Intracellular recordings from nonspiking interneurons in a semiintact,tethered walking insect

Nonspiking interneurons were investigated in a tethered, walking insect, Carausius morosus, that was able to freely perform walking movements. Experiments were carried out with animals walking on a lightweight, double-wheel treadmill. Although the animal was opened dorsally, the walking system was left intact. Intracellular recordings were obtained from the dorsal posterior neuropil of the mesothoracic ganglion. Nonspiking inter-neurons, in which modulations of the membrane potential were correlated with the walking rhythm, were described physiologically and stained with Lucifer Yellow. Interneurons are demonstrated in which membrane potential oscillations mirror the leg position or show correlation with the motoneuronal activity of the protractor and retractor coxae muscles during walking. Other interneurons showed distinct hyperpolarizations at certain important trigger points in the step cycle, for example, at the extreme posterior position. Through electrical stimulation of single, nonspiking interneurons during walking, the motoneuronal activity in two antagonistic muscles—protractor and retractor coxae—could be reversed and even the movement of the ipsilateral leg could be influenced. The nonspiking interneurons described appear to be important premotor elements involved in walking. They receive, integrate, and process information from different leg proprioceptors and drive groups of leg motoneurons during walking.     

Patch-clamp recordings of spiking and nonspiking interneurons from rabbit olfactory bulb slices: Membrane properties and ionic currents

Summary Physiological and morphological properties of rabbit, Oryctolagus cuniculus, olfactory bulb interneurons were characterized by using a thin slice preparation in combination with patch-clamp measurements and Lucifer Yellow fills. Two types of interneurons, periglomerular (PG) and juxtaglomerular (JG) cells, were unequivocally distinguished in the glomerular layer. Their properties were compared to those of mitral cells. PG cells closely resembled previously described periglomerular cells in their morphology. During current clamp recording these neurons were characterized by their lack of action potentials upon depolarization. Consistent with these results no Na⁺ currents could be elicited in voltage clamp experiments. Two types of outward K⁺ currents were distinguished: one which inactivated and one which did not. From their morphology JG cells appear to be either short axon cells or external tufted cells. JG cells always responded with a single, TTX-blockable action potential in response to maintained current injection. Two types of membrane currents were identified in JG cells during voltage clamp: a fast, inactivating Na⁺ current that was fully activated at — 80 mV, and a sustained outward current that shared some properties with a delayed rectifier K⁺ current. The particular relationship between the voltage dependence of the Na⁺ and K⁺ currents appeared to preclude repetitive spike activity.Abbreviations JG juxtraglomerular interneuron - LOT lateral olfactory tract - M/T mitral/tufted (cells) - PG periglomerular - SA short axon     

Patch-clamp recordings of spiking and nonspiking interneurons from rabbit olfactory bulb slices: GABA- and other transmitter receptors

Summary Transmitter receptor ion channels from previously identified rabbit olfactory bulb neurons were studied by using a thin slice preparation in combination with patch-clamp measurements. PG cells, which closely resembled previously described periglomerular interneurons in their morphology, responded to microapplication of GABA, acetylcholine, norepinephrine and glycine with the activation of distinct ionic currents. JG cells, which belong either to the class of short axon cells or external tufted cells, never showed GABA responses. In mitral cells ionic currents activated by GABA, acetylcholine, norepinephrine and glutamate could be elicited. Further measurements of GABA-activated currents of PG cells were made and indicated that these cells expressed two different types of GABA receptors: one which showed fast desensitization with a decay time constant of about 5 s, and one which slowly desensitized with a decay time constant of about 20–30 s. Both types were completely inhibited by bicuculline methiodide (50 M). GABA receptors were not blocked by Zn²⁺ (0.1 mM). From the dose-response relationship of the peak GABA-activated currents, an apparent dissociation constant of 50 M was derived. From single channel measurements in excised outside-out patches, a single channel conductance of GABA-activated Cl^– currents of 24 pS was obtained during continuous application of the agonist. Single channel events had a mean open time of 1.9 ms.     

