Sonometric measurements of motor-neuron-evoked movements of an internal feeding structure (the radula) in Aplysia.

In many systems used to study rhythmic motor programs, the structures that generate behavior are at least partially internal. In these systems, it is often difficult to directly monitor neurally evoked movements. As a consequence, although motor programs are relatively well characterized, it is generally less clear how neural activity is translated into functional movements. This is the case for the feeding system of the mollusk Aplysia. Here we used sonomicrometry to monitor neurally evoked movements of the food-grasping organ in Aplysia, the radula. Movements were evoked by intracellular stimulation of motor neurons that innervate radula muscles that have been extensively studied in reduced preparations. Nevertheless our results indicate that the movements and neural control of the radula are more complex than has been assumed. We demonstrate that motor neurons previously characterized as radula openers (B48) and closers (B8, B15, B16) additionally produce other movements. Moreover, we show that the size of the movement evoked by a motor neuron can depend on the preexisting state of the radula. Specifically, the motor neurons B15 and B16 produce large closing movements when the radula is partially open but produce relatively weak closing movements in a preparation at rest. Thus the efficacy of B15 and B16 as radula closers is context dependent.

Encoding of forces by cockroach tibial campaniform sensilla: implications in dynamic control of posture and locomotion.

Forces exerted by a leg in support and propulsion can vary considerably when animals stand upon or traverse irregular terrains. We characterized the responses of the cockroach tibial campaniform sensilla, mechanoreceptors which encode force via strains produced in the exoskeleton, by applying forces to the leg at controlled magnitudes and rates. We also examined how sensory responses are altered in the presence of different levels of static load. All receptors exhibit phasico-tonic discharges that reflect the level and rate of force application. Our studies show that: (1) tonic discharges of sensilla can signal the level of force, but accurate encoding of static loads may be affected by substantial receptor adaptation and hysteresis; (2) the absolute tonic sensitivities of receptors decrease when incremental forces are applied at different initial load levels; (3) phasic discharges of sensilla accurately encode the rate of force application; and (4) sensitivities to changing rates of force are strictly preserved in the presence of static loads. These findings imply that discharges of the sensilla are particularly tuned to the rate of change of force at all levels of leg loading. This information could be utilized to adapt posture and walking to varying terrains and unexpected perturbations.

Active signaling of leg loading and unloading in the cockroach.

The ability to detect changes in load is important for effective use of a leg in posture and locomotion. While a number of limb receptors have been shown to encode increases in load, few afferents have been demonstrated to signal leg unloading, which occurs cyclically during walking and is indicative of slipping or perturbations. We applied mechanical forces to the cockroach leg at controlled rates and recorded activities of the tibial group of campaniform sensilla, mechanoreceptors that encode forces through the strains they produce in the exoskeleton. Discrete responses were elicited from the group to decreasing as well as increasing levels of leg loading. Discharges of individual afferents depended on the direction of force application, and unit responses were correlated morphologically with the orientation of the receptor's cuticular cap. No units responded bidirectionally. Although discharges to decreasing levels of load were phasic, we found that these bursts could effectively encode the rate of force decreases. These discharges may be important in indicating leg unloading in the step cycle during walking and could rapidly signal force decreases during perturbations or loss of ground support.

Load signalling by cockroach trochanteral campaniform sensilla.

A major problem in sensory motor integration is to delineate how forces acting upon a leg are encoded and regulated in the control of posture and locomotion. We have studied responses of the trochanteral campaniform sensilla, the largest array of force detecting mechanoreceptors in the cockroach leg. Afferents from two groups of sensilla (Groups 3 and 4) encode forces applied to the leg in the plane of joint movement of the coxo-trochanteral joint. The receptors within Group 3 exhibit fixed patterns of recruitment that could differentially indicate when force levels are adequate to provide support and propulsion during walking.

Gating of afferent input by a central pattern generator.

Intracellular recordings from the sole proprioceptor (the oval organ) in the crab ventilatory system show that the nonspiking afferent fibers from this organ receive a cyclic hyperpolarizing inhibition in phase with the ventilatory motor pattern. Although depolarizing and hyperpolarizing current pulses injected into a single afferent will reset the ventilatory motor pattern, the inhibitory input is of sufficient magnitude to block afferent input to the ventilatory central pattern generator (CPG) for approximately 50% of the cycle period. It is proposed that this inhibitory input serves to gate sensory input to the ventilatory CPG to provide an unambiguous input to the ventilatory CPG.

