Rate Of Spontaneous Release Research Articles

In vitro dopamine release from the rat striatum: diurnal rhythm and its modification by the estrous cycle.

In the present experiment we examined spontaneous endogenous in vitro dopamine release from the corpus striatum of female rats during the morning (09.00-09.30 h) and afternoon (15.00-15.30 h) photoperiod on each day of the estrous cycle. In the morning, the spontaneous dopamine release rates of D-1, D-2 and proestrous female rats were characterized by initial low values which gradually increased (approximately 3-fold) over the 2.5-hour in vitro perifusion. In the afternoon, spontaneous release rates of D-1, D-2 and estrous females gradually declined (approximately 2.5-fold) over the perifusion period. This rhythmic diurnal fluctuation was disrupted in the afternoon of proestrus and morning of estrus when release rate profiles remained stable over the entire perifusion period. These results suggest that changes in spontaneous in vitro dopamine release of corpus striata derived from rats in different phases of the estrous cycle may reflect novel in vivo interactions of both photoperiodic and hormonal cues.

Effects of dopamine and apomorphine on gonadotropin release from the transplanted pars distalis in goldfish

Male goldfish bearing pars distalis transplants from other male goldfish have increased serum gonadotropin (GtH) levels, due to the high spontaneous release rate of GtH by the transplants. Intraperitoneal injection of dopamine or its agonist, apomorphine, each reduced the elevated serum GtH levels caused by the release from the transplanted pars distalis. These results suggest that dopamine has GtH-release-inhibitory activity and acts directly on gonadotrophs to inhibit spontaneous secretion of GtH.

Interactions of lead and cadmium on acetylcholine release at the frog neuromuscular junction

The interactive effects of Cd 2+ and Pb 2+ on evoked and spontaneous transmitter release were studied in the sciatic nerve-sartorius muscle preparation of the frog ( Rana pipiens). Either Pb 2+ or Cd 2+ competitively inhibited the actions of Ca 2+ in bringing about evoked release, as measured by the endplate potential (EPP) amplitude. Combinations of Pb 2+ and Cd 2+ were additive in their effects on the EPP. The rate of spontaneous transmitter release was measured as the miniature endplate potential (MEPP) frequency. In contrast to their effects on the EPP, exposure of preparations to combinations of Pb 2+ and Cd 2+ actually increased the MEPP frequency less than exposures of the preparations to Pb 2+ alone. The degree of reduction in MEPP frequency produced by Cd 2+ depended upon the relative ratio of Pb 2+ and Cd 2+ ions. These results suggest that Pb 2+ and Cd 2+ ions competed for a common presynaptic receptor site during evoked release. They also suggest that Pb 2+ ions may enter the nerve terminal, possibly through the Ca 2+ channel, and that this entry is inhibited by Cd 2+.

Cadmium: Effects on transmitter release at the frog neuromuscular junction

Cd 2+ competitively antagonizes Ca 2+ in the evoked release of acetylcholine from nerve terminals in the frog sciatic-sartorius neuromuscular junction. The dissociation constant between Cd 2+ and its receptor sites was calculated to be 1.7 μM. In agreement with previous work we found that brief exposures to 100–500 μM Cd 2+ had little or no effect on spontaneous transmitter release. However, in contrast to other reports, prolonged exposures to 100–1000 μM Cd 2+ or tetanic nerve stimulation during brief exposures to high concentrations of Cd 2+; produced substantial increases in spontaneous transmitter release. We conclude that Cd 2+, when present at low concentrations, is a specific Ca 2+ channel blocker. At higher concentrations, Cd 2+ is either relatively impermeable to the presynaptic nerve membrane or, if it does enter the nerve terminal, it less effective than such metals as Pb 2+, La 3+ or Hg 2+ in increasing the rate of spontaneous transmitter release.

