Frog Spinal Cord Research Articles

An examination of frog myelinated axons using intracellular microelectrode recording: the role of voltage-dependent and leak conductances on the steady-state electrical properties.

1. Intracellular microelectrode current-clamp technique was used to study the steady-state membrane properties of single intact large primary afferent axons (conduction velocity > 10 m/s) attached to isolated hemisected frog spinal cord. 2. Hyperpolarizing electrotonic potentials (ETPs) had a slow complex multiphasic charging. This complex charging could be approximated by two time constants: one in the range of 70-210 ms, the other of < 20 ms. 3. Two regions of outward and inward rectification hyperpolarized to the resting membrane potential were observed, in addition to the previously characterized outward rectification active at potentials depolarized to resting membrane potential. The peak and steady-state input resistance of these axons in tetrodotoxin Ringer solution was on average 65.6 +/- 21.1 and 31.1 +/- 10.8 M omega, mean +/- SE, respectively. 4. Application of external tetraethylammonium (10-20 mM) significantly depolarized the axon and decreased the outward rectification just hyperpolarized to the resting membrane potential. This outward rectification could also be blocked by external barium ions (2-10 mM). 5. Activation of an inward or anomalous rectification in these axons was observed 300-600 ms after the start of a current pulse. In addition, a depolarizing afterpotential (DAP) (1-3 mV in amplitude, 500 ms-10 s in duration) was evident after a current pulse in which inward rectification had been activated. This DAP most likely reflected the slow inactivation of the inwardly rectifying conductance. 6. Inward rectification was blocked by external application of cesium ions (1-3 mM) but it was insensitive to external application of barium ions (2-10 mM). The blockade of the voltage attenuation was accompanied by a disappearance of the DAP and an increase in the charging time constant of the axon. This blockade resulted in a single linear voltage-current (V-I) relationship. Axons now had, on average, an input resistance of 114 +/- 19.1 M omega. 7. Reducing the concentration of external potassium ions increased both the peak and steady-state slope resistance. Reducing the external sodium concentration altered the ETPs and the V-I relationship little but it consistently reduced the magnitude and length of the DAP. These results are compatible with the hypothesis that anomalous rectification is a mixed ionic conductance dependent on potassium and sodium ions in the external media. 8. Overall, the V-I relationship of these intact axons had both linear and nonlinear regions reflecting the activity of numerous slowly activating and inactivating conductances. (ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of monosynaptic excitatory postsynaptic potentials by glutamate in frog spinal motoneurons

We analyzed glutamate-induced enhancement of the amplitude of monosynaptic excitatory postsynaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Low concentrations (0.1–0.3 mM) of glutamate, which produced small depolarization, often enhanced EPSP associated with inhibition of a paired pulse facilitation and increased occurrence of spontaneous EPSPs. With 1 mM glutamate, transient enhancement of EPSP was seen in some cells during the early phase or prior to large depolarization, even when input conductance was increased. Transient or sustained enhancement of EPSP was occasionally seen with N-methyl- d-aspartate , kainate and quisqualate, but not with l-2-amino-4-phosphonobutyrate. The results suggest that glutamate enhanced release of excitatory transmitters at low concentrations that apparently did not affect the postsynaptic membrane.

Stizolobic acid on frog spinal cord; possible species dependent activation of excitatory amino acid receptors

1. We examined the effects of stizolobic acid, an amino acid isolated from a plant, Stizolobium hassjoo, on the binding of [ 3H]glutamic acid and [ 3H]kainic acid to synaptosomes from frog spinal cords and on the depolarization at the ventral roots of frog spinal cords. 2. Stizolobic acid inhibited the binding of [ 3H]kainic acid more potently than that of [ 3H]glutamic acid. 3. Among Stizolobic acid derivatives, 3-Br-stizolobic acid was the most potent inhibitor of the binding of [ 3H]kainic acid, but the inhibitory potency was 100 times weaker than that of kainic acid. 4. Stizolobic acid and its derivatives could cause depolarization of the ventral root of frog spinal cord in a dose dependent manner, and 3-Br-stizolobic acid was a more potent inducer of depolarization than kainic acid, but the dose dependency of 3-Br-stizolobic acid was a little different from that of kainic acid. 5. The results suggest that Stizolobic acid and its analogues act as a kainic acid agonist in frog spinal cord. 6. The present results and others indicate that Stizolobic acid may interact with the different types of excitatory amino acid receptors dependent on the species of animals.

