Frog Nerve Research Articles

Astragaloside depresses compound action potential in sciatic nerve of frogs involved in L-type Ca2+-channel dependent mechanism

The sciatic nerve is the largest sensorimotor nerve within the peripheral nervous system (PNS), possessing the ability to produce endogenous neurotrophins. Compound nerve action potentials (CNAPs) are regarded as a physiological/pathological indicator to identify nerve activity in signal transduction of the PNS. Astragaloside (AST), a small-molecule saponin purified from Astragalus membranaceus, is widely used to treat chronic disease. Nonetheless, the regulatory effects of AST on the sciatic nerve remain unknown. Therefore, the present investigation was undertaken to study the effect of AST on CNAPs of frog sciatic nerves. Here, AST depressed the conduction velocity and amplitude of CNAPs. Importantly, the AST-induced responses could be blocked by a Ca2+-free medium, or by applying all Ca2+ channel antagonists (CdCl2/LaCl3) or L-type Ca2+ channel blockers (nifedipine/diltiazem), but not the T-type and P-type Ca2+ channel antagonist (NiCl2). Altogether, these findings suggested that AST may attenuate the CNAPs of frog sciatic nerves in vitro via the L-type Ca2+-channel dependent mechanisms.

Compatibility of eugenol anesthesia with classroom physiology experiments on nerve and muscle of frogs

Frogs serve as valuable model organisms for studying physiological responses of nerve, skeletal muscle and the heart in undergraduate biology labs. Heretofore, induced hypothermia has been the preferred technique for pain and stress reduction prior to euthanasia by pithing. However, AVMA guidelines discourage hypothermia for restraint or euthanasia of amphibians and advocate anesthetic overdose as preferable to pithing alone. Unfortunately, the primary anesthetic used in fish and other aquatic invertebrates, MS-222, blocks normal nerve and muscle function in leopard frogs and renders them useless for classroom physiology experiments, as demonstrated by Medler (2019). Eugenol is a safe and affordable anesthetic that is effective in amphibians. We sought to test the hypothesis that eugenol anesthesia is compatible with classroom nerve and muscle experiments on frogs. Bullfrogs and leopard frogs were immersed in a bath of eugenol suspended in tap water at 275 to 350 mg/L. At various time points, a four step reflex test was performed including tests of righting response, superficial pain reflex, deep pain reflex, and corneal blink reflex. Once all reflexes were absent, the frog was euthanized by pithing and the gastrocnemius muscle was prepared for force recordings. The sciatic nerve was stimulated with a bipolar stimulating electrode while gastrocnemius force was measured using an isometric force transducer. The threshold voltage eliciting a muscle contraction and the peak isometric twitch force were recorded at 15 minute intervals for 2 hours. Control frogs were sedated by immersion in ice water for 20 to 30 minutes, pithed when reflexes were substantially weakened, and then subjected to the same stimulation protocol and force measurements. At these doses, eugenol reliably induces loss of reflexes in 20 to 35 minutes and neuromuscular preparations maintain the ability to respond to electrical stimulation. Preliminary results suggest that eugenol may produce a modest elevation of threshold voltage and a modest reduction in maximum twitch force compared to control frogs euthanized without chemical anesthesia. Eugenol may be an effective anesthetic to prepare bullfrogs and leopard frogs for classroom experiments on nerve and muscle. Funding was provided by the Bill and Linda Frost Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of compound action potentials in the frog sciatic nerve by inchinkoto, a traditional Japanese medicine used for oral mucositis

