Intracellular recording of the frog dorsal root single fibres' responses mediated by the GABAA and 5-HT-receptors

The effects of GABA, bicuculline and 5-HT on primary afferents in the isolated spinal cord of the frog Rana ridibunda were studied. Bath application of GABA (1 mM) reduced the primary afferent depolarisation (PAD) in IX segment of the spinal cord evoked by X dorsal root stimulation (57 +/- 8% of initial level, n = 5, p < 0.05). The action potentials (AP) recorded in dorsal root afferents was also suppressed under the GABA action (74 +/- 9%, p < 0.05). Bath application of bicuculline (50 microM) reduced the PAD (21 +/- 7%), n = 6, p < 0.05), meanwhile the AP in dorsal root afferents was resistant against the bicuculline action. Bath application of 5-HT (25 microM) depressed the PAD (34 +/- 7%, n = 7, p < 0.05) and the amplitude of the AP recorded from the single afferent fibre in dorsal column (76 +/- 6%, n = 7, p < 0.05). In contrast to GABA, 5-HT more effectively suppressed the late phase of the PAD evoked by X dorsal root stimulation and caused (76 +/- 6%, n = 7, p < 0.05) an alteration of the AP shape. All effects induced by these drugs were reversible. The mechanisms of GABA and 5-HT modulation of spinal cord afferent income are discussed.     

Supersensitivity of the GABA receptor in the frog spinal cord, as induced by kainic acid

In the isolated frog spinal cord perfused with kainic acid (KA, 5 X 10(-4) M) containing Ringer's solution, within 2 hr there were increases in the amplitude of the dorsal root depolarization, as induced by the GABA-agonists. KA perfusion produced increases in the specific binding of [3H]muscimol to crude synaptic membranes and incubation with KA for 3 hr did not increase [3H]muscimol binding. [3H]GABA was released from KA-treated spinal cord slices in the presence of high K+. KA-induced supersensitivity of the dorsal root to GABA may relate to direct actions on primary afferent terminals and not to denervation of GABAergic neurons.     

Functional expression of AMPA receptors on central terminals of rat dorsal root ganglion neurons and presynaptic inhibition of glutamate release

No direct evidence has been found for expression of functional AMPA receptors by dorsal root ganglion neurons despite immunocytochemical evidence suggesting they are present. Here we report evidence for expression of functional AMPA receptors by a subpopulation of dorsal root ganglion neurons. The AMPA receptors are most prominently located near central terminals of primary afferent fibers. AMPA and kainate receptors were detected by recording receptor-mediated depolarization of the central terminals under selective pharmacological conditions. We demonstrate that activation of presynaptic AMPA receptors by exogenous agonists causes inhibition of glutamate release from the terminals, possibly via primary afferent depolarization (PAD). These results challenge the traditional view that GABA and GABA(A) receptors exclusively mediate PAD, and indicate that PAD is also mediated by glutamate acting on presynaptically localized AMPA and kainate receptors.     

The dorsal root reflex in isolated mammalian spinal cord

1. The dorsal root reflex has been investigated in an isolated preparation of adult mammalian spinal cord. 2. Both evoked and spontaneous activity can be recorded from the cord in the dorsal spinal roots. 3. The spontaneous activity has a characteristic pattern of firing in bursts of action potentials. Spontaneous and evoked activity are optimum at temperatures between 25 and 27 degrees C; little activity can be detected above 35 degrees C. 4. The spontaneous dorsal root activity has been shown to be correlated with negative potentials in the dorsal horn of the cord, and intracellular recordings made from primary afferent fibres have shown spontaneous primary afferent depolarizations (PAD) which underlie the generation of the spontaneous dorsal root activity. 5. The evoked dorsal root reflex has been shown to spread up to 16 spinal segments both rostrally and caudally from the stimulated dorsal root, and to the contralateral side of the cord. 6. The spontaneous dorsal root activity in widely separated segments has been shown by cross-correlation analysis to be linked both ipsi- and contra-laterally. 7. The significance of such a widespread system for the generation of PAD is discussed.     