A nonrandom interneuronal pattern in the developing frog spinal cord.

In the developing spinal cord of the frog, Xenopus laevis, a population of interneurons assumes a pattern that represents a previously undescribed level of organization. Glyoxylic acid treatment and immunocytochemistry show that the neurons contain catecholamines and their synthetic enzyme, tyrosine hydroxylase. Cells are located within the ependymal layer of the floor plate region of the larval spinal cord. The cells have several processes including a long one that projects toward the brain without fasciculating with other labeled processes. In addition, the cytoplasm of the catecholaminergic cells extends into the central canal, showing that they are a population of cerebrospinal fluid-contacting neurons. The spatial domain of catecholaminergic neurons starts abruptly at the boundary between the hindbrain and spinal cord and continues to the tip of the tail. The neurons occupy two longitudinal columns within the sheet of floor plate cells, which includes cells that do not exhibit the catecholaminergic phenotype. Unlabeled cells are intercalated between catecholaminergic cells in each column, giving the labeled cells the appearance of being spaced along the length of the spinal cord. This general arrangement is evident at the time of hatching. Spatial analysis showed that the position of cells along a column is not random. The nonrandom behavior is due to cells being excluded from the area immediately surrounding other catecholaminergic cells. Further analysis showed that the cellular pattern lacks segmental or other periodic repeats. Ultimately, the location of a cell within a column depends upon the position of its closest catecholaminergic neighbor.

Convergent force fields organized in the frog's spinal cord

Microstimulation of the gray matter of the frog's spinal cord was used to elicit motor responses. Force responses were recorded with the frog's ankle clamped while EMG activity was monitored. The collections of force patterns elicited at different leg configurations were summarized as force fields. These force fields showed convergence to an equilibrium point. The equilibrium paths were calculated from the force fields with the leg clamped. These paths predicted free limb motion in 75% of trials. The force fields were separated into active and prestimulation resting responses. The active force field responses had a fixed position equilibrium. These active force fields were modulated in amplitude over time, although the balance and orientations of forces in the pattern remained fixed. The active fields grouped into a few classes. These included both convergent and parallel fields. The convergent force fields (CFFS) could be observed in deafferented preparations. Motoneuron (MN) activity underlying the force fields was marked using sulforhodamine. The marked activity covered several segments. Several simulations and MN stimulations show that topography, limb geometry, and random activation could not account for the results. It is likely that propriospinal interneurons distribute the activity that underlies the responses observed here. Experiments showed that CFFs that resemble those elicited by microstimulation also underlie natural behaviors. The full variety of fields revealed by microstimulation was larger than the repertoire elicited by cutaneous stimulation. It was concluded that fixed-pattern force fields elicited in the spinal cord may be viewed as movement primitives. These force fields could form building blocks for more complex behaviors.

Structure of recurrent axon collaterals of frog lumbar motoneurons as revealed by intracellular HRP labelling

Iontophoretical injection of horseradish peroxidase (HRP) into lumbar motoneurons of the isolated perfused frog spinal cord allowed to reveal recurrent axon collaterals of motoneurons and to study their structural organization. The axons of about 50% of stained motoneurons gave rise to recurrent collaterals. Motoneurons emitted usually one and in a few cases two recurrent axon collaterals. Recurrent collaterals exhibited a complex ramification pattern with numerous swellings presumably corresponding to synaptic boutons both en passant and terminaux. Swellings of recurrent axon collaterals were observed exclusively in the gray matter. Most of the boutons were located in the neuropil, while some of them were found in close apposition to the soma of ventral horn neurons, in particular, motoneurons. The present data provide direct evidence for structural basis involved in recurrent actions of motoneurons activity in the amphibian spinal cord.

