The neuronal targets for GABAergic reticulospinal inhibition that stops swimming in hatchling frog tadpoles.

In most animals locomotion can be started and stopped by specific sensory cues. We are using a simple vertebrate, the hatchling Xenopus tadpole, to study a neuronal pathway that turns off locomotion. In the tadpole, swimming stops when the head contacts solid objects or the water's surface meniscus. The primary sensory neurons are in the trigeminal ganglion and directly excite inhibitory reticulospinal neurons in the hindbrain. These project axons into the spinal cord and release GABA to inhibit spinal neurons and stop swimming. We ask whether there is specificity in the types of spinal neuron inhibited. We used single-neuron recording to determine which classes of spinal neurons receive inhibition when the head skin is pressed. Ventral motoneurons and premotor interneurons involved in generating the swimming rhythm receive reliable GABAergic inhibition. More dorsal inhibitory premotor interneurons are inhibited less reliably and some are excited. Dorsal sensory pathway interneurons that start swimming following a touch to the trunk skin do not appear to receive such inhibition. There is therefore specificity in the formation of descending inhibitory connections so that more ventral neurons producing swimming are most strongly inhibited.

Defining classes of spinal interneuron and their axonal projections in hatchling Xenopus laevis tadpoles.

Neurobiotin was injected into individual spinal interneurons in the Xenopus tadpole to discern their anatomical features and complete axonal projection patterns. Four classes of interneuron are described, with names defining their primary axon projection: Dorsolateral ascending and commissural interneurons are predominantly multipolar cells with somata and dendrites exclusively in the dorsal half of the spinal cord. Ascending interneurons have unipolar somata located in the dorsal half, but their main dendrites are located in the ventral half of the spinal cord. Descending interneurons show bigger variance in their anatomy, but the majority are unipolar, and they all have a descending primary axon. Dorsolateral commissural interneurons are clearly defined using established criteria, but the others are not, so cluster analysis was used. Clear discriminations can be made, and criteria are established to characterize the three classes of interneuron with ipsilateral axonal projections. With identifying criteria established, the distribution and axonal projection patterns of the four classes of interneuron are described. By using data from gamma-aminobutyric acid immunocytochemistry, the distribution of the population of ascending interneurons is defined. Together with the results from the axonal projection data, this allows the ascending interneuron axon distribution along the spinal cord to be estimated. By making simple assumptions and using existing information about the soma distributions of the other interneurons, estimates of their axon distributions are made. The possible functional roles of the four interneuron classes are discussed.

Intrinsic and extrinsic modulation of a single central pattern generating circuit.

Intrinsic and extrinsic neuromodulation are both thought to be responsible for the flexibility of the neural circuits (central pattern generators) that control rhythmic behaviors. Because the two forms of modulation have been studied in different circuits, it has been difficult to compare them directly. We find that the central pattern generator for biting in Aplysia is modulated both extrinsically and intrinsically. Both forms of modulation increase the frequency of motor programs and shorten the duration of the protraction phase. Extrinsic modulation is mediated by the serotonergic metacerebral cell (MCC) neurons and is mimicked by application of serotonin. Intrinsic modulation is mediated by the cerebral peptide-2 (CP-2) containing CBI-2 interneurons and is mimicked by application of CP-2. Since the effects of CBI-2 and CP-2 occlude each other, the modulatory actions of CBI-2 may be mediated by CP-2 release. Although the effects of intrinsic and extrinsic modulation are similar, the neurons that mediate them are active predominantly at different times, suggesting a specialized role for each system. Metacerebral cell (MCC) activity predominates in the preparatory (appetitive) phase and thus precedes the activation of CBI-2 and biting motor programs. Once the CBI-2s are activated and the biting motor program is initiated, MCC activity declines precipitously. Hence extrinsic modulation prefacilitates biting, whereas intrinsic modulation occurs during biting. Since biting inhibits appetitive behavior, intrinsic modulation cannot be used to prefacilitate biting in the appetitive phase. Thus the sequential use of extrinsic and intrinsic modulation may provide a means for premodulation of biting without the concomitant disruption of appetitive behaviors.

Actions of a pair of identified cerebral-buccal interneurons (CBI-8/9) in Aplysia that contain the peptide myomodulin.