Primary afferent depolarization in frog spinal cord is associated with an increase in membrane conductance

The mechanism of primary afferent depolarization (PAD) was studied in the isolated frog spinal cord using intrafibre recording (microelectrodes filled with 0.6 M potassium sulfate) from large myelinated axons of dorsal roots. Standard current-clamp technique was used to obtain voltage-current (V-I) relationship. It was found that: (i) PAD is voltage dependent: its amplitude and rate of rise are increased with hyperpolarization; (ii) the slope of the linear part of the V-I curve obtained during PAD is decreased compared with the V-I curve at rest; (iii) the PAD equilibrium potential, estimated by extrapolation, ranged from -66 to -40 mV. These results suggest that PAD is associated with an increase in conductance of primary afferent terminals and thus seem to provide the first experimental evidence for the hypothesis that shunting of primary afferent membrane is the mechanism of presynaptic inhibition in the vertebrate nervous system.     

Hypothalamic substance P as a candidate for transmitter of primary afferent neurons.

A peptide that exerts a depolarizing action on frog spinal motoneurons was found in the dorsal root of bovine spinal nerve. Pharmacological, chemical, and immunological properties of this motoneuron-depolarizing peptide were investigated and the results indicated that the peptide is identical with an undecapeptide, substance P, recently isolated from bovine hypothalamus by M.M.Chang and S.E.Leeman. The amount of hypothalamic substance P in bovine dorsal root determined by bioassay or radioimmunoassay was 24-130 pmole/g wet wt, whereas that in the ventral root was 9-27 times less. The effects of synthetic hypothalamic substance P on the isolated spinal cord of the frog and the newborn rat were studied. The peptide exerted a powerful depolarizing action on the motoneurons, its potency being about 200 times higher than that of L-glutamate. Distribution of substance P in the cat spinal cord was studied. The concentration of the peptide was highest in the dorsal part of dy lowered. When the dorsal root of the cat was ligated, substance P accumulated in a high concentration on the ganglion side of the ligature. These results, taken together, support the hypothesis that hypothalamic substance P is an excitatory transmitter of primary afferent neurons.     

Effects of Serotonin on Primary Afferents in the Frog Spinal Cord

We studied, on isolated preparations of the frog spinal cord, the effects of serotonin in different concentrations on the amplitude-temporal parameters of action potentials (AP) in primary afferent fibers, on the potentials reflecting depolarization of primary afferents (DPA), and on the properties of the membrane of these fibers. It was demonstrated that in a part of the dorsal root afferent fibers serotonin caused a drop in the AP amplitude (by 15-20%) and an increase in the AP duration (by 8-13%). Serotonin also significantly (by 70-90%) decreased the amplitude of DPA induced by stimulation of a neighboring dorsal root and noticeably reduced the input membrane resistance of afferent fibers. Serotonin-induced modulation of the AP parameters in the afferents and suppression of DPA under the influence of this amine are postulated as possible factors involved in the central control of afferentation.     

A Study of the Role of GABAA- and GABAB-Receptors in Presynaptic Inhibition of Primary Afferents in the Spinal Cord of the Frog Rana ridibunda

The role of GABA_A- and GABA_B-receptors in presynaptic inhibition of primary afferent fibers was studied on an isolated preparation of the spinal cord of the frog Rana ridibunda. It is shown that the inhibitory effect of GABA on synaptic transmission from afferent fiber to motoneuron is caused by activation of both GABA_A- and GABA_B-receptors. A temporal correlation (± 5 min) was shown between the blocking action of bicuculline (a specific antagonist of GABA_A-receptors) on primary afferent fiber depolarization (PAD) and its potentiating effect on the excitatory postsynaptic potential (EPSP) at parallel intracellular recording of EPSP in motoneuron and PAD in axons of the dorsal root. As a basis of this correlation, the single GABA_A-receptor mechanism is discussed, which mediates the effect of bicuculline on PAD and EPSP. When a specific agonist of GABA_B-receptor, baclofen, and an antagonist of GABA_B-receptor, 2(OH)-saclofen, were applied, the obtained data indicated an involvement of GABA_B-receptors in inhibition of synaptic transmission from afferent fibers to the motoneuron. Analysis of parameters of the unitary synaptic responses recorded in the control experiments and of their changes under the effect of (– )-baclofen indicates that the inhibitory action caused by activation of GABA_B-receptors develops at the presynaptic level.     