Intracellular recordings from phycomyces

Intracellular recordings from the giant sporangiophore of Phycomyces stage II were obtained. The mean transmembrane potential for 30 observations was −119.9 millivolts (negative inside), and it did not change either as a result of a light stimulus or during dark adaptation. Injected depolarizing and hyperpolarizing step currents and steady currents did not produce any avidence of spike activity. We conclude that light transduction and dark adaptation in Phycomyces are not based on alterations of the transmembrane potential.     

Role of local nonspiking interneurons in the generation of rhythmic motor activity in the stick insect

Local nonspiking interneurons in the thoracic ganglia of insects are important premotor elements in posture control and locomotion. It was investigated whether these interneurons are involved in the central neuronal circuits generating the oscillatory motor output of the leg muscle system during rhythmic motor activity. Intracellular recordings from premotor nonspiking interneurons were made in the isolated and completely deafferented mesothoracic ganglion of the stick insect in preparations exhibiting rhythmic motor activity induced by the muscarinic agonist pilocarpine. All interneurons investigated provided synaptic drive to one or more motoneuron pools supplying the three proximal leg joints, that is, the thoraco-coxal joint, the coxa-trochanteral joint and the femur-tibia joint. During rhythmicity in 83% (n=67) of the recorded interneurons, three different kinds of synaptic oscillations in membrane potential were observed: (1) Oscillations were closely correlated with the activity of motoneuron pools affected; (2) membrane potential oscillations reflected only certain aspects of motoneuronal rhythmicity; and (3) membrane potential oscillations were correlated mainly with the occurrence of spontaneous recurrent patterns (SRP) of activity in the motoneuron pools. In individual interneurons membrane potential oscillations were associated with phase-dependent changes in the neuron's membrane conductance. Artificial changes in the interneurons' membrane potential strongly influenced motor activity. Injecting current pulses into individual interneurons caused a reset of rhythmicity in motoneurons. Furthermore, current injection into interneurons influenced shape and probability of occurrence for SRPs. Among others, identified nonspiking interneurons that are involved in posture control of leg joints were found to exhibit the above properties. From these results, the following conclusions on the role of nonspiking interneurons in the generation of rhythmic motor activity, and thus potentially also during locomotion, emerge: (1) During rhythmic motor activity most nonspiking interneurons receive strong synaptic drive from central rhythm-generating networks; and (2) individual nonspiking interneurons some of which underlie sensory-motor pathways in posture control, are elements of central neuronal networks that generate alternating activity in antagonistic leg motoneuron pools. © 1995 John Wiley & Sons, Inc.     

Unique,identifiable local nonspiking interneurons in the locust mesothoracic ganglion

The hypothesis that local nonspiking interneurons are unique and identifiable has been tested rigorously for a neuron in the mesothoracic ganglion of the locust. Neurons were physiologically characterized and subsequently stained with cobalt ions. The resulting preparations were examined in whole mounts and serial sections. It is concluded that at least three neurons are unique, based upon a combination of their function, gross morphology, and the location and size of their main processes relative to other neurons. It is strongly suggested that there are other local nonspiking interneurons that are unique and identifiable. A classification system for local nonspiking interneurons is proposed. The implications of this finding for future neuroethological studies are discussed.     

Intracellular recordings from spinal neurons during 'swimming' in paralysed amphibian embryos

Intracellular microelectrode recordings have been made from probable motoneurons in the spinal cord of Xenopus laevis embryos during fictive 'swimming' in preparations paralysed with the neuromuscular blocking agent tubocurarine. These cells had resting potentials of -50 mV or more. During spontaneous or stimulus-evoked 'swimming' episodes: (a) the cells were tonically excited; the level of tonic synaptic excitation and the conductance increase underlying it were both inversely related to the 'swimming' cycle period; (b) the cells usually fired one spike per cycle in phase with the motor root burst on the same side; spikes did not overshoot zero and were evoked by phasic excitatory synaptic input on each cycle, superimposed on the tonic excitation; (c) in phase with motor root discharge on the opposite side of the body, the cells were hyperpolarized by a chloride-dependent inhibitory postsynaptic potential. The nature of synaptic potentials during 'swimming' was evaluated by means of intracellular current injections. The 'swimming' activity could be controlled by natural stimuli. The results provide clear evidence on the relation of tonic excitation to rhythmic locomotory pattern generation, and indirect evidence for reciprocal inhibitory coupling between antagonistic motor systems.     

