel conductance was 12.8 3.8(S.D.) pS for kittens and 10.4 +/- 1.5 pS for rats. Dividing these values by the conductance for unitary postsynaptic currents, we calculated the number of non-N-methyl-D-aspartate receptor channels activated during the postsynaptic currents. The obtained estimates were 52 (kittens) and 41 (rats). To further estimate the number of channels involved in each quantal event, we analysed amplitude histograms of miniature and spike-evoked excitatory postsynaptic currents. The derived number of estimates from these two kinds of histograms agreed quite well; about 20 channels were required for individual quantal events. Assuming open probability of non-N-methyl-D-aspartate receptor channels to be 0.7, our results suggest that the number of channels available for synaptic transmission between individual pyramidal cells would be 74 (kittens) and 59 (rats). We propose that at pyramidal-pyramidal synapses, the number of open channels is several times smaller than that previously reported for the synapses between geniculo-cortical afferent and layer IV spiny stellate cells.     

Matching of facilitation at the neuromuscular junction of the lobster: a possible case for influence of muscle on nerve

1. The facilitation of neuromuscular transmission, which occurs during repetitive activation, was examined in the proximal accessory flexor muscle in walking legs of the lobster using electrophysiological techniques.2. Post-synaptic potentials (p.s.p.s) in different muscle fibres facilitated to markedly different degrees. P.s.p.s in some fibres did not facilitate at all, while in others they increased in size by 20-30 times during stimulation at 20 Hz even though all the excitatory neuromuscular synapses are made by a single axon.3. Stimulation of widely separated groups of synapses on any single muscle fibre evoked p.s.p.s with closely matched facilitation properties. Extracellular p.s.p.s recorded from single synaptic spots showed the same characteristics of facilitation as those of intracellular p.s.p.s in the same muscle fibre, suggesting that individual synaptic contacts on any single fibre are similar to each other.4. Facilitation can be accounted for by an increase in the number of quanta released from the nerve terminals. There is no evidence for an increase in post-synaptic membrane sensitivity.5. Low Ca solutions reduce transmitter release with comparatively little change in facilitation, while Cs solutions increase the size of p.s.p.s without increasing the amplitude of spontaneous miniature potentials. Thus, at poorly facilitating synapses it is unlikely that the absence of facilitation is caused by the saturation of some post-synaptic process.6. It is concluded that the excitatory presynaptic nerve terminals on a single muscle fibre have matching facilitation characteristics. Some interaction between individual muscle fibres and their associated nerve endings may be required to establish or maintain this matching.     

The effects of calcium and magnesium on statistical release parameters at the crayfish neuromuscular junction

1. Transmitter release at excitatory neuromuscular junctions of crayfish muscle was studied at low temperature by recording synaptic potentials with extracellular micro-electrodes.2. Increasing the Ca concentration in the bathing solution produced an increase in the average number of quanta released per nerve stimulus (m). Increasing the Mg concentration resulted in a decrease in m.3. Statistical analysis of fluctuations in the quantal release from trial to trial, assuming binomial statistics, indicated that both the changes in m were due to changes in the average quantal release probability (p).     

Correlation of presynaptic and postsynaptic events during establishment of long-term facilitation at crayfish neuromuscular junction

Repetitive stimulation (10-20 Hz) of the motor axon supplying the opener muscle in the crayfish leg produces long-lasting enhancement of excitatory postsynaptic potentials. This long-term facilitation (LTF) was investigated by recording simultaneously from the presynaptic nerve terminal and from the innervated muscle fiber with intracellular microelectrodes. On cessation of stimulation, the facilitated postsynaptic potential declines in amplitude when monitored with low-frequency test stimuli. A rapid decline (phase I) occurs over the first 30 s and is succeeded by a more gradual decline lasting several minutes (phase II). Finally, a residual potentiation with a very slow decay (phase III) persists for several hours. Simultaneous pre- and postsynaptic recordings were made during induction of LTF with stimuli delivered at 20 Hz for 10 min. During the tetanus, excitatory postsynaptic potentials were enhanced 20-fold, while action potentials in the presynaptic terminal declined in amplitude from 108.6 to 97.2 mV, and the presynaptic membrane became hyperpolarized by 6.4 mV. The Na+ pump inhibitor ouabain (0.5-1.0 mM) abolished the hyperpolarization, indicating that the latter resulted from activation of an electrogenic Na+ pump. The reduction in amplitude of the presynaptic action potential was consistent with a reduced transmembrane concentration gradient for Na+. Thus, it is suggested that a significant accumulation of Na+ occurs during repetitive stimulation of crayfish motor axons. Decay of phase II of LTF, but not of phases I or III, had approximately the same time course as the decay of Na+ accumulation in the terminals, monitored by changes in the presynaptic action potential. Thus it is probable that in crayfish this phase of LTF is linked to an increased intraterminal Na+ concentration. Injection of Na+ from a microelectrode into the presynaptic terminal produced enhancement of the excitatory postsynaptic potential lasting for many minutes, as well as changes in presynaptic membrane potential and action potential similar to those seen during repetitive stimulation. The results provide the first direct measurements of electrical and ionic changes in axonal terminals during prolonged periods of activity leading to LTF, and support the hypothesis that accumulation of intraterminal Na+ is associated with one phase of LTF.     