Evidence that [ 3H]glutamate binding sites are masked by biologically relevant endogenous factor on cell membranes of frog spinal cord

We identified the possible endogenous factor effective to modulate the binding of [ 3H]-labeled excitatory amino acid agonists and antagonists in the 100,000 × g supernatant of Triton X-100 (0.01%)-treated cell membranes from frog spinal cords. The factor inhibited the binding of [ 3H]glutamate to Triton X-100-treated cell membranes, to which the binding capacity of [ 3H]glutamate increased much more than that to intact cell membranes. The binding capacities of [ 3H]AMPA (an AMPA type agonist) and [ 3H]CPP (an NMDA type antagonist) to cell membranes remained low by Triton treatment, but they were enhanced significantly by the addition of the factor. The effect of the factor on the [ 3H]kainate binding was hardly observable. The factor may provide key information on receptor structures and the classification of receptor types concerning excitatory amino acids in the mammalian central nervous system.

Does the nervous system use equilibrium-point control to guide single and multiple joint movements?

The hypothesis that the central nervous system (CNS) generates movement as a shift of the limb's equilibrium posture has been corroborated experimentally in studies involving single- and multijoint motions. Posture may be controlled through the choice of muscle length-tension curve that set agonist-antagonist torque-angle curves determining an equilibrium position for the limb and the stiffness about the joints. Arm trajectories seem to be generated through a control signal defining a series of equilibrium postures. The equilibrium-point hypothesis drastically simplifies the requisite computations for multijoint movements and mechanical interactions with complex dynamic objects in the environment. Because the neuromuscular system is springlike, the instantaneous difference between the arm's actual position and the equilibrium position specified by the neural activity can generate the requisite torques, avoiding the complex "inverse dynamic" problem of computing the torques at the joints. The hypothesis provides a simple, unified description of posture and movement as well as contact control task performance, in which the limb must exert force stably and do work on objects in the environment. The latter is a surprisingly difficult problem, as robotic experience has shown. The prior evidence for the hypothesis came mainly from psychophysical and behavioral experiments. Our recent work has shown that microstimulation of the frog spinal cord's premotoneural network produces leg movements to various positions in the frog's motor space. The hypothesis can now be investigated in the neurophysiological machinery of the spinal cord.

Glutamate-induced inhibition of paired pulse facilitation of monosynaptic excitatory post-synaptic potentials in frog spinal motoneurons

To evaluate actions of glutamate on excitatory synaptic transmission in the central nervous system, we examined glutamate-induced changes in the paired pulse facilitation of monosynaptic excitatory post-synaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Glutamate (1 mM) depolarized motoneurons both in the presence and absence of Mg 2+. In most cells perfused with Mg 2+-free or high Ca 2+-Mg 2+ solutions, the glutamate potential was accompanied by a reduction in pak amplitude of EPSPs, although the degree of change varied with the cells. Glutamate enhanced the EPSP amplitude in a few cells with Mg 2+-free and high Ca 2+-Mg 2+ solutions, and in most cells with high Mg 2+ medium. In 3/5 cells tested, the paired pulse facilitation of EPSPs was reduced by glutamate when the EPSP amplitude either increased or decreased. NMDA (50 μM), kainate (50–100 μM), quisqualate (5–50 μM) and l-2-amino-4-phosphonobutyrate ( l-AP4, 1 mM) also decreased the facilitation in about half of the cells tested. The glutamate-induced decrease in the facilitation was observed in both the presence and absence of Mg 2+ and was not affected by the concomitant application of glutamate and antagonists for non-NMDA or NMDA receptors, such as 6-cyano-7-nitro-quinoxalinediones (CNQX, 60 μM) or 2-amino-5-phosphonovalerate (APV, 250 μM). Glutamate reduced the facilitation of excitatory post-synaptic currents (EPSCs) recorded at a constant membrane potential under voltage clamp, when the EPSC amplitude either increased or decreased and when the input conductance either increased or decreared. Thus changes in membrane potential or input conductance of the post-synaptic membrane were not essential to the glutamate-induced inhibition of the facilitation. These results suggest that glutamate modulates release of excitatory transmitters via mechanisms insensitive to Mg 2+, CNQX and APV.