ObjectiveThis study aimed to determine the effects of traditional Japanese (Kampo) medicines used to treat oral mucositis on nerve conduction. MethodsThe effects of Kampo medicines, crude drugs, and chemical compounds on compound action potentials (CAPs) were analyzed using extracellular recordings in frog sciatic nerves. ResultsAmong the Kampo medicines, inchinkoto demonstrated the most significant reduction in CAP amplitude, with a half-maximal inhibitory concentration (IC50) of 5.4 mg/mL. Hangeshashinto, shosaikoto, hochuekkito, and juzentaihoto also showed a significant reduction. Regarding inchinkoto, Artemisiae Capillari Spica (artemisia) was the most effective crude drug, with an IC50 of 4.2 mg/mL for CAP amplitude reduction, whereas Gardeniae Fructus (gardenia) exerted no significant effect. However, the combined use of artemisia and gardenia reduced the CAP amplitude more effectively than artemisia alone, indicating a synergistic interaction. The chemical ingredient eugenol from artemisia administered at 1 and 3 mmol/L reduced CAP amplitude, whereas other chemical ingredients administered at 0.1 and 1 mmol/L had no significant effects. ConclusionsInchinkoto exhibited the most effective reduction in CAP amplitude in the sciatic nerve of frogs, primarily through the action of artemisia, with potential synergistic interaction between artemisia and gardenia.

Impact of cannabidiol and lamotrigine on parameters of action potentials in sciatic nerves of frogs

Cannabidiol causes inhibitory effects on sodium channels, but its action on nerve impulses is not understood. Lamotrigine blocks the nerve impulses, however, it is not known if this action is of extracellular or intracellular origin. With the use of nerve chambers, we are hoping to elucidate this unknown mechanism. We hypothesize that these two chemicals may modify components of action potentials of nerve fibers in frogs. Action potential recording was made possible by the IWorx Data Quest system and a nerve chamber. Action potential data was analyzed by looking at two different components—the peaks of depolarization and the difference between the peak of depolarization and the nadir of repolarization. For every 50 minutes of recording, the data was divided into three clusters—before chemicals, after 10 minutes of treatment, and 30 minutes after treatment. In frogs, we found the high dosage (1.0 milligram/milliliter (mg/mL)) of cannabidiol had no effect on the maximum amplitude of depolarization among the three data clusters—saline, 10 minutes after cannabidiol, and 30 minutes after cannabidiol. The high dosage of cannabidiol also had no effect on the repolarization of action potentials when compared among the above three data clusters. In addition, in frogs we found the low dosage (0.1 mg/mL) of cannabidiol showed a significant difference in the maximum amplitude of depolarization when the saline data cluster was compared to the 10-minute cluster after cannabidiol cluster. Likewise, there was a significant difference on the repolarization of action potentials when the saline data cluster was compared to the 10-minute cluster after cannabidiol cluster and when the 10-minute cluster was compared to the 30-minute cluster after cannabidiol cluster. High dosage of lamotrigine showed a significant difference on the maximum amplitude of depolarization when the saline cluster was compared to the 10-minute cluster after lamotrigine on action potentials. Low dosage of lamotrigine data is currently being collected to be analyzed and presented cumulatively with all the data. This research is being supported and funded by the Department of the Biological Sciences at the University of Tennessee at Martin. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Effect of Pronase on Mollusk, Leech, and Frog Nerve Ganglia Causes the Formation of Neuron–Neuronal Gap Junctions

The Effect of Pronase on Mollusk, Leech, and Frog Nerve Ganglia Causes the Formation of Neuron–Neuronal Gap Junctions

Transmission Mysteries: Art and Technophilia

Transmission Mysteries: Art and Technophilia

Calretinin Immunoreactivity in the VIIIth Nerve and Inner Ear Endorgans of Ranid Frogs.

Calcium-binding proteins are essential for buffering intracellular calcium concentrations, which are critical for regulating cellular processes involved in neuronal computations. One such calcium-binding protein, calretinin, is present in many neurons of the central nervous system as well as those which innervate cranial sensory organs, although often with differential distributions in adjacent cellular elements. Here, we determined the presence and distribution of calretinin-immunoreactivity in the peripheral vestibular and auditory system of ranid frogs. Calretinin-immunoreactivity was observed in ganglion cells innervating the basilar and amphibian papilla, and in a subpopulation of ganglion cells innervating the saccular epithelium. In contrast, none of the ganglion cells innervating the lagena, the utricle, or the three semicircular canals were calretinin-immunopositive, suggesting that this calcium-binding protein is a marker for auditory but not vestibular afferent fibers in the frog. The absence of calretinin in vestibular ganglion cells corresponds with the lack of type I hair cells in anamniote vertebrates, many of which in amniotes are contacted by the neurites of large, calyx-forming calretinin-immunopositive ganglion cells. In the sensory epithelia of all endorgans, the majority of hair cells were strongly calretinin-immunopositive. Weakly calretinin-immunopositive hair cells were distributed in the intermediate region of the semicircular canal cristae, the central part of the saccular macula, the utricular, and lagenar striola and the medial part of the amphibian papilla. The differential presence of calretinin in the frog vestibular and auditory sensory periphery might reflect a biochemical feature related to firing patterns and frequency bandwidths of self-motion versus acoustic stimulus encoding, respectively.