Intracellular recordings from gecko photoreceptors during light and dark adaptation

Intracellular recordings were obtained from rods in the Gekko gekko retina and the adaptation characteristics of their responses studied during light and dark adaptation. Steady background illumination induced graded and sustained hyperpolarizing potentials and compressed the incremental voltage range of the receptor. Steady backgrounds also shifted the receptor's voltage-intensity curve along the intensity axis, and bright backgrounds lowered the saturation potential of the receptor. Increment thresholds of single receptors followed Weber's law over a range of about 3.5 log units and then saturated. Most of the receptor sensitivity change in light derived from the shift of the voltage-intensity curve, only little from the voltage compression. Treatment of the eyecup with sodium aspartate at concentrations sufficient to eliminate the beta-wave of the electroretinogram (ERG) abolished initial transients in the receptor response, possibly indicating the removal of horizontal cell feedback. Aspartate treatment, however, did not significantly alter the adaptation characteristics of receptor responses, indicating that they derive from processes intrinsic to the receptors. Dark adaptation after a strongly adapting stimulus was similarly associated with temporary elevation of membrane potential, initial lowering of the saturation potential, and shift of the voltage-intensity curve. Under all conditions of adaptation studied, small amplitude responses were linear with light intensity. Further, there was no unique relation between sensitivity and membrane potential suggesting that receptor sensitivity is controlled at least in part by a step of visual transduction preceding the generation of membrane voltage change.     

Neuropeptides in interneurons of the insect brain

A large number of neuropeptides has been identified in the brain of insects. At least 35 neuropeptide precursor genes have been characterized in Drosophila melanogaster, some of which encode multiple peptides. Additional neuropeptides have been found in other insect species. With a few notable exceptions, most of the neuropeptides have been demonstrated in brain interneurons of various types. The products of each neuropeptide precursor seem to be co-expressed, and each precursor displays a unique neuronal distribution pattern. Commonly, each type of neuropeptide is localized to a relatively small number of neurons. We describe the distribution of neuropeptides in brain interneurons of a few well-studied insect species. Emphasis has been placed upon interneurons innervating specific brain areas, such as the optic lobes, accessory medulla, antennal lobes, central body, and mushroom bodies. The functional roles of some neuropeptides and their receptors have been investigated in D. melanogaster by molecular genetics techniques. In addition, behavioral and electrophysiological assays have addressed neuropeptide functions in the cockroach Leucophaea maderae. Thus, the involvement of brain neuropeptides in circadian clock function, olfactory processing, various aspects of feeding behavior, and learning and memory are highlighted in this review. Studies so far indicate that neuropeptides can play a multitude of functional roles in the brain and that even single neuropeptides are likely to be multifunctional.The original research in the authors’ laboratories was supported by DFG grants HO 950/14 and 950/16 (U.H.) and Swedish Research Council grant VR 621-2004-3715 (D.R.N).     

Multimodal responses of the nonspiking giant interneurons in the brain of the crayfish Procambarus clarkii

Three pairs of nonspiking giant interneurons (NGIs; G1, G2, and G3) of the crayfish brain responded with depolarizing and hyperpolarizing graded potentials to body tilt in roll to the ipsi- and contralateral sides in the dark. The higher and the larger the angle of body tilt, the larger was the amplitude of the geotactic responses. In ipsilaterally statocystectomized animals, all the NGIs responded with hyperpolarizing potentials only to the contralateral side-down tilt, whereas in contralaterally statocystectomized animals, they responded with depolarizing potentials only to the ipsilateral side-down tilt. In bilaterally statocystectomized animals, none of the NGIs responded to body tilt in the dark, but in the presence of an overhead light, they exhibited depolarizing and hyperpolarizing potentials in response to body tilt to the ipsi-and contralateral sides, respectively. All the NGIs responded with depolarizing and hyperpolarizing graded potentials to illumination of the contra- and ipsilateral eyes, respectively. The amplitude of these visual responses, however, varied in association with the amplitude of the geotactic response produced by body tilt. These results indicate that the NGIs integrate the sensory inputs from eyes and statocysts and that the interaction between sensory inputs from the left and right sensory organs with either the same modality or with different modalities enhance the directional sensitivity of NGIs as premotoneurons in the compensatory oculomotor system.     