Synchronous exocytosis in Paramecium cells : III. Rearrangement of membranes and membrane-associated structural elements after exocytosis performance

Aminoethyldextran (AED) was used to trigger the synchronous release of trichocysts from Paramecium tetraurelia cells (see [8]) by a mechanism involving exocytotic membrane fusion and resealing (see [5]). Ultrastructural changes were analyzed by quantitative evaluation of ultrathin sections. In resting cells the percentage of potential trichocystdocking sites which are actually occupied by a trichocyst was 58%; 36% of potential docking sites contained ghosts and 6% a “plug” of electron-dense material. We derived from our data that paramecia would discharge permanently and spontaneously trichocysts (without AED) at a rate of 2–3 per min (which we then also verified by counting the spontaneous release rate) and that this value is equivalent to the docking rate. For the synchronous expulsion of trichocysts in response to AED we had determined that the degree of synchrony is more than a hundred times better than in most other systems (see [8]). We have determined the half-lives (HL) for different events involved in exocytosis and re-docking as follows: ~3 sec for trichocyst discharge, ~3 sec for the formation of ghosts, 8 min for the clearing of ghosts from the cell surface, 4 min for the formation of “plugs”. Trichocysts are docked with a HL of 40 min and “plugs” (considered as receptor-type structures for trichocyst docking) disappear with a concomitant HL of 50 min. Evidently the clearing of ghosts allows for re-formation of “plugs” but the respective HL values signal that “plugs” may also be formed anew. The relatively slow decline of the percentage of “plugs” (after their azimuth 15 min after AED triggering) may also indicate the synthesis of new docking sites. After a period of over ~3 h following AED triggering, the original situation is roughly re-established and maintained over the whole period of population growth analyzed.

Anion effects on in vitro sarcoplasmic reticulum function. The relationship between anions and calcium flux.

Isolated sarcoplasmic reticulum vesicles exhibited different functional characteristics in the presence of zwitterionic as compared to anionic buffers. In the absence of oxalate, dicarboxylic anions (e.g. maleate, succinate) in a dose-dependent manner enhanced ATP-supported Ca2+ accumulation, the ensuing spontaneous Ca2+ release, and Ca2+-dependent ATPase activity compared to zwitterionic buffers (e.g. piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Hepes). This was not attributed to ionic strength and osmotic effects. The additional anion-dependent Ca2+ accumulation was linked to augmented Ca2+-dependent ATPase activity, and both could be induced by the addition of anion at any time during Ca2+ accumulation as long as ATP was present. Since the initial Ca2+ accumulation rates and acyl phosphoenzyme formation were the same between the two buffer classes, and the presence of either oxalate (a Ca2+-precipitating anion) or A23187 (a Ca2+ ionophore) abolished differences in Ca2+-dependent ATPase activity between the two buffer classes, it is likely that conditions favoring high intravesicular Ca2+ concentration allow the expression of the observed effect of the anions. Initial spontaneous Ca2+ release in the presence of maleate was not caused by ATP depletion, and it was virtually absent in Pipes buffer. The rate of spontaneous release was also stimulated in a dose-dependent manner by the dicarboxylic anions, with the time of release being related to the time of anion addition and not ATP addition. A later, more rapid release phase in either maleate or Pipes buffer corresponded to ATP depletion, and could be duplicated at any time in the Ca2+ accumulation/release cycle by the addition of an ATP trap. With an ATP-regenerating system present or with very high ATP concentrations, the maximal peak Ca2+ accumulation in Pipes buffer could approach that in maleate buffer. The data suggest that dicarboxylic anions stimulate the filling of a Ca2+ compartment from which spontaneous Ca2+ release occurs.

Quercetin stimulation of calcium release from rabbit skeletal muscle sarcoplasmic reticulum

To elucidate the mechnism by which quercetin enhances the rate of tension development in skinned muscle fibers, effects on calcium release from longitudinal tubule-derived SR (LSR) after phosphate-supported calcium uptake were examined. In all studies, 100 μM quercetin (which inhibits initial calcium uptake velocity 85%) was added at or shortly after the time calcium content reached a maximum at various extravesicular Ca 2+ concentrations (Ca o). At moderate Ca o (0.2–1.0 μM). where spontaneous calcium release rate depended on Ca o, quercetin caused a marked stimulation of calcium release. This was accompanied by a 60% reduction in calcium influx and a 30-fold increase in calcium efflux. Thus, the previously reported quercetin-induced increase in the rate of tension development by skinned muscle fibers may result, at least in part, from sensitization of Ca 2+-triggered calcium release to lower Ca o.

Changes in enzymes of the cholinergic system and acetylcholine release in the cerebra of aging male Fischer rats.