Early activation of the primary somatosensory cortex without conscious awareness of somatosensory stimuli in tumor patients

While G-proteins are involved in the synaptic release machinery and also can mediate inhibition of presynaptic Ca2+ channels, we find that pertussis toxin (PTX) does not affect the amount and the time course of quantal release from motor nerve terminals on crayfish or mouse muscle. Monoquantal excitatory currents (qEPSCs) were recorded that were elicited by constant depolarisation pulses to a terminal by means of a perfused macro-patch electrode. Although presynaptic effects of PTX on output and time course of release of quanta were absent, postsynaptically the rise time of qEPCs was increased and their decay time constant reduced. Adenosine (Ad) is known to inhibit quantal release in vertebrate motor nerve terminals via PTX sensitive G-proteins, and Ad is generated during nicotinic synaptic transmission by breakdown of the co-transmitter adenosine triphosphate (ATP). As reported by others, we found in mouse muscle inhibition of quantal release after application of Ad, but in addition late facilitation. Both these effects of Ad were blocked when the muscle was pre-incubated with PTX.     

Presynaptic plasticity at two giant auditory synapses in normal and deaf mice

Large calyceal synapses are often regarded as simple relay points, built for high-fidelity and high-frequency synaptic transmission and a minimal requirement for synaptic plasticity, but this view is oversimplified. Calyceal synapses can exhibit surprising activity-dependent developmental plasticity. Here we compare basal synaptic transmission and activity-dependent plasticity at two stereotypical calyceal synapses in the auditory pathway, the endbulb and the calyx of Held. Basal synaptic transmission was more powerful at the calyx than the endbulb synapse: the amplitude of evoked AMPA receptor-mediated excitatory postsynaptic currents (eEPSCs) was significantly greater at the calyx, as were the release probability, and the number of release sites. The quantal amplitude was smaller at the calyx, consistent with the smaller amplitude of spontaneous miniature EPSCs at this synapse. High-frequency trains of stimuli revealed that the calyx had a larger readily releasable pool of vesicles (RRP), less tetanic depression and less asynchronous transmitter release. Activity-dependent synaptic plasticity was assessed in congenitally deaf mutant mice ( dn/dn ). Previously we showed that a lack of synaptic activity in deaf mice increases synaptic strength at the endbulb of Held via presynaptic mechanisms. In contrast, we have now found that deafness does not affect synaptic transmission at the calyx synapse, as eEPSC and mEPSC amplitude, release probability, number of release sites, size of RRP, tetanic depression and asynchronous release were unchanged compared to normal mice. Synaptic transmission at the calyx synapse is more powerful and has less capacity for developmental plasticity compared to the endbulb synapse.     

Unitary conductance changes at teleost Mauthner cell glycinergic synapses: a voltage-clamp and pharmacologic analysis

1. The magnitude and kinetics of inhibitory postsynaptic currents (IPSCs) evoked in the goldfish Mauthner (M-) cell by intracellular stimulation of identified presynaptic interneurons (unitary responses) and by activation of the recurrent collateral network were determined with single-and double electrode voltage-clamp techniques. 2. The peak magnitude of the inhibitory conductance changes were 5610 +/- 4800 nS (mean +/- SD; n = 13) for the collateral response, and 144 +/- 44 nS (n = 7) for the unitary IPSCs. These synaptic conductances, which are due to the opening of Cl- channels, were independent of the degree of Cl- -loading of the M-cell. 3. The peak amplitude of the collateral inhibitory postsynaptic potential (IPSP) was a constant fraction (0.52 +/- 0.06) of the driving force, which was determined from current-voltage plots for both types of IPSCs and ranged from 10 to 37 mV. These findings confirm indirect measurements from previous current-clamp studies and validate the normalization procedure used to previously calculate synaptic conductances from IPSP amplitudes, a method that therefore may be applicable to other central neurons. 4. At the resting membrane potential, the rise time of the unitary IPSCs was 0.34 +/- 0.07 ms (n = 18), whereas their decay was exponential, with a time constant of 5.7 +/- 1.1 ms (n = 16). 5. Iontophoretic and intramuscular applications of the glycine antagonist strychnine reduced or blocked M-cell inhibitory responses, without altering the excitability of the presynaptic neurons, or the driving force. 6. Amplitude fluctuations of unitary IPSPs recorded during partial blockade by strychnine were analyzed according to a binomial model of quantal transmitter release. In one experimental series, comparison of the binomial parameters before and after applying the antagonist indicated that only quantal size, q, was reduced, whereas n, the number of available release units, and p, the probability of release, were unaffected by strychnine. In a second series, the individual presynaptic cells were injected with horseradish peroxidase (HRP), and it was found that the correlation between n and the number of stained presynaptic boutons and, therefore, of active zones, was maintained in the presence of the drug. No evidence was found for silent synapses in these conditions. 7. The quantal conductance, gq, was estimated from the binomially derived quantal size, in millivolts, and the voltage-clamp measurements of the IPSP driving force and M-cell input conductance. gq averaged 21.5 nS in control conditions and 12.3 nS in the presence of strychnine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ammonium decreases excitatory synaptic transmission in cat spinal cord in vivo