Effects of scaphognathite nerve stimulation on the acutely deafferented crab ventilatory central pattern generator.

1. Sensory axons from crab (Carcinus maenas) scaphognathites enter the thoracic ganglion primarily via the LNb branch of the levator nerve. The LNa branch of the levator nerve and the depressor nerve each contain relatively few sensory axons. 2. Acutely deafferented ventilatory central pattern generators show a free running burst rate which is lower than that observed in intact crabs. Electrical stimulation of the levator nerve, or of its LNb branch, increases the burst rate in a frequency dependent manner. Stimulation at high enough intensity to recruit afferents will restart a paused motor rhythm. Stimulation of the levator nerve with short pulse trains phase resets and can entrain the rhythm. 3. In addition to increasing the burst rate, LNb stimulation also causes a progressive elimination of motor neurons from the bursts as the stimulating frequency increases, probably due to depolarization of the 3 oval organ 'giant' afferent axons in this branch. Intracellular depolarization of single oval organ afferents will also inhibit some motor neurons as well as slow or stop the rhythm. 4. Continuous stimulation of the depressor nerve does not affect the ganglionic burst rate and this nerve contains only a few small diameter afferent axons; however, brief trains of stimuli can reset the rhythm in a phase-dependent manner.

Electrical coupling of mechanoreceptor afferents in the crayfish: a possible mechanism for enhancement of sensory signal transmission.

1. Central electrical coupling between primary afferent axons was investigated in an in vitro preparation of the crayfish thoracic locomotor system by using intracellular recordings. 2. Intracellular injection of the dye Lucifer yellow in single afferents resulted in staining of one to three additional afferents through dye-coupling. Three-dimensional confocal imaging of dye-coupled axons revealed a large zone of close apposition that may correspond to the gap junction site. 3. A depolarization preceding the spike in one sensory terminal was shown to facilitate the excitatory postsynaptic potential occurring in postsynaptic motoneurons. Further, a spike in one afferent axon can depolarize other, electrically coupled, axons above spike threshold, resulting in an increased number of active afferents. 4. The electrical coupling occurred between sensory afferents of similar function. It may therefore serve to facilitate sensory signal transmission from functionally homologous afferents onto postsynaptic target neurons.

Afferents to the medial nucleus of the trapezoid body and their collateral projections.

Cells and axons that supply direct afferent input to the medial nucleus of the trapezoid body are described. Afferents were intracellularly labeled in brainstem tissue slices of two rodent and two bat species. The main afferents are calyciferous axons from globular bushy cells of the ventral cochlear nucleus. Calyciferous axons were highly consistent across species, projecting directly from the cochlear nucleus, across the midline in the trapezoid body, to the contralateral medial nucleus of the trapezoid body. Within the target nucleus, a typical axon turned sharply away from horizontal to form a large ending, the calyx of Held, around the soma of a single principal cell. Three groups of calyciferous axons were classified based on the path taken from bend to calyx. In subjects younger than four weeks, single axons often formed two calyces, each on a different cell. These calyx pairs were often found on adjacent or vertically aligned cells. In older animals, calyx pairs were more closely aligned, but fewer double calyx axons were seen. A secondary focus of this study was the system of thin collateral branches that characterizes calyciferous axons in all species. The projection patterns of these collaterals suggest that calyciferous axons may provide ascending input to periolivary cell groups with descending projections. In addition to calyciferous afferents, labeled cells that provide input to the medial nucleus of the trapezoid body from adjacent periolivary cell groups are described. Also described is a type of afferent that descends from the level of the lateral lemniscus to the medial nucleus of the trapezoid body.

Monosynaptic Interjoint Reflexes and their Central Modulation During Fictive Locomotion in Crayfish.