A combination of biocytin back-fills of the cerebral-buccal connectives and immunocytochemistry of the cerebral ganglion demonstrated that of the 13 bilateral pairs of cerebral-buccal interneurons in the cerebral ganglion, a subpopulation of 3 are immunopositive for the peptide myomodulin. The present paper describes the properties of two of these cells, which we have termed CBI-8 and CBI-9. CBI-8 and CBI-9 were found to be dye coupled and electrically coupled. The cells have virtually identical properties, and consequently we consider them to be "twin" pairs and refer to them as CBI-8/9. CBI-8/9 were identified by electrophysiological criteria and then labeled with dye. Labeled cells were found to be immunopositive for myomodulin, and, using high pressure liquid chromatography, the cells were shown to contain authentic myomodulin. CBI-8/9 were found to receive synaptic input after mechanical stimulation of the tentacles. They also received excitatory input from C-PR, a neuron involved in neck lengthening, and received a slow inhibitory input from CC5, a cell involved in neck shortening, suggesting that CBI-8/9 may be active during forward movements of the head or buccal mass. Firing of CBI-8 or CBI-9 resulted in the activation of a relatively small number of buccal neurons as evidenced by extracellular recordings from buccal nerves. Firing also produced local movements of the buccal mass, in particular a strong contraction of the I7 muscle, which mediates radula opening. CBI-8/9 were found to produce a slow depolarization and rhythmic activity of B48, the motor neuron for the I7 muscle. The data provide continuing evidence that the small population of cerebral buccal interneurons is composed of neurons that are highly diverse in their functional roles. CBI-8/9 may function as a type of premotor neuron, or perhaps as a peptidergic modulatory neuron, the functions of which are dependent on the coactivity of other neurons.

C-PR neuron of Aplysia has differential effects on "Feeding" cerebral interneurons, including myomodulin-positive CBI-12.

Head lifting and other aspects of the appetitive central motive state that precedes consummatory feeding movements in Aplysia is promoted by excitation of the C-PR neuron. Food stimuli activate C-PR as well as a small population of cerebral-buccal interneurons (CBIs). We wished to determine if firing of C-PR produced differential effects on the various CBIs or perhaps affected all the CBIs uniformly as might be expected for a neuron involved in producing a broad undifferentiated arousal state. We found that when C-PR was fired, it produced a wide variety of effects on various CBIs. Firing of C-PR evoked excitatory input to a newly identified CBI (CBI-12) the soma of which is located in the M cluster near the previously identified CBI-2. CBI-12 shares certain properties with CBI-2, including a similar morphology and a capacity to drive rhythmic activity of the buccal-ganglion. Unlike CBI-2, CBI-12 exhibits myomodulin immunoreactivity. Furthermore when C-PR is fired, CBI-12 receives a polysynaptic voltage-dependent slow excitation, whereas, CBI-2 receives relatively little input. C-PR also polysynaptically excites other CBIs including CBI-1 and CBI-8/9 but produces inhibition in CBI-3. In addition, firing of C-PR inhibits plateau potentials in CBI-5/6. The data suggest that activity of C-PR may promote the activity of one subset of cerebral-buccal interneurons, perhaps those involved in ingestive behaviors that occur during the head-up posture. C-PR also inhibits some cerebral-buccal interneurons that may be involved in behaviors in which C-PR activity is not required or may even interfere with other feeding behaviors such as rejection or grazing, that occur with the head down.

Compartmentalization of information processing in an aplysia feeding circuit interneuron through membrane properties and synaptic interactions.

We describe a pair of cerebral-to-buccal interneurons, CBI-5/6, which have outputs and inputs in two ganglia. The soma in the cerebral ganglion received synaptic inputs during buccal motor programs (BMPs) and after mechanical stimulation of the lips. During BMPs the soma received antidromic spikes generated in processes in the buccal ganglion. The soma was driven into a plateau potential by each of these inputs, during which it fired orthodromically at 0-5 Hz. The soma had outputs in the cerebral ganglion consisting of electrical coupling to the adjacent CBI-5/6 and to a cerebral-to-pedal neuron (CPN1). The buccal terminals of CBI-5/6 received inputs that generated rhythmic barrages (up to 25 Hz) of antidromic spikes during BMPs. The buccal terminals had chemical and electrical outputs to motor and premotor elements of feeding circuitry. This combination of synaptic interactions and endogenous properties mean that CBI-5/6 can process information in a number of ways. During the barrage of antidromic spikes, high-frequency firing will produce strong inputs to buccal followers and on their arrival at the soma will transfer excitation electrotonically to CPN1. Subthreshold input to the soma will be transferred electrotonically to cerebral followers but will not be relayed to postsynaptic buccal neurons. Plateau potentials after the antidromic spikes or local cerebral inputs will locally excite CPN1 via electrical coupling but will have little influence on buccal events because of the low orthodromic firing rate. Thus, CBI-5/6 may transmit information locally within the cerebral ganglion or more extensively in both buccal and cerebral ganglia simultaneously.