Activation of 5-HT 1C/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord

Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and α-methyl-5-HT (α-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 μM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or α-Me-5-HT (both 100 μM). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of l-glutamate and l-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists N-methyl- d-aspartate (NMDA), quisqualate, α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate. α-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HT-induced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HT 1C/2 antagonist ketanserin and the 5-HT 1A/2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA NMDA-depolarizations. The relatively selective 5-HT 1A antagonist spiroxatrine and the selective 5-HT 3 blocker MDL 72222 were without effect against the depressive effects of 5-HT. These observations support the notion that 5-HT putatively released from descending bulbospinal fibers can alter frog motoneuronal output. The data indicate that 5-HT 1C/2 receptors affect the afferent input to motoneurons and do so by attenuation of NMDA receptor-mediated synaptic processes.

Serotonin 1A facilitation of frog motoneuron responses to afferent stimuli and to N-methyl- d-aspartate

The effects of serotonin and excitatory amino acids on motoneurons were examined by sucrose gap recordings from the ventral root of the isolated, hemisected frog spinal cord superfused with magnesium-free, carbonate-buffered Ringer solution. Low concentrations of serotonin (0.1 μM) and the serotonin 1A agonist8-hydroxy-2-( n-dipropylamino)tetralin (8-OH-DPAT; 0.01 μM) significantly increased the duration and amplitude of the polysynaptic components of ventral root potentials produced by dorsal root stimulation. The facilitations of the ventral root potentials were blocked by the serotonin 1A antagonist spiroxatrine, but were unaffected by the serotonin 2 antagonist ketanserin or the serotonin 3 antagonist 1αH,3α,5αH-tropan-3-yl-3,-dichlorobenzoate (MDL 72222). The actions of 0.1 μM serotonin on motoneuron depolarizations evoked by the putative excitatory amino acid transmitters l-glutamate and l-aspartate were quite variable, but in the presence of ketanserin (20 μM), small consistent increases in amino acid-induced motoneuron depolarizations were observed. 8-OH-DPAT significantly enhanced motoneuron depolarizations elicited by the selective excitatory amino acid agonist N-methyl- d-aspartate in both normal and tetrodotoxin-containing Ringer solution. Quisqualate-induced motoneuron depolarizations were also facilitated by 8-OH-DPAT in normal Ringer solution, but these increases were eliminated by addition of either tetrodotoxin or the N-methyl- d-aspartate antagonist d(−)-2-amino-5-phosphonovalerate to the Ringer superfusate. Kainate-depolarizations were not altered by low concentrations of serotonin or 8-OH-DPAT. Prior exposure of the cord to spiperone, but not ketanserin or MDL 72222 blocked the enhancement of N-methyl- d-aspartate-induced motoneuron depolarizations by 8-OH-DPAT. In the presence of saturating concentrations of glycine, 8-OH-DPAT facilitated N-methyl- d-aspartate responses. Increases in extracellular potassium activity, measured with ion-selective microelectrodes inserted in the intermediate gray matter, evoked by repetitive stimulation of myelinated afferent fibers in the sciatic nerve and by application of N-methyl- d-aspartate were significantly enhanced by 8-OH-DPAT. These findings are compatible with the notion that serotonergic activation of serotonin 1A receptors plays a role in regulating the responsiveness of frog spinal motoneurons to excitatory afferent inputs and to N-methyl- d-aspartate-mediated synaptic transmission.

Bulbospinal inhibition of PAD elicited by stimulation of afferent and motor axons in the isolated frog spinal cord and brainstem.