An in vitro preparation of the crayfish nervous system has been utilized to study an interjoint reflex pathway and its variability during rhythmic locomotor activity. The coxo-basal chordotonal organ (CBCO) is a joint stretch receptor spanning the second joint of walking legs in crayfish, where it encodes joint movements and position. Mechanical stimulation (stretch and release) of the CBCO and electrical stimulation of the CBCO nerve elicits reflex responses in promotor and remotor motor neurons innervating muscles moving the basal thoraco-coxal (TC) leg joint. Promotor and remotor motor neurons receive monosynaptic excitatory inputs from at least four CBCO afferents, including both stretch- and release-sensitive CBCO afferents. In a tonic preparation, in which there is no tendency to produce alternating bursts of activity in antagonistic motor neurons, the reflex responses were evoked during each cycle of imposed movement. However, when the preparation became rhythmic and produced bouts of fictive locomotion, the reflex responses were unstable and their gain was phasically modulated. Paired recordings indicate that such a modulation of the monosynaptic interjoint reflex could be due to both a phasic change in the excitability of the motor neurons and presynaptic inhibition that reduces the excitatory input from CBCO primary afferents.

An interneurone mediating motor programme switching in the ventilatory system of the crab.

The central pattern generator controlling ventilation in the crab can generate two distinct motor programmes, which determine the direction of water flow during irrigation of the gills. An interneurone has been identified that depolarizes when the ventilatory motor output switches from forward to reverse ventilation and remains depolarized for the duration of the reverse motor programme. Depolarization of this neurone by intracellular current injection causes a switch in the motor programme from forward to reverse ventilation, which persists for the duration of the current step. Hyperpolarization of this cell during reverse ventilation terminates the reverse motor programme. The possible role of this reversal switch interneurone is considered in the context of the observed changes in the activity of other ventilatory interneurones and motor neurones during reverse ventilation.

Nonspiking interneurons in the ventilatory central pattern generator of the shore crab, Carcinus maenas.

Eight nonspiking interneurons were identified that are elements of the central pattern generator controlling ventilation in the shore crab, Carcinus maenas. Intracellular recordings from these neurons in an isolated ganglion preparation revealed that these cells exhibit large amplitude oscillations in their membrane potentials, which are in-phase with the ventilatory motor pattern. These oscillations are present during the expression of the two distinct ventilatory motor output patterns corresponding to forward and reversed ventilation, and the oscillations stopped during pauses in the ventilatory rhythm. Injection of intracellular current pulses into these interneurons caused a resetting of the ongoing ventilatory rhythm, indicating that these cells are part of the ventilatory central pattern generator. The structure of each interneuron was determined by the intracellular injection of Lucifer Yellow dye. These neurons have a large diameter main neurite ranging from 10 to 20 microns in diameter with very restricted primary and secondary branching from the main neurite. All of the interneurons are restricted to a single hemiganglion and perturbation of these cells with intracellular current pulses only affect the motor output of the hemiganglion containing the interneuron. These eight nonspiking interneurons appear to be the primary components of the central pattern generator underlying ventilation in the crab.

An inexpensive strain gage amplifier and signal conditioner for biological applications.

The design and operation of a strain gage signal conditioning amplifier is described. The complete amplifier is based on the Analog Devices 2B30 module and features adjustable gain, bridge excitation voltage, bridge balance, d.c. offset and low-pass filtering. The ease of construction and low cost of this amplifier make it ideal for applications requiring a large number of signal channels.

Intracellular labeling of afferents to the lateral superior olive in the bat, Eptesicus fuscus.

An in vitro tissue slice preparation of the bat brain stem was used to label intracellularly individual axons projecting to the lateral superior olive from two different sources: the medial nucleus of the trapezoid body (MNTB) and the anteroventral cochlear nucleus (AVCN). The tracing of individually labeled MNTB axons into the lateral superior olive reaffirms the long accepted indirect route by which information from the contralateral ear reaches the lateral superior olive. While the MNTB appears to relay input from the contralateral AVCN, information from the ipsilateral ear reaches the lateral superior olive via a direct projection from the ipsilateral AVCN. Axons from the contralateral and ipsilateral pathways have different distribution patterns upon the fusiform cells of the lateral superior olive. Axon terminals of MNTB principal cells have a perisomatic and proximal dendritic distribution pattern. Axon terminal varicosities from the ipsilateral anteroventral cochlear nucleus are distributed primarily to more distal dendrites.

Neural control of ventilation in the shore crab, Carcinus maenas. II. Frequency-modulating interneurons.