5-HT7 receptors are involved in mediating 5-HT-induced activation of rat primary afferent neurons

The purpose of this study was to determine whether the 5-hydroxytryptamine7 (5-HT7) receptor is expressed by nociceptor-like neurons in the rat PNS and whether 5-HT activates these nociceptors via the 5-HT7 receptor subtype. Using a polyclonal antibody and the method of immunofluorescence staining, we demonstrated that the 5-HT7 receptor appears predominately on "nociceptor-like" neurons of the rat lumbar dorsal root ganglia. Using immunocytochemical methods, we showed that the immunoreactivity of the 5-HT7 receptor antibody complex is localized in the superficial layers of the spinal cord dorsal horn, which corresponds with laminae I, IIouter and IIinner. Furthermore, we demonstrated that noxious stimulation produced by knee injection of 5-HT or a 5-HT7 agonist dose-dependently increases c-Fos production of the rat spinal cord dorsal horn. This effect was significantly inhibited by the preinjection of a 5-HT7 antagonist. We conclude that the 5-HT7 receptor is expressed by rat primary afferent nociceptors which terminate in the superficial layers of the spinal cord dorsal horn and that the 5-HT7 receptor subtype is involved in nociceptor activation by 5-HT.     

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.     

Electrophysiological study of the effects of cardiodilatin 1-16 on the frog spinal cord in vitro

Using the isolated spinal cord of the frog, hemisected and further divided into two distinct quadrants, we studied electrophysiological changes produced by peptides present in the atrial natriuretic factor (ANF) preprohormone. ANF and related peptides (atriopeptin I and atriopeptin III) did not affect the frog spinal cord. The 1-16 fragment from cardiodilatin (10(-5) M) induced slow depolarization in ventral and dorsal nerve stumps. The depolarization was associated with an increase of the evoked dorsal root potentials and depression of the fast component of the reflex responses. When depolarization approached its maximum value, spontaneous slow potentials appeared progressively similar to the evoked potentials, and became rhythmic until they reached a frequency of one potential every 15-20 seconds. The effects of cardiodilatin 1-16 are localized at dorsal horn level. It is suggested that this substance exerts a modulatory effect on frog cord physiology.     

Effects of vasopressin and oxytocin on dorsal root potentials in perfused spinal cord isolated from newborn rats

Dorsal root potentials before and after adding vasopressin or oxytocin to the perfusing fluid were investigated during experiments on one or two perfused spinal cord segments isolated from 12- to 16-day-old rats. It was found that both neuropeptides reversibly inhibited the amplitude of dorsal root potentials produced by stimulating the adjoining dorsal root. The effect was dependent on concentration and time of peptide action on the brain. Both vasopressin and oxytocin were found to produce slow, reversible, dose-dependent depolarization at primary afferent fiber terminals. Depolarization persists when trans-synaptic transmission has been completely blocked owing to substitution of calcium by manganese ions in the perfusing solution. Synaptic contacts are thought to exist between peptidergic hypothalamospinal fibers and dorsal root afferent fiber terminals. The functional significance of these connections is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 6, pp. 757–763, November–December, 1988.     

Primary afferent depolarization distribution of the γ-aminobutyric acid system in frog spinal cord

In the frog spinal cord primary afferent depolarization (PAD) constitutes a powerful inhibitory control mechanism. It has been suggested that -aminobutyric acid (GABA) is the transmitter substance involved in the genesis of PAD. In these studies we show that maximal glutamic acid decarboxylase activity is localized roughly 400–600 m from the dorsal surface, and that correlates well with the intraspinal distribution of field potentials associated with PAD. Measurement of GABA in serial spinal cord sections cut in a dorsal-ventral direction shows that high levels of GABA are seen at 400–600 m, with a peak at 800 m from the dorsal surface. Stimulation at frequencies shown to produce PAD augments the release of endogenous GABA from a superfused frog hemicord preparation.     