Calcium Transients and Transmitter Secretion in Different Parts of Frog Nerve Endings in Different Conditions of Calcium Ion Influx

Experiments on frog neuromuscular preparations were performed to study the characteristics of the calcium response and the quantum secretion of acetylcholine in different pats of extended nerve terminals in different conditions of calcium influx. A calcium-sensitive fluorescent dye was used to analyze Ca2+ influx (Ca2+ transients) into the proximal and distal parts of nerve endings in conditions of increased K+ ion content, in response to blockers of N- and L-type calcium channels, and on blockade of calcium-activated potassium channels. These studies showed that at a uniform distribution density of voltage-gated calcium channels along nerve endings, the proximal-to-distal decrement in calcium transients and quantum secretion intensity persisted in conditions of additional opening of voltage-gated calcium channels by potassium depolarization, on “thinning” of these channels using specific blockers, but changed on blockade of calcium-activated potassium channels.

Anesthetic MS-222 eliminates nerve and muscle activity in frogs used for physiology teaching laboratories

Frogs are routinely used in physiology teaching laboratories to demonstrate important physiological processes. There have been recent directives that promote the use of the anesthetic MS-222 (tricaine methanesulfonate), rather than lowering body temperature with a cold water bath to prepare reptiles and amphibians for physiological experiments or euthanasia. Indeed, the most recent edition of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals proclaims that chilling in water is not an appropriate method and advocates for the usage of MS-222 or other anesthetics. However, prominent researchers have responded to this position by highlighting evidence that cooling ectothermic vertebrates is, in fact, an effective and appropriate method. Furthermore, MS-222 is a known voltage-gated Na+ channel blocker, and this anesthetic's impact on the physiology of excitable tissues suggests that its use might be incompatible with experiments on nerve and muscle tissues. In the present study, I examined the effects of MS-222 at a concentration of 1.5 g/l on nerve, skeletal muscle, and cardiac muscle physiology of frogs. I found that immersion of frogs in this anesthetic blocked basic nerve and muscle physiology, making the frogs unsuitable for laboratory experiments. Applying MS-222 directly to the sciatic nerve dramatically blocked normal excitation-contraction coupling in skeletal muscle preparations, and direct application to the heart caused the organs to stop contracting. Based on these results, I conclude that MS-222 at the concentration studied may be incompatible with physiological preparations that rely on electrically excitable tissues for their normal function. Physiology educators who must use MS-222 with frogs should empirically determine an appropriate dosage and recovery time before using the anesthetic in the teaching laboratory.

Вклад кальциевых каналов L-типа в секрецию ацетилхолина в нервно-мышечных соединениях лягушки и мыши при активных и инактивированных потенциал-зависимых калиевых каналах

In this work, the contribution of L-type Ca2+ channels to the evoked secretion of acetylcholine from the frog and mouse motor nerve endings with active and inactivated potential-dependent K+ channels was studied. The effects of the specific Ca2+ blocker of the L-type channels nitrendipine on the quantal content of endplate currents and the timing of acetylcholine quanta secretion in intact preparations and after the preliminary blockade of potential-activated K+ channels with 4-aminopyridine (4-AP) were evaluated under conditions of a decreased and physiological level of ambient Ca2+. Fluorescent measurement of calcium transient reflecting the integral input of Ca2+ into the nerve ending was performed as well as the computer simulation of the processes underlying exocytosis assuming the presence of two types of Ca2+ channels (N- and L-types) and varying duration of the nerve action potential. It was shown that in the frog synapses, L-type calcium channels contribute to the evoked secretion of acetylcholine at active K+ channels only under conditions of decreased Ca2+ level; after inactivation of potential-dependent K+ channels, the contribution of L-type channels to the secretory process becomes less significant. At the physiological level of ambient Ca2+, the involvement of L-type channels in the evoked acetylcholine secretion, as in mice synapses, is manifested only under conditions of inactivated potential-dependent K+ channels.