The functional decline of memory in the aging human brain has been partially attributed to defects in cholinergic transmission. Therefore, we have investigated various components of the cholinergic system in cerebra of Fischer 344 male rats, ages 3-33 months. Choline acetyltransferase (ChA), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) activities were determined in homogenates of the cerebra using specific radiometric assays. For measuring the release of acetylcholine (ACh), cerebral slices were incubated for one hour in Krebs buffer containing 3H-choline chloride to label ACh formed in situ, washed, and transferred to a microbath for superfusion. 3H-ACh released into the superfusate was determined. The levels of ChA in the cerebra of 9- to 27-month-old rats were lower (33%) than those in 3-month-old rats. Only 1% of these rats survive to the age of 33 months. In rats of this age, there was no decrease in ChA levels. AChE decreased while BChE increased with advancing age. The rate of spontaneous release of 3H-ACh decreased gradually by 63% from 3 to 33 months of age. The evoked release of ACh decreased by 50% in 33-month-old rats. Alterations in the levels of ChA, AChE (or BChE) and cholinergic receptors are not large enough to account for losses in cholinergic transmission in the cerebrum. The large decreases in the rates of spontaneous or evoked release of ACh in the aging cerebrum indicates that the functional defect in the cholinergic transmission of the aging cerebrum is possibly due to a defective release mechanism of this transmitter.

Lysosomal enzyme secretion by cystic fibrosis fibroblasts is normal

Lysosomal enzyme secretion by skin fibroblasts derived from cystic fibrosis homozygotes and normal donors of the same range of age, sex and culture passage number were compared. No differences were detected between these groups in their rates of spontaneous lysosomal enzyme release. Incubation of either normal or cystic fibrosis fibroblasts with cystic fibrosis serum did not alter these secretion rates. The lysosomal enzyme secretion of both normal and cystic fibrosis fibroblasts responded similarly to incubation with NH 4Cl, monensin, A23187, trifluoperazine or Cytochalasin B. It was concluded that lysosomal enzyme hypersecretion, which has previously been suggested to occur in cystic fibrosis, is unlikely to provide a reliable diagnostic test for the disease.

Effects of hypertonic solutions on quantal transmitter release at the crayfish neuromuscular junction.

1. The effects of a hypertonic bathing medium, containing either NaCl or melezitose, on the average number (m) and the time course of quantal release following an action potential were studied using focal extracellular recording methods at synaptic sites on the opener muscle of the crayfish leg.2. After the application of hypertonic saline, the rate of spontaneous quantal release increased but m decreased to a new level within 1 min; the extent of depression depended on the magnitude of the increase in tonicity until the osmolarity was 50% greater than the normal value of 0.43 osmol/l (Osm), but greater increases in tonicity exerted little further effect.3. The synaptic delays were increased and distributed over a longer range of time in hypertonic solutions; also, the latency between the first and second quantal releases in a multiple response to a single nerve impulse was also increased.4. Hypertonicity had no significant effect on the conduction velocity of the action potential, the independence of successive quantal releases in the same response, or the uniformity of the rise and fall of the probability of quanta release following an action potential, alpha(t).5. The time course of alpha(t) is prolonged in hypertonic solutions; this was manifested as an increase in the time constant of the exponential decline in alpha(t) from its peak value following the nerve impulse.5. When the potentiating agent 5-hydroxytryptamine (5-HT) was added to the hypertonic saline, m was increased, and the time course of alpha(t) was prolonged further than in the hypertonic solution alone; 5-HT produced no change in the time course of alpha(t) when it was added to normal saline, although m was increased.6. It is concluded that in this preparation hypertonicity decreases the rate of release of each quantum from the nerve terminal following a nerve impulse.

Parallels between spontaneous release of transmitter at the neuromuscular junction and subcellular damage of muscle. Evidence for the underlying common involvement of intracellular Ca 2+?

1. 1. Parallels between the rate of spontaneous release of transmitter at the neuromuscular junction and the triggering of ultrastructural damage in cardiac and skeletal muscle cells are reviewed. 2. 2. Agents that increase release at the presynaptic terminals promote damage; treatments that may protect muscle cells also suppress transmitter release. 3. 3. It is suggested that changes in [Ca 2+] i are involved in both tissues and the possible implications for dystrophic muscle are discussed.