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons in the crayfish Procambarus clarkii Girard

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons of crayfish have been studied using intradendritic recording and current injection. The input resistance of phasic motoneurons (5.20 ± 0.5 M; mean ± standard error) measured by injecting constant hyperpolarizing current was significantly lower than that of tonic motoneurons (10.3 ± 2.6 M; 0.02 < P < 0.05). The membrane time constant of phasic motoneurons (7.3 ± 0.9 ms) was also significantly shorter than that of tonic motoneurons (24.3 ± 2.5 ms; P < 0.001). Both types of motoneurons behaved linearly during hyperpolarization and sub-threshold depolarization. Nonspiking interneurons showed outward rectification upon depolarization. During hyperpolarization, their membrane behaved linearly and showed significantly higher input resistance (19.5 ± 2.5 M) than phasic and tonic motoneurons (P < 0.001). Their membrane time constant (38.0 ± 5.7 ms) was significantly longer than that of phasic motoneurons (P < 0.001) but not than that of tonic motoneurons (P > 0.05). In response to intracellular injection of sinusoidally oscillating current, phasic motoneurons showed one or two spikes per depolarization period irrespective of oscillating frequency ranging from 1 to 16 Hz. Tonic motoneurons showed larger numbers of spikes per stimulus period at lower frequencies. Nonspiking interneurons also showed phase-locked effects on the motoneuron spike activity. The effective frequency range over which injected oscillating current could modulate motoneuron spike activity was similar for tonic motoneurons and nonspiking interneurons.     

GABAergic and glutamatergic inhibition of nonspiking local interneurons in the terminal abdominal ganglion of the crayfish

Nonspiking local interneurons in the terminal abdominal ganglion of the crayfish Procambarus clarkii receive inhibitory inputs from mainly glutamatergic spiking local interneurons and GABAergic nonspiking interneurons. In this study, the inhibitory responses of nonspiking interneurons to local application of glutamate and GABA into the neuropil were compared. Glutamate and GABA injection mediated the hyperpolarization of the nonspiking interneurons with an increase in membrane conductance. The glutamate-mediated membrane hyperpolarization was reversed by injection of 1 or 2 nA hyperpolarizing current. By contrast, more than 3 nA hyperpolarizing current was frequently necessary to reverse the GABA-mediated hyperpolarization. Bath application of a chloride channel blocker, 50 microM picrotoxin (PTX), reduced the glutamate-mediated hyperpolarization, but had no effect on the GABA-mediated hyperpolarization. The GABA-mediated hyperpolarization was not consistently affected by bath application of low chloride solution. These results suggest that the glutamate-mediated inhibition was related to the gating of a Cl(-) conductance, while the GABA-mediated inhibition was not. Electrical stimulation of sensory afferents innervating the exopodite elicited ipsps in uropod opener motor neurons. These sensory-evoked ipsps were also PTX-insensitive, suggesting GABAergic nonspiking interneurons could be the predominant premotor elements in organizing the uropod motor control system.     

Intracellular calcium recordings from isolated cells of the mammalian central nervous system