On local anesthetic action of some dimethylacetamide compounds

The study aim was to explore local anesthetic properties of some tertiary and quaternary derivatives of dimethylacetamide. Materials and methods. The study was performed on white laboratory mice and rats of both sexes, male Agouti guinea pigs, and isolated sciatic nerves of lake frog. In the focus of the study there were two quaternary and eight tertiary compounds of dimethylacetamide with substituted anion with some amino and carbonic acids residue. A local anesthetic property was predicted by computational analysis. Acute toxicity of the most promising substances was studied in mice through subcutaneous route. Local anesthetic activity of tertiary compounds LKhT-3-00, LKhT-4-00 and quaternary LKhT-12-02 was studied on models of terminal, infiltration and conduction anesthesia. The influence of substances on mixed nerve conduction was investigated on lake frog’s isolated sciatic nerves. Results and discussion. The greatest probability of the local anesthetic activity during computational analysis was estimated for the tertiary derivatives of dimethylacetamide LKhT-3-00 and LKhT-4-00 and for the quaternary compound LKhT-12-02. According to their toxicological profile, the compounds belong to moderately toxic substances (class 3). On the model of terminal and infiltration anesthesia, substances LKhT-3-00 and LKhT-4-00 at concentrations of 0.5-1% rapidly cause deep and prolonged anesthesia. On the models of conduction anesthesia, the quaternary derivative of dimethylacetamide LKhT-12-02 has the greatest analgesic effect. The duration of the effect of the substance is over 3 hours. All the investigated compounds block sciatic nerve conduction. The longest effect is registered for LKhT-12-02. Conclusions. Dimethylacetamide derivatives at concentrations of 0.5-1.0% exhibit a local anesthetic activity, and are effective for terminal, conduction and infiltration anesthesia. Their effect is due to blockade of nerve conduction.

MRI-based assessment of function and dysfunction in myelinated axons

Repetitive electrical activity produces microstructural alteration in myelinated axons, which may afford the opportunity to noninvasively monitor function of myelinated fibers in peripheral nervous system (PNS)/CNS pathways. Microstructural changes were assessed via two different magnetic-resonance-based approaches: diffusion fMRI and dynamic T2 spectroscopy in the ex vivo perfused bullfrog sciatic nerves. Using this robust, classical model as a platform for testing, we demonstrate that noninvasive diffusion fMRI, based on standard diffusion tensor imaging (DTI), can clearly localize the sites of axonal conduction blockage as might be encountered in neurotrauma or other lesion types. It is also shown that the diffusion fMRI response is graded in proportion to the total number of electrical impulses carried through a given locus. Dynamic T2 spectroscopy of the perfused frog nerves point to an electrical-activity-induced redistribution of tissue water and myelin structural changes. Diffusion basis spectrum imaging (DBSI) reveals a reversible shift of tissue water into a restricted isotropic diffusion signal component. Submyelinic vacuoles are observed in electron-microscopy images of tissue fixed during electrical stimulation. A slowing of the compound action potential conduction velocity accompanies repetitive electrical activity. Correlations between electrophysiology and MRI parameters during and immediately after stimulation are presented. Potential mechanisms and interpretations of these results are discussed.