A cerebral central pattern generator in Aplysia and its connections with buccal feeding circuitry.

Different feeding-related behaviors in Aplysia require substantial variations in the coordination of movements of two separate body parts, the lips and buccal mass. The central pattern generators (CPGs) and motoneurons that control buccal mass movements reside largely in the buccal ganglion. It was previously thought that control of the cerebral neuronal circuitry and motoneurons that generate lip movements was coordinated directly by feedback from buccal interneurons. Here, we describe cerebral lip motoneuron C15, which drives rhythmic activity in the isolated cerebral ganglion. Other lip motoneurons are active during this program, so we define it as a cerebral motor program (CMP). The C15 in each cerebral hemiganglion drives the CMP in ipsilateral neurons only, suggesting there are independent CPGs in each hemiganglion. The cerebral and buccal CPGs interact at several points. For example, cerebral-to-buccal interneurons (CBIs), which can drive the buccal CPG, receive excitatory input when the cerebral CPG is active. Likewise, C15, which can drive the cerebral CPG, is excited when the buccal CPG is active. This excitation is simultaneous in both C15s, coupling the activity in the two hemiganglionic cerebral CPGs. Therefore, there are independent cerebral and buccal CPGs, which can produce distinct rhythms, but which interact at several points. Furthermore, the connections between the cerebral and buccal CPGs alter during different forms of motor program. We suggest that such alterations in the interactions between these CPGs might contribute to the generation of the various forms of coordination of lip and buccal mass movements that are necessary during different feeding-related behaviors.

Composition of the excitatory drive during swimming in two amphibian embryos: Rana and Bufo.

It has recently been shown that spinal neurons in Xenopus embryos receive cholinergic and electrotonic excitation during swimming, in addition to the well documented excitatory amino acid (EAA)-mediated excitation. We have now examined the composition of the excitatory drive during swimming in embryos of two further amphibian species, Rana and Bufo, which have somewhat different motor patterns. Localised applications of antagonists show that presumed motoneurons in Rana and Bufo embryos receive both cholinergic and FAA input during swimming. There is also a further chemical component which is blocked by Cd2+ and a small Cd(2+)-insensitive component, which is usually non-rhythmic. Rhythmic Cd(2+)-insensitive components, presumed to be phasic electrotonic potentials, were only seen in a small proportion of Bufo neurons and in no Rana neurons. While EAA and cholinergic inputs therefore appear to be consistent features of excitatory drive for swimming in amphibian embryo motoneurons, electrotonic input apparently occurs less commonly. Antagonist specificity was tested using applied agonists in Rana. Results of these tests also suggested that the further, unidentified Cd(2+)-sensitive component seen during swimming could represent an incomplete block of AMPA receptor-mediated excitation.

Local effects of glycinergic inhibition in the spinal cord motor systems for swimming in amphibian embryos.

1. We have studied the effects of locally applying the glycinergic antagonist strychnine to rhythmically active spinal neurons in amphibian embryos during fictive swimming. Intracellular recordings were made from motoneurons and premotor interneurons in Xenopus laevis, a well-studied model system, and from motoneurons in three other species (Rana temporaria, Bufo bufo, and Triturus vulgaris). Overall, these embryos cover a range of swimming patterns from the short-cycle-period, brief-motor-root bursts of Xenopus, to the long-cycle-period, long-motor-root bursts of Rana, which are more typical of adult patterns. 2. Local strychnine application had no significant effect on the gross pattern of swimming; episode duration and the burst duration in rostral ventral roots away from the application site were unaltered, and left-right alternation was preserved. We have therefore been able to examine the effects of inhibition on individual neurons, uninfluenced by overall changes in the operation of the swimming neural circuitry. 3. In all cases strychnine blocked midcycle inhibition and significantly increased the peak on-cycle depolarization during swimming. In Rana, Bufo, and Triturus motoneurons, and in Xenopus interneurons, strychnine significantly increased the reliability of firing during swimming. In Xenopus motoneurons, where spiking was 100% reliable anyway, the timing of the spikes was advanced relative to rostral ventral root activity. These results do not provide support for postinhibitory rebound as a factor in the spike-generating process during swimming. In addition to midcycle inhibition, Xenopus motoneurons can also show a smaller, additional on-cycle inhibition that is blocked by strychnine. 4. In both Rana and Bufo the duration of caudal ventral root bursts close to the site of drug application was increased by strychnine, showing that the increased motoneuron reliability not only leads to more intense, but also more extensive, ventral root activity. 5. At the level of single neurons, glycinergic inhibition effectively reduces on-cycle excitation and in turn controls the reliability, extent, and precise timing of motoneuron firing. These changes may be the individual components underlying broader effects of inhibition described previously, such as locomotor frequency control. They also show how any modulation of inhibition in localized regions of the spinal cord could produce localized control of neuronal firing properties.