In vitro Release of LHRH from the Hypothalamus of Female Rats during Prepubertal Development

In this study changes in serum FSH concentration, hypothalamic LHRH content and in vitro release of LHRH from hypothalami of female Wistar rats during prepubertal development are described. The concentration of FSH in serum was determined by radioimmunoassay. Extremely high levels (1.800 ng/ml) were found between 12 and 16 days of age. Thereafter, FSH levels rapidly fell to low levels (135 ng/ml) on day 32. LHRH was measured by radioimmunoassay in extracts of mediobasal hypothalami (MBH) and showed a gradual increase during the first 10 days of life. Between 10 and 16 days of age, hypothalamic LHRH content did not change. After day 16, LHRH content sharply increased to a maximum found on day 32. The release of LHRH from MBH of developing rats was studied during in vitro incubation in Krebs-Ringer bicarbonate buffer. LHRH in the medium was measured by radioimmunoassay. The spontaneous release rate of LHRH was maximal from MBH of 12-day-old rats and minimal from those of 32 days. Depolarization of neural membranes, by medium containing 45 mM K+ induced a stimulation of LHRH release. This K+ -induced release of LHRH was also most prominent on day 12. These observations are consistent with the hypothesis that the high circulating FSH levels found in female rats between 12 and 16 days of age are at least in part caused by a high secretion rate of LHRH during this period.

Cytolytic T lymphocyte mediated chromium-51 release versus spontaneous release from blast and spleen cell targets at low temperatures

The rate of spontaneous 51Cr release from spleen cell and LPS blast target cells is strongly temperature dependent. Between 32 and 37 °C the rate of spontaneous release increases dramatically with temperature. Cytolytic T-lymphocyte-mediated lysis of these target cells is also temperature dependent, but lysis does not increase greatly above 32 °C. The ratio of specific 51Cr release to spontaneous release can be significantly improved by doing 51Cr-release assays at temperatures below 37 °C.

In vivo release of met-enkephalin in the cat brain

Push-pull cannulae were implanted into the globus pallidus (anterior part), the caudate nucleus (medio-dorsal part) and the substantia nigra of halothane-anaesthesized cats to study the characteristics of the in vivo release of met-enkephalin. Met-enkephalin was measured by radioimmunoassay (sensitivity: 0.1 pg) and identified by Bio-Gel P 2 chromatography and high-pressure liquid chromatography. Under resting conditions, met-enkephalin was detected in perfusates from these three regions; in the globus pallidus, the rate of spontaneous release of met-enkephalin was 5–6 times higher than that observed in the caudate nucleus and the substantia nigra. The local application of either an excess (60 mM) of K + or of glutamate (10 μM) produced a marked increase in the rate of met-enkephalin release both from the globus pallidus and the caudate nucleus. Further analyses in the globus pallidus indicated that the depolarizing agents, veratridine (50 μM) and batrachotoxin (0.1 μM), produced a large increment in the rate of met-enkephalin release; this effect was completely prevented by the local application of tetrodotoxin (2 μM). The chemical (with 60 mM K + or 10 μM glutamate), electrical or mechanical stimulation of the dorsomedial part of the caudate nucleus failed to significantly alter the rate of met-enkephalin release in the ipsilateral globus pallidus, in spite of the high sensitivity of enkephalinergic nerve terminals in the globus pallidus itself to local stimuli. This observation argues against the existence of a major caudatopallidal enkephalinergic pathway.

Blocking of frog endplate channels by the organic calcium antagonist D600.

The actions of the organic ‘calcium antagonist’ D600 were examined on the frog sartorius nerve-muscle preparation. D600 blocked voltage-dependent Na + and K + membrane channels in nerve and muscle, and blocked also endplate channels induced by acetylcholine (ACh). The rate of spontaneous transmitter release from motor nerve terminals was increased by D600, independently of external Ca 2+ , although the drug had little effect on transmitter release evoked by nerve impulses. Postsynaptically, miniature endplate currents were reduced in size and their decay time constant became shorter and relatively independent of membrane potential. D600 reduced the increase in ACh-induced endplate current seen with membrane hyperpolarization, and with paired ACh pulses a marked depression and slow recovery of ACh sensitivity were observed. These actions of D600 on the postsynaptic membrane suggest that D600 may act by blocking open endplate channels.