ATP Reduces the Entry of Calcium Ions into the Nerve Ending by Blocking L-type Calcium Channels

At neuromuscular junctions, ATP inhibits both the evoked and spontaneous acetylcholine release and inward calcium current operating via presynaptic P2Y receptors. It was shown in the experiments with the frog neuromuscular synapse using specific calcium-sensitive dye Oregon Green Bapta 1 that exogenous ATP reduces the amplitude of calcium transient, which reflects the changes in the entry of calcium ions in response to the nerve pulse. The depressing effect of ATP on the transient was prevented by suramin, the blocker of P2 receptors. Nitrendipine, a specific blocker of L-type calcium channels, per se decreased the calcium transient amplitude and significantly attenuated the effect of ATP on the calcium signal. Contrariwise, the preliminary application of ATP to the neuromuscular junction completely eliminated the depressing effect of nitrendipine on the calcium response. The obtained data suggest that an essential component in the inhibitory action of ATP on the calcium transient amplitude is provided by reduction of the entry of calcium ions into a frog nerve ending via L-type voltage-gated calcium channels.

Electro-Physiology of Coupling Model and Its Impact on Naja Kaouthia Venom Treated Sciatic Nerves of Toad.

Demyelination in peripheral nerves causes dysfunction of slowing down and stoppage of nerve impulses causing many neurological diseases, such as chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, etc. This paper aims to develop a recovery model having interaction of a demyelinated nerve with a normal myelinated nerve. We validated the model by coupling between peripheral nerve of toad (demyelinated with Naja kaouthia venom) and a normal nerve of toad. An increase in both nerve conduction velocity as well as compound action potential amplitude is observed in the repetition of the experiments indicating gradual recovery of the patients. The significance behind this work is to suppress the malfunctioning of the demyelinated nerve by the normal electro-physiological activity of the normal nerve for speedy recovery using coupling model. The recovery model will be used in the treatment of neurological disorders with the influence of normal neuro physiological properties of a normal adjacent nerve.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2–10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians.

Action of ATP on Ca2+-Transient in Different Parts of the Frog Motor Nerve Ending

Electrophysiological evidence indicates a difference in neurotransmitter secretion along the motor nerve terminals of the frog neuromuscular junction. This includes a decrease of the minimal synaptic delay value and a reduction in the quantal content of the evoked endplate currents from the proximal to distal portion of the motor nerve ending. Besides, various physiologically active compounds may have different effects on the acetylcholine secretion in the proximal and distal parts of the nerve terminal. Here, we explored the effects of ATP on Ca2+-transient using optical detection methods with high-speed camera in different parts of the frog nerve terminal. There was shown a significant inhibitory effect of ATP on Ca2+-transient in both the proximal and distal regions of nerve terminals. However, in different parts of nerve endings, any significant differences in ATP effects were not found. Thus, ATP decreases the Ca2+-transient along the entire presynaptic terminal.

An implantable ENG detector with in-system velocity selective recording (VSR) capability.

Detection and classification of electroneurogram (ENG) signals in the peripheral nervous system can be achieved by velocity selective recording (VSR) using multi-electrode arrays. This paper describes an implantable VSR-based ENG recording system representing a significant development in the field since it is the first system of its type that can record naturally evoked ENG and be interfaced wirelessly using a low data rate transcutaneous link. The system consists of two CMOS ASICs one of which is placed close to the multi-electrode cuff array (MEC), whilst the other is mounted close to the wireless link. The digital ASIC provides the signal processing required to detect selectively ENG signals based on velocity. The design makes use of an original architecture that is suitable for implantation and reduces the required data rate for transmission to units placed outside the body. Complete measured electrical data from samples of the ASICs are presented that show that the system has the capability to record signals of amplitude as low as 0.5μV, which is adequate for the recording of naturally evoked ENG. In addition, measurements of electrically evoked ENG from the explanted sciatic nerves of Xenopus Laevis frogs are presented.

Quantifying Demyelination in NK venom treated nerve using its electric circuit model.

Reduction of myelin in peripheral nerve causes critical demyelinating diseases such as chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, etc. Clinical monitoring of these diseases requires rapid and non-invasive quantification of demyelination. Here we have developed formulation of nerve conduction velocity (NCV) in terms of demyelination considering electric circuit model of a nerve having bundle of axons for its quantification from NCV measurements. This approach has been validated and demonstrated with toad nerve model treated with crude Naja kaouthia (NK) venom and also shows the effect of Phospholipase A2 and three finger neurotoxin from NK-venom on peripheral nerve. This opens future scope for non-invasive clinical measurement of demyelination.