1. In the isolated spinal cord and brainstem of the frog, stimulation of the brainstem (BS) with trains of 3-4 pulses at 60-400 Hz produced dorsal root potentials (DRPs). The lowest threshold sites eliciting DRPs were located at the level of the obex up to about 2.5 mm rostrally, 0.5-1.2 mm laterally, between 0.5 and 1.6 mm depth. This region corresponds to the bulbar reticular formation (RF). 2. Stimulation of the RF with strengths below those required to produce DRPs, very effectively inhibited the DRPs produced by stimulation of a neighboring dorsal root (DR-DRPs) as well as the DRPs produced by antidromic stimulation of the central end of motor nerves (VR-DRPs). The inhibition was detectable 20 ms after the first pulse of the conditioning train, attained maximal values between 50 and 100 ms and lasted more than 250 ms. 3. Stimulation of the bulbar RF increased the negative response (N1 response) produced in the motor pool by antidromic activation of motoneurons. The time course of the facilitation of the N1 response resembled that of the reticularly-induced inhibition of the VR-DRPs and DR-DRPs. 4. The present series of observations supports the existence of reticulo-spinal pathways that are able to inhibit the depolarization elicited in afferent fibers by stimulation of other afferent fibers or by antidromic activation of motor axons. This inhibition appears to be exerted on the PAD mediating interneurons and is envisaged as playing an important role in motor control.

Synapses on motoneuron dendrites in the brachial section of the frog spinal cord: a computer-aided electron microscopic study of cobalt-filled cells

Cobalt-labelled motoneuron dendrites of the frog spinal cord at the level of the second spinal nerve were photographed in the electron microscope from long series of ultrathin sections. Three-dimensional computer reconstructions of 120 dendrite segments were analysed. The samples were taken from two locations: proximal to cell body and distal, as defined in a transverse plane of the spinal cord. The dendrites showed highly irregular outlines with many 1-2 microns-long 'thorns' (on average 8.5 thorns per 100 microns 2 of dendritic area). Taken together, the reconstructed dendrite segments from the proximal sites had a total length of about 250 microns; those from the distal locations, 180 microns. On all segments together there were 699 synapses. Nine percent of the synapses were on thorns, and many more close to their base on the dendritic shaft. The synapses were classified in four groups. One third of the synapses were asymmetric with spherical vesicles; one half were symmetric with spherical vesicles; and one tenth were symmetric with flattened vesicles. A fourth, small class of asymmetric synapses had dense-core vesicles. The area of the active zones was large for the asymmetric synapses (median value 0.20 microns 2), and small for the symmetric ones (median value 0.10 microns 2), and the difference was significant. On average, the areas of the active zones of the synapses on thin dendrites were larger than those of synapses on large calibre dendrites. About every 4 microns 2 of dendritic area received one contact. There was a significant difference between the areas of the active zones of the synapses at the two locations. Moreover, the number per unit dendritic length was correlated with dendrite calibre. On average, the active zones covered more than 4% of the dendritic area; this value for thin dendrites was about twice as large as that of large calibre dendrites. We suggest that the larger active zones and the larger synaptic coverage of the thin dendrites compensate for the longer electrotonic distance of these synapses from the soma.

Macrophages direct process elongation from adult frog motorneurons in culture

Motorneurons and macrophages have been isolated and identified in primary cultures from adult frog (Rana pipiens) spinal cord. Time-lapse video microscopy revealed that during the first two weeks migrating macrophages contact the growth cones of motorneurons. As they continue to migrate, the motorneuron processes elongate in close association with the moving macrophages. Elongating motorneuron processes are thereby brought into contact with other motorneurons and networks are formed. At later stages, the macrophages die but the motorneurons and the networks survive for at least another two weeks. These experiments show that macrophages can promote a directed elongation of motorneuron processes and suggest that they play a similar role during regeneration in vivo.

Release of endogenous glutamate and aspartate from the frog spinal cord in vitro

The release of the endogenous excitatory amino acids aspartate and glutamate from an in vitro slice preparation of the frog spinal cord was investigated by using high performance liquid chromatography (HPLC). Spinal slices were incubated at 4°C to minimize amino acid uptake; samples of the bathing solution were collected at 10 min intervals for amino acid assay. In each experiment electrophysiological responses were recorded continuously from ventral roots after stimulation of an adjacent dorsal root, so that changes in the profile of amino acid release could be correlated with electrophysiological responses. At rest the release of glutamate and aspartate was 52.9 ± 5.8 and 66.9 ± 5.6 pmol/10 min, respectively, and was unaffected by low Ca 2+ media or tetrodotoxin. After trains of high frequency stimulation applied to a dorsal root the release of glutamate and aspartate was significantly and reproducibly enhanced by 33 ± 13 and 49 ± 18%, respectively. The stimulus-dependent release was blocked by low Ca 2+ media, tetrodotoxin or topical application of the neuropeptide thyrotropin releasing hormone. The present study provides direct evidence to support the role of glutamate and aspartate as neurotransmitters in the spinal cord.