1. Glutamine is thought to be a precursor of the pool of glutamate that is used as synaptic transmitter. NH4+ inhibits glutaminase, the enzyme presumed to cleave glutamine into glutamate in synaptic terminals. Therefore a decrease by NH4+ of excitatory synaptic transmission in hippocampus was suggested to be due to the inability to utilize glutamine as a precursor for glutamate and subsequent transmitter depletion. This study reexamines the effects of NH4+ on excitatory synaptic transmission. 2. The effects of NH4+ on excitatory synaptic transmission from low-threshold afferent fibers, presumably Ia-afferent fibers, to motoneurons was investigated in the spinal cord of anesthetized cats in vivo. 3. Action potentials of low-threshold afferent fibers were recorded at the entry of the dorsal roots into the spinal cord. An extracellular electrode within a motoneuron nucleus recorded the action potential of low-threshold afferent fibers and the extracellular monosynaptic excitatory postsynaptic potential, i.e., the focal synaptic potential (FSP). This extracellular electrode also recorded the antidromic field potential (AFP) in response to ventral root stimulation. Electrodes on the ventral roots recorded the monosynaptic reflex (MSR) and the monosynaptic excitatory postsynaptic potential in motoneurons electrotonically conducted into the ventral roots (VR-EPSP). 4. Intravenous infusion of ammonium acetate (AA) reversibly decreased MSR, VR-EPSP, and FSP, i.e., decreased excitatory synaptic transmission. 5. The decrease of VR-EPSP and FSP was accompanied initially by a decrease of conduction and, eventually, a conduction block in presynaptic terminals of low-threshold afferent fibers. 6. The decreases of VR-EPSP and FSP were also accompanied by the transient appearance of a reflex discharge, triggered by VR-EPSPs of decreased amplitude, and changes of the AFP indicating increased invasion of motoneuron somata by antidromic action potentials. 7. It is suggested that NH4+ depolarizes intraspinal Ia-afferent fibers and motoneurons. This depolarization initially decreases and then blocks conduction of action potentials into the presynaptic terminals of Ia-afferent fibers. The conduction block prevents the release of excitatory transmitter and decreases excitatory synaptic transmission. 8. The suggested depolarizing action of NH4+ may be due to K+-like ionic properties of NH4+ and/or an inhibition of K+-uptake into astrocytes. 9. The conduction block in presynaptic terminals of low-threshold afferent fibers can fully explain the decrease of excitatory synaptic transmission by NH4+. Because of the conduction block in presynaptic terminals, this study does not permit a conclusion as to an inhibition by NH4+ fo the utilization of glutamine as a precursor for glutamate used as synaptic transmitter.     

Heterogeneous reallocation of presynaptic efficacy in recurrent excitatory circuits adapting to inactivity

Recurrent excitatory circuits face extreme challenges in balancing efficacy and stability. We recorded from CA3 pyramidal neuron pairs in rat hippocampal slice cultures to characterize synaptic and circuit-level changes in recurrent synapses resulting from long-term inactivity. Chronic tetrodotoxin treatment greatly reduced the percentage of connected CA3-CA3 neurons, but enhanced the strength of the remaining connections; presynaptic release probability sharply increased, whereas quantal size was unaltered. Connectivity was decreased in activity-deprived circuits by functional silencing of synapses, whereas three-dimensional anatomical analysis revealed no change in spine or bouton density or aggregate dendrite length. The silencing arose from enhanced Cdk5 activity and could be reverted by acute Cdk5 inhibition with roscovitine. Our results suggest that recurrent circuits adapt to chronic inactivity by reallocating presynaptic weights heterogeneously, strengthening certain connections while silencing others. This restricts synaptic output and input, preserving signaling efficacy among a subset of neuronal ensembles while protecting network stability.