Calcium dependence of evoked transmitter release at very low quantal contents at the frog neuromuscular junction.

1. The relationship between the rate of evoked transmitter release and the extra-cellular concentration of Ca ions, [Ca2+]o, was studied at surface neuromuscular junctions of the frog cutaneous pectoris muscle. The average quantal content of the end-plate potential was reduced to low levels by reducing [Ca2+]o and adding 2 mM-Mn2+, 45 MM-Co2+ or 10 mM-Mg2+. 2. When the motor nerve was stimulated at a low frequency (0.5--2 Hz) in 2 mM-Mn2+ or 4 mM-Co2+, the average quantal content of evoked release was proportional to the fourth power of [Ca2+]o down to the lowest measurable quantal contents, around 2--4 quanta per 1000 stimuli. Combined with previous studies, this result indicates that evoked transmitter release has a steep, nonlinear dependence on [Ca2+]0 over our orders of magnitude of evoked release. 3. Calculations predict that if evoked and spontaneous release have the same fourth power dependence on intracellular [Ca2+], then the curve relating evoked release and [Ca2+]o should become much less steep as the evoked release rate approaches the spontaneous release rate. Our observation that the relationship between evoked release and [Ca2+]o remains fourth power down to very low release rates suggests that most spontaneous quantal release does not have the same dependence on intracellular [Ca2+], or does not use the same intracellular Ca2 pool, as evoked release. 4. In 2--10 mM-Mg2+, the lowest average quantal contents were markedly higher than the fourth power prediction. This discrepancy may occur either because Mg2+ somehow elevates intracellular [Ca2+], or because Mg2+ is itself a weak activator of transmitter release. 5. Even at very low rates of evoked release, increasing the stimulus frequency to 5--50 Hz caused a progressive increase in both evoked release and the rate of 'background' quantal release during the interstimulus interval. The frequency-dependent enhancement of both evoked and background release was more pronounced in solutions containing 10 mM-Mg2+ than in solutions containing 2 mM-Mn2+.

Release of enkephalin-like immunoreactive material from isolated bovine chromaff in cells

The enkephalin-like immunoreactive material which is stored in primary cultures of bovine chromaffin cells is released spontaneously when these cells are transferred from the culture medium to the Krebs-Ringer buffer. Potassium (56 mM) and acetylcholine (10 −4M) increase this rate of spontaneous release in a Ca 2+ dependent fashion. Preliminary evidence indicates that nicotinic receptors are operative in mediating the release of enkephalin-like peptides elicited by acetylcholine.

Mechanisms of regrowth of the bulbospinal serotonin system following 5,6-dihydroxytryptamine induced axotomy. I. Biochemical correlates

The regrowth of the bulbospinal serotonin neurons after 5,6-dihydroxytryptamine (5,6-DHT)-induced axotomy has been followed between 1–2 weeks and 8 months after neurotoxin injection, using determinations of endogenous serotonin (5-HT) and [ 3H]5-HT uptake, and measurements of the conversion of [ 3H]tryptophan ([ 3H]TP) into [ 3H]5-HT and [ 3H]5-HIAA in vitro. In the spinal cord there was, at 2 weeks after the 5,6-DHT treatment (75 μg), a drastic reduction in all three parameters throughout the cord signifying that the 5,6-DHT-induced axotomy causes an almost complete denervation of the spinal cord from the lumbar up to the most cranial cervical segments. With time there was a gradual recovery in the [ 3H]5-HT uptake, which appeared to progress in a cranio-caudal direction. By 2 months the recovery was noted mainly in the upper cervical segments; by 4 months it was evident also in the lower cervical segments, and by 8 months it had reached the thoracic and lumbar segments. By 7–8 months, when the [ 3H]5-HT uptake had recovered to 60% of normal in the upper cervical segments and to 25% of normal in the lumbar cord, endogenous 5-HT had recovered to 34% of the age-matched vehicle-treated controls in the cervical cord and to 13% in the lumbar cord, while the [ 3H]5-HT synthesis from [ 3H]TP had recovered to about 40–50%. The [ 3H]5-HIAA/[ 3H]5-HT ratio (reflecting the rate of metabolism of the newly synthesized transmitter) and the medium/tissue ratio (reflecting the rate of spontaneous release under the in vitro conditions) had undergone a more than two-fold increase in the long-term 5,6-DHT-treated rats, indicating an increased turnover of the transmitter in the partially reinnervated spinal cord. In medulla oblongata, where the cell bodies of origin of the bulbospinal serotonin neurons, as well as proximal terminal areas are located, there was acutely a partial (40–50%) reduction in [ 3H]5-HT uptake and [ 3H]5-HT synthesis capacity, whereas the endogenous 5-HT content was increased by 30% (due to accumulation of the transmitter in the lesioned axon stumps). There was a rapid and extensive recovery, resulting in supranormal values in all three parameters by 7–8 months. This was accompanied by a marked retardation of the in vitro turnover of newly-synthesized [ 3H]5-HT (reflected in marked reductions in the [ 3H]5-HIAA/[ 3H]5-HT and the medium/tissue ratios). The results demonstrate an extensive recovery of the neurotransmitter biosynthetic machinery during regeneration of the chemically axotomized serotonin neurons. Moreover, the observations of increased transmitter turnover in the incompletely reinnervated areas in the spinal cord and decreased turnover in the hyperinnervated brain stem areas suggest that regional compensatory mechanisms may alleviate the functional consequences of the abnormal distribution of the regenerated terminals. It is proposed that with a combination of axonal regeneration, increased utilization of the transmitter, and development of receptor supersensitivity also an incomplete regeneration, resulting in a partial reinnervation of an area, can become efficient in restoring normal function in the CNS.