1. We have identified a class of nonspiking interneurons which can control the frequency of ventilation in a graded manner. These frequency modulating interneurons (FMis) also receive synaptic inputs in-phase with the ventilatory motor output providing a functional positive feedback loop in the ventilatory system. The class of FMis is composed of three morphologically and physiologically distinct interneurons, FMi1, FMi2 and FMi3. 2. Depolarization of FMi1 increases the rate of ventilation, while hyperpolarization decreases the rate (Fig. 1). This control is restricted to a single ventilatory central pattern generator (CPG), (Fig. 2), although FMi1 sends processes into the neuropils of both hemiganglionic CPGs (Fig. 3). 3. Hyperpolarization of FMi2 increases the rate of both ventilatory CPGs while depolarization of this cell slows and eventually arrests the rhythm (Figs. 5 and 6). FMi2 receives a synaptic input correlated with the motor output of each of the ventilatory CPGs (Fig. 4). During periods of reversed ventilation, this cell is abruptly hyperpolarized and continues to be driven in-phase with the ventilatory motor output (Fig. 7). 4. Hyperpolarization of FMi3 increases the rate of ventilation and depolarization decreases the rate of ventilation produced by both CPGs (Fig. 10). This control of the ventilatory rate by FMi3 is graded (Fig. 11). There is no apparent change in the membrane potential of FMi3 during reversed ventilation and it is morphologically distinct from FMi2. 5. FMi2 and FMi3 may be involved in the switch in ventilatory motor pattern from forward to reversed ventilation. Hyperpolarization of FMi2 and depolarization of FMi3 can elicit bouts of reversed ventilation from both CPGs (Fig. 13). 6. These results suggest that the FM interneurons act in parallel to control the frequency of ventilation and may act as integrating elements between spiking 'command' fibers in the circumesophageal connectives and the nonspiking interneurons of the ventilatory CPG.

A freeze-fracture and concanavalin A-binding study of the membrane of cleaving Xenopus embryos.

The freeze-fracture appearance and concanavalin A-binding capacity of the plasma membrane of cells of the cleaving Xenopus embryo have been examined up to the 16-cell stage. It was found that membrane on the outer surface of the embryo, which faces the vitelline membrane and is remote from cleavage furrows, and membrane in the shallow regions of the furrow possessed a high population of intramembranous particles on the PF-face (1171 per mum2). The EF-face of these membranes showed a lower particle population (245 per mum2). By contrast, membrane deep in the furrow and bounding the blastocoel did not display a face with high particle numbers. Both faces of this membrane, which is newly exposed as the furrow grows, were relatively poorly supplied with particles (93 per mum2). Therefore it appears that, in this tissue, newly added membrane possesses fewer intramembranous particles than the pre-existing membrane. Concanavalin A, as detected cytochemically using peroxidase and haemocyanin techniques, bound extensively to both particle-rich and particle-poor membrane. Thus there was no correlation between intramembranous particle frequency and degree of concanavalin A binding.

Intercellular junctions in the Xenopus embryo prior to gastrulation.

A freeze-fracture study of Xenopus embryos at the eight-cell stage revealed the presence of tight junctions and small aggregations of 120 A diameter intramembranous particles. These aggregations consisted of up to ten particles and resembled forming gap junctions. Examination of early blastulae showed that fully developed gap junctions were present, apparently occurring very infrequently. These results support the contention that specialized junctions mediate electrical coupling during early amphibian development.

On the mechanism of electrical coupling between cells of early Xenopus embryos.

The mechanism of electrical coupling between cells of early Xenopus embryos has been studied by examination of the nonjunctional membrane resistances and capacitances as a function of cleavage stage, the junctional and nonjunctional membrane resistances as functions of time during the first cleavage, and the electrical properties of the primitive blastocoel. The changes in membrane resistances and capacitances during the first two cleavages may be completely explained by the addition of new membrane, identical in specific resistance and capacitance to the original membrane, at a constant rate to furrows which are electrically connected to the perivitelline space. Microelectrode recording from the primitive blastocoel indicates that there is no electrical difference detectable between it and the perivitelline space. These results are discussed in the context of current theories of the mechanism of intercellular electrotonic coupling.