Strength-duration and activity-dependent excitability properties of frog afferent axons and their intraspinal projections.

1. Excitability properties of afferent axons and terminal regions in frog dorsal roots (DR) and spinal cords in vitro were investigated by antidromic activation from three sites--the root, the entry zone (dorsal white matter or DW), and deep within the dorsal horn (DH)--while recordings were made from the DR. 2. Two approaches were used to assess physiological differences between telodendria and trunk axons. Rheobases and strength-duration time constants (tau sd) of single DR fibers were measured by stimulation in the DH or in the DW. Conduction velocity was estimated on the basis of onset latencies of evoked spikes (the time from stimulation to action potential arrival at the recording electrodes). Population supernormality was evaluated on the basis of responses to conditioned and unconditioned submaximal stimuli delivered to the DH or to the proximal end of isolated DRs. 3. Single-fiber action potentials occurred at longer latencies after DH stimulation than after DW stimulation. Estimated intraspinal conduction velocity was congruent to 0.6 m/s. Extraspinal conduction velocity in these fibers averaged 22.2 m/s. Average tau sd was longer in the DH than in the DW (670 microseconds vs. 204 microseconds). 4. DH and DR test responses evoked 10-150 ms after a conditioning stimulus had increased areas relative to unconditioned test responses. Conditioning-associated changes in evoked responses were greater with the DH stimulation site than with the DR stimulation site, and these changes were not altered by treatment designed to block synaptic transmission. 5. We conclude that membrane properties determining tau sd differ between large afferent axons and fine terminal regions of those axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Dorsal root potentials in the isolated frog spinal cord: Amino acid neurotransmitters and magnesium ions

Sucrose gap techniques recorded dorsal root potentials evoked by supramaximal dorsal root stimulation in in vitro, hemisected frog spinal cords. In 0 mM Mg 2+ large (mean 13.0 mV), long lasting (mean 8.1 s) dorsal root potentials were recorded which consisted of two components: (1) an early component sensitive to picrotoxin, bicuculline, and low [Cl −] 0 and presumably produced by activation of GABA A receptors; and (2) a long-duration second component enhanced and lengthened by picrotoxin, bicuculline and low [Cl −] 0 and thought to result from increased intemeuron discharges resulting from depression of GABA-mediated pre- and postsynaptic inhibition. Both the early and late components were reduced by over 90% in amplitude and duration by 20 mM Mg 2+ or by kynurenate and bicuculline. The early component of the dorsal root potential may depend mainly upon activation of non-N-methyl- d-aspartate receptors, but the late component requires bothN-methyl- d-aspartate and non-N-methyl- d-aspartate receptors. Thus, theN-methyl- d-aspartate antagonist d-(−)-2-amino-5-phosphonovalerate caused only a modest reduction in the amplitude of the early dorsal root potential component while the nonN-methyl- d-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione caused a much more substantial reduction. Exposure of the spinal cord to a “physiological” concentration of Mg 2+ (1.0 mM) greatly reduced the duration and somewhat reduced the amplitude of the dorsal root potential. The reduction of dorsal root potentials by 1.0 mM Mg 2+ appears to be caused by both pre- and postsynaptic factors. These include: (1a) reduction of evoked transmitter release; the reduction of the dorsal root potential by 1.0 mM Mg 2+ was partly reversed by doubling [Ca 2+] 0; (2a) block of theN-methyl- d-aspartate receptor ion channels; (3a) decrease of GABA-depolarization of primary afferent fiber terminals; GABA-depolarizations of terminals were depressed by 1.0 mM Mg 2+; and (4a) decreased release of K + by afferent volleys; exposure to 1.0 mM Mg 2+ reduced the increment in [K +] 0 produced by repetitive afferent stimulation. d-(−)-2-amino-5-phosphonovalerate had only minimal effects on dorsal root potentials in 1.0 mM Mg 2+ presumably because theN-methyl- d-aspartate receptor-ion channel complex is largely inactivated by the Mg 2+. In contrast, kynurenate and 6-cyano-7-nitroquinoxaline-2,3-dione substantially diminished dorsal root potential amplitude and duration.