Cholinergic and electrical synapses between synergistic spinal motoneurones in the Xenopus laevis embryo.

1. To investigate central motoneurone synapses within the spinal cord of a simple vertebrate, the Xenopus embryo, simultaneous intracellular recordings were made from fifty-five pairs of spinal motoneurones. 2. Chemical synapses were found between seventeen out of thirty-five pairs on the same side of the spinal cord. Current-evoked spikes in the presynaptic neurone led to fast depolarizing postsynaptic potentials (PSPs) in the postsynaptic neurone at latencies of 0.5-1.5 ms. The PSPs had an average amplitude of 7 mV, a rise time of 8 ms and a half-fall time of 18 ms. 3. The presynaptic motoneurone was always the more rostral of the pair. No excitatory connections were found which crossed the cord. The fast PSPs were blocked by 10 microM mecamylamine but not by 1 mM kynurenic acid, so were mediated by nicotinic acetylcholine receptors (nAChRs). These are the first unitary excitatory postsynaptic potentials (EPSPs) mediated by nAChRs recorded intracellularly within the vertebrate central nervous system. 4. Bidirectional electrical synapses were found between five pairs of motoneurones. All these pairs were on the same side of the spinal cord and less than 70 microns apart. Each neurone responded in a graded manner to either hyperpolarizing or depolarizing current injected into the other. 5. Since motoneurones are rhythmically active during swimming, both chemical and electrical synapses will add to the fast on-cycle excitation underlying spiking activity in other motoneurones. This may increase the reliability and local synchrony of synergistic motoneurone firing during locomotion.

Cholinergic and electrical motoneuron-to-motoneuron synapses contribute to on-cycle excitation during swimming in Xenopus embryos.

1. We have previously shown that Xenopus spinal motoneurons make both chemical and electrical synapses with neighboring motoneurons. Because motoneurons are active during swimming, these synapses would be expected to contribute excitation to their neighbors. The significance of central motoneuron to motoneuron synapses was therefore investigated by analyzing the composition of the fast on-cycle excitation underlying spiking activity during fictive swimming in spinal motoneurons. To accomplish this we developed a method for very local application of drugs around a caudal recorded neuron while still being able to evoke and record essentially unaltered fictive swimming rostrally. 2. Intracellular recordings were made from spinal motoneurons during fictive swimming. Bicuculline (40 microM) and strychnine (2 microM) were used continuously to block inhibitory potentials locally around the motoneurons. The amplitude and duration of the fast excitation underlying spiking activity was measured before and during local applications of excitatory antagonists. 3. The nicotinic antagonists d-tubocurarine (10 microM) and dihydro-beta-erythroidine (10 microM) reduced the amplitude of this excitation by approximately 20%. Nicotinic antagonists also reduced the duration of this fast on-cycle excitation. The kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) reduced the amplitude (by approximately 30%) but not the duration of the on-cycle excitation. In the presence of 100 microM Cd2+, which blocks all chemically mediated transmission, a considerable amount (50%) of on-cycle excitation remained. 4. These results suggest that 20% of the on-cycle excitation comes from activation of nicotinic receptors by naturally released acetylcholine (ACh), presumably from other motoneurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic contribution to excitation in a spinal locomotor central pattern generator in Xenopus embryos.