Inhibitory action of Ca2+ on spontaneous transmitter release at motor nerve terminals in a high K+ solution.

The inhibitory effect of a high external Ca2+ ([Ca2+]o) on spontaneous transmitter release in a high K+ solution (Gage and Quastel 1966; Birks et al. 1968) was studied at the frog neuromuscular junction, based on the hypothesis that an increased intracellular free Ca2+ ([Ca2+]i) in the nerve terminal plays a key role in the depression. Three procedures were employed to increase [Ca2+]i; increasing [Ca2+]o, application of caffeine and tetanic nerve stimulation. All of these procedures increased m.e.p.p. frequency in normal Ringer. However, as the basic m.e.p.p. frequency was increased by raising the external K+ concentration (7--15 mM), their facilitatory effects on m.e.p.p. frequency decreased, dissapeared and eventually reversed to depressant actions. Since a rise in the external K+ concentration would increase the steady state level of [Ca2+]i, it is suggested that when the [Ca2+]i is preset at a high level, manipulations so as to further increase [Ca2+]i depress spontaneous release of transmitter. Possible mechanisms for this inhibition was discussed in relation to a question whether or not the rate of spontaneous transmitter release is a monotonic function of [Ca2+]i.

Inhibitory effects of cholinergic agents on the release of transmitter at the frog neuromuscular junction.

1. The cholinesterase inhibitors neostigmine, edrophonium and eserine (7 x 10(-7) M) reduced m.e.p.p. frequency by some 50% at the frog neuromuscular junction. Neostigmine also produced a small reduction in quantal content. 2. Tetraisopropylpyrophosphoramide, with a high specificity for non-specific cholinesterase, has a similar effect on m.e.p.p. frequency but ambenonium, with a high specificity for acetylcholinesterase, was markedly less effective in this respect. 3. Carbachol (10(-5) M) and the muscarinic agonists muscarine and metacholine (7 x 10(-7) M) also reduced the rate of spontaneous release. 4. The action of neostigmine was antagonized by atropine, but not by D-tubocurarine. Muscarine did not have any further effect when m.e.p.p. frequency was reduced with neostigmine. 5. Experiments with reduced extracellular Ca2+ concentration suggest that the cholinergic agents reduce Ca2+ permeability directly at the presynaptic terminals and that they do not act via a change in PNa or PK. It is suggested that the consequent reduction in Ca2+ entry causes a fall in both evoked release and in intracellular Ca2+ concentration, thereby reducing m.e.p.p. frequency. 6. It is concluded that non-specific cholinesterases present on the presynaptic terminals can act as inhibitory muscarinic cholinergic receptors. This form of presynaptic inhibition at the amphibian neuromuscular junction contrasts with that described in the mammalian preparation in which the sites are blocked by D-tubocurarine and are excitatory.