Measurements made with two different techniques of intracellular calcium levels from small isolated cells of the mammalian central nervous system are described and compared. Recordings in cultured mouse embryo spinal cord and dorsal root ganglion neurons, made with double-barrelled borosilicate Ca2+-selective microelectrodes yielded a mean Ca2+ level of 2.3 (SE +/- 0.54) microM for the lowest values recorded in 24 out of 46 cells. Intracellular Ca2+ dependence on membrane potential was apparent with levels of calcium greater than or equal to 4 microM (r = 0.371, n = 29). Both cyclic fluctuations induced by tetraethylammonium and an apparent increase in Ca2+ evoked by the depolarizing excitatory amino acid, L-aspartate, were observed. In contrast, estimates of intracellular Ca2+ obtained by spectrofluorimetry of suspensions of mouse embryo brain cells, loaded with the intracellular Ca-binding fluorescent probe, quin2 provided a approximately equal to 10-fold lower value, 152 (SE +/- 7) nM. This more closely resembles levels reported for large neurons where large-tip microelectrodes with greater sensitivity were used, and in spite of the heterogeneity of the cells this value is presumed to be a more accurate estimate of intraneuronal Ca2+ concentration. In these fluorescence studies KCl readily evoked increases in intracellular Ca2+ which could be blocked by verapamil and Cd2+ and were not induced in the absence of Ca2+. Increases were also produced by N-methyl-D-aspartate, but not by the kainate-like Lathyrus neurotoxin, L-3-oxalylamino-2-aminopropionic acid. These results provide preliminary evidence for both voltage-sensitive and receptor-activated Ca channels in embryonic brain cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular voltage recordings in the opercular epithelium of Fundus heteroclitus

Opercular epithelial cells of Fundus heteroclitus were investigated using conventional microelectrodes. The area of interest was the cells lining the inside of the opercular epithelium closest to the gill arches, an area with a high density of chloride cells. Only one cell type could be discerned from the values of 60 opercular cells measured with the opercular epithelium in open circuit conditions. A mean apical voltage of -18.0 +/- 0.6 mV was observed with intracellular values ranging from -10 to -30 mV. The predicted intracellular chloride content was 59 mM/liter. Apical fractional resistance (faR) was 0.78 +/- 0.02. The intracellular potential measurements were typically difficult to maintain for extended periods (longer than 3 min). The opercular cells depolarize with serosal isoproterenol treatment (10(-6) M) corresponding to the increase in opercular transepithelial potential. The opercular cell apical fR decreased with isoproterenol treatment. These data indicate the observed opercular cells were involved in opercular chloride transport.     

Calcium spikes in a leech nonspiking neuron

The NS neurons are nonspiking cells, present as pairs in each midbody ganglion of the leech nervous system, which display a very extensive arborization. They were shown to regulate the coactivation of motoneurons. Here we have investigated the electrophysiological properties of these neurons under the hypothesis that transmission along the extensive neurites requires the aid of voltage-dependent conductances. The results indicate that NS neurons respond to electrical stimulation with a spike-like event, which was not an all-or-none but rather a graded phenomenon that depended on the intensity and duration of the electrical stimulus. The spike-like response was activated at a membrane potential of approximately −50 mV; its amplitude was a logarithmic function of the extracellular Ca²⁺ concentration and was unaffected by a broad range of changes in the extracellular Na⁺ concentration; intracellular application of tetraethylammonium (TEA) caused a large increase in its amplitude and duration. These data indicate that NS neurons bear voltage-dependent low-threshold Ca²⁺ and TEA-sensitive K⁺ conductances that could contribute to shaping synaptic signals, or transmission along the extensive neuritic tree.     

Physiological changes of premotor nonspiking interneurons in the central compensation of eyestalk posture following unilateral sensory ablation in crayfish

We investigated how the physiological characteristics and synaptic activities of nonspiking giant interneurons (NGIs), which integrate sensory inputs in the brain and send synaptic outputs to oculomotor neurons innervating eyestalk muscles, changed after unilateral ablation of the statocyst in order to clarify neuronal mechanisms underlying the central compensation process in crayfish. The input resistance and membrane time constant in recovered animals that restored the original symmetrical eyestalk posture 2 weeks after operation were significantly greater than those immediately after operation on the operated side whereas in non-recovered animals only the membrane time constant showed a significant increase. On the intact side, both recovered and non-recovered animals showed no difference. The frequency of synaptic activity showed a complex pattern of change on both sides depending on the polarity of the synaptic potential. The synaptic activity returned to the bilaterally symmetrical level in recovered animals while bilateral asymmetry remained in non-recovered ones. These results suggest that the central compensation of eyestalk posture following unilateral impairment of the statocyst is subserved by not only changes in the physiological characteristics of the NGI membrane but also the activity of neuronal circuits presynaptic to NGIs.