1. We have investigated whether in Xenopus embryos, spinal interneurons of the central pattern generator (CPG) receive cholinergic or electrical excitatory input during swimming. The functions of cholinergic excitation during swimming were also investigated. 2. Intracellular recordings were made from rhythmically active presumed premotor interneurons in the dorsal third of the spinal cord. After locally blocking inhibitory potentials with 2 microM strychnine and 40 microM bicuculline, the reliability of spike firing and the amplitude of fast, on-cycle, excitatory postsynaptic potentials (EPSPs) underlying the single on-cycle spikes were measured during fictive swimming. 3. The nicotinic antagonists d-tubocurarine and dihydro-beta-erythroidine (DH beta E, both 10 microM) reversibly reduced the reliability of the spike firing during swimming and reduced the amplitude of the on-cycle EPSP by 16%. DH beta E also reduced the EPSP amplitude in spinalized embryos by 22%. These results indicate that interneurons receive rhythmic cholinergic excitation from a source within the spinal cord. 4. Combined applications of nicotinic and excitatory amino acid (EAA) antagonists or cadmium (Cd2+, 100-200 microM) resulted in complete block of the fast EPSP, suggesting that interneurons do not receive electrical excitation. 5. The nicotinic antagonists mecamylamine and d-tubocurarine (both 5 microM) reduced the duration of episodes of fictive swimming recorded from the ventral roots, in spinal embryos. When applied in the middle of a long episode, d-tubocurarine decreased the swimming frequency, ruling out an effect on the initiation pathway. The cholinesterase inhibitor eserine (10 microM) increased the duration of swimming episodes.(ABSTRACT TRUNCATED AT 250 WORDS)

Positive feedback as a general mechanism for sustaining rhythmic and non-rhythmic activity.

Our aim is to reassess our proposal that various states of motor output may be sustained by positive feedback generated within the premotor neural circuitry. The evidence for this proposal came from the Xenopus embryo which when touched can swim for many seconds even after all movement has been prevented by a neuromuscular blocking agent. Experiments showed that even the spinal cord could sustain its own swimming activity for a few seconds after stimulation. We proposed that this was the result of the glutamatergic excitatory spinal interneurons synapsing with each other. Because this excitation is of long duration compared to the swimming cycle period it can sum from cycle to cycle to sustain swimming by a form of positive feedback. We have tested the plausibility of these ideas by making realistic computer simulations of the spinal networks and have shown that positive feedback can sustain stable swimming activity. Pharmacological evidence recently suggested that acetylcholine contributes to the excitation underlying swimming in spinal embryos so we investigated the central synapses made by motoneurons. Recordings from pairs of synergistic motoneurons then showed: a) cholinergic chemical synapses from more rostral motoneurons activate nicotinic receptors and produce excitation; and b) local intrasegmental electrical synapses also lead to mutual excitation. The presence of central motoneuron synapses suggested that they could contribute to excitation during swimming. We therefore used local drug applications to see if spinal neurons received cholinergic or electrical excitation during fictive swimming. The results show that motoneurons received both types of excitation while interneurons received only cholinergic excitation. This evidence suggests that when motoneurons are active during swimming they contribute positive feedback excitation not only to themselves but also to the premotor interneurons of the spinal rhythm generating network. This excitation would sum with that from 'glutamatergic' excitatory interneurons. We conclude that in addition to our original proposal of feedback between excitatory interneurons, there are other forms of positive feedback during swimming in the Xenopus embryo spinal cord. Motoneurons feed excitation back to each other. They may also contribute cholinergic excitation to premotor interneurons which could sum with the excitation from 'glutamatergic' interneurons and help to sustain swimming. If they do this, motoneurons may be a component part of the central pattern generator for swimming. Since central motoneuron synapses are a feature of most vertebrate groups, these results suggest a reevaluation of such synapses in these groups also.

Nicotinic and muscarinic ACh receptors in rhythmically active spinal neurones in the Xenopus laevis embryo.

1. Intracellular recordings were made from presumed motoneurones in the Xenopus embryo spinal cord, and their response to cholinergic agents was investigated. Nicotine and 1,1-dimethyl-4-phenylpiperazinium (DMPP; both 1-10 microM) strongly depolarized, and muscarine and oxotremorine (2-20 microM) weakly hyperpolarized, these neurones. Tetrodotoxin (1 microM), which blocks action potentials in Xenopus neurones, did not affect either of these responses. 2. The extrapolated reversal potential of the nicotinic depolarization was -12.1 +/- 8.3 mV (mean +/- S.E.M.) suggesting the opening of a mixed conductance. The nicotinic response was antagonized by dihydro-beta-erythroidine, d-tubocurarine and mecamylamine (10-20 microM) but not by alpha-bungarotoxin (10 microM). 3. The muscarinic response was not reversed when recorded with electrodes filled with potassium chloride but was antagonized by atropine (0.1 microM). 4. Acetylcholine (ACh, 10 microM) caused a strong depolarization of the neurones which was blocked by d-tubocurarine and dihydro-beta-erythroidine, suggesting that its effects are mediated predominantly by nicotinic ACh receptors. 5. ACh and nicotinic agonists applied to the spinal cord produced a barrage of IPSPs that were blocked by TTX and strychnine.