Long‐term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs

Motoneurons of adult frog spinal cord have been retrogradely labeled with the carbocyanine derivative diI. Spinal cords were then dissociated and the labeled motoneurons partially purified by centrifugation over a bovine serum albumin (BSA) density gradient. The resulting cell suspension was plated on a substrate of innervated muscle extracellular matrix (ECM) to which the motoneurons attached and extended processes. Labeled adult motoneurons survived for more than 4 weeks in a defined medium in the absence of added serum or growth factors. These cultures of adult motoneurons provide a favorable preparation for studying molecular factors that influence process outgrowth and synapse formation.

Serotonin and GABA-induced depolarizations of frog primary afferent fibers

The interaction of γ-aminobutyric acid (GABA) and serotonin (5-HT) on primary afferent terminals of the isolated frog spinal cord was investigated by sucrose gap recordings from dorsal roots. Application of 5-HT (1.0–100 μM) to the Ringer's solution significantly reduced afferent terminal depolarizations elicited by concentrations of GABA ranging from 0.1 to 1.0 mM. The reductions of GABA-depolarizations which were produced by 1.0 μM 5-HT were mimicked by the 5-HT 1A agonists 8-OH-DPAT (8-hydroxy-2-( n-dipropylamino)tetralin) and ipsapirone. The effects of ipsapirone were reversed by the 5-HT 1A antagonists spiperone. The decreases of GABA-depolarizations produced by high doses of 5-HT were duplicated by application of α-methyl-5-HT 1 c/2 agonist and reversed by superfusion of the cord with mianserin, a 5-HT 1C/2 antagonist. The presumptive 5-HT 1A receptor-mediated effects of 1.0 μM 5-HT and 8-OH-DPAT appeared to result from a direct action on afferent terminals because the reduction of GABA responses was unchanged by addition of TTX to the Ringer's solution. In contrast, the putative 5-HT 1C/2 receptor actions of 100 μM 5-HT and α-methyl-5-HT were substantially reduced by TTX and are presumably caused by activation of receptors located on interneurons. GABA B receptors did not appear to be affected by addition of 5-HT at low or high concentrations because baclofen-induced afferent terminal hyperpolarizations remained unchanged during exposure to 5-HT. The effects of 5-HT on GABA-depolarizations were not caused by changes in GABA uptake or changes in the rate of desensitization of GABA receptors because: (1) afferent terminal depolarizations produced by the non-transportable GABA A receptor agonist muscimol were significantly reduced by 8-OH-DPAT and (2) the decrement in depolarization produced by repetitive exposure to GABA, which is caused by both ‘receptor’ desensitization and by neuronal transport of GABA, was unaffected by 5-HT. These observations are important in understanding the action of 5-HT on transmission of sensory information by afferent fibers.

Recurrent dorsal root potentials and motoneuron morphology in the frog spinal cord

About one third of motoneurons stimulated intracellularly evoked dorsal root potentials (DRP) in the lumbar segments of the isolated and perfused frog spinal cord. Axon collaterals were found in one of the 22 motoneurons filled with HRP (horseradish peroxidase) through the stimulating electrode. In further experiments injecting individual motoneurons with cobalt, and filling the ventral roots with HRP or cobalt, the frequency of occurrence of axon collaterals was about 2% of the number of labelled motor cells. It is suggested that the presence of motor axon collaterals is not indispensable in the generation of the DRP evoked by ventral root or motor cell stimulation.