Depolarizing effects of dopamine on the primary afferent fibers of a segment isolated from the spinal cord of newborn rats

Effects of dopamine on dorsal root potentials were investigated during experiments on a segment of spinal cord isolated from 12- to 18-day-old rats. Applying dopamine to the brain was found to produce a slow, reversible, dose-dependent depolarization at primary afferent fiber terminals. This dopamine-induced depolarization was retained during complete blockade of synaptic transmission brought about by exchanging calcium ions in the perfusing fluid by magnesium or manganese ions. Minimum dopamine concentration required to produce this effect was 1·10^–10–1·10^–9 M. Peak amplitude of depolarization equaled 1.5 mV. Duration of this reaction ranged from 5.5 to 36.7 min, depending on the duration and concentration of dopamine application. Depolarizing response to dopamine differed considerably from GABA-induced dorsal root depolarization in amplitude and rate of rise. Haloperidol, a dopamine antagonist, reduced dopamine-induced dorsal root depolarization. Findings indicate that dopamine acts directly on the membrane of primary afferent fiber terminals, shifting membrane potential toward depolarization. This raises the possibility that dopaminergic brainstem-spinal pathways may exert an effect on sensory information transmission in segmental reflex arcs already traveling to the spinal cord.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 741–748, November–December, 1987.     

The role of calcium ion in the L-glutamate-induced depolarization in the frog spinal cord

1. The role of Ca2+ in L-glutamate-induced depolarization was investigated in the isolated frog spinal cord. 2. The size of a depolarization induced by L-glutamate (3 mM) was inversely related to the extracellular Ca2+ concentration, but was reduced in a Ca2+-free medium containing EGTA (0.3 mM). 3. L-Glutamate caused a marked depolarization in both ventral and dorsal roots, even in a NaCl-deficient medium (Ca2+, 2.0 mM). The size of the depolarization was attenuated by a prolonged or repeated application of L-glutamate. Ca2+ can be replaced by Sr2+ or Mg2+. 4. Concanavalin A (1 microM) prevents the development of desensitization to L-glutamate. 5. Present results suggest that Ca2+ plays the role of a charge carrier for L-glutamate-induced depolarization and of a regulator of modulator for L-glutamate-receptor sensitivity. The roles are exaggerated in NaCl-free medium.     

Slow excitatory transmission in rat spinal dorsal horn and the effects of capsaicin

High intensity repetitive stimulation of a dorsal root elicited slow depolarization in more than half of the dorsal horn neurons examined in the rat spinal cord slice preparation. There was a significantly smaller group of neurons showing slow hyperpolarization as well. Slow depolarization was not observed when synaptic activity was blocked by perfusing the slice with a TTX- or a low-Ca2+ high-Mg2+ solution. This result is consistent with a presynaptic origin of the slow response. Capsaicin treatment of neonatal rats significantly reduced the incidence of slow depolarization, suggesting that the slow depolarization was generated by small diameter afferent fibres, probably unmyelinated afferents. DR-evoked slow depolarization and SP-induced depolarization were similar in several important aspects: a) Both responses caused depolarization and increased the excitability of dorsal horn neurons; b) They were frequently associated with similar membrane conductance changes; c) The size of both responses varied in parallel when the membrane potential was shifted over a wide range; d) Both responses were markedly reduced or abolished by an analogue of SP having antagonist properties, and by polyclonal and monoclonal antibodies to SP; e) The depression of the DR-elicited slow depolarization during and after the SP-induced depolarization suggested that SP and the natural transmitter for the DR-elicited slow depolarization were bound to the same receptors. The results suggest that SP or, SP-like peptide, is an agonist that mimics in some aspects the action on the natural transmitter for the slow depolarizing potential.     

Depolarization of primary afferents during fictitious scratching in thalamic cats

The dorsal cord and dorsal root potentials were recorded in immobilized thalamic cats during fictitious scratching evoked by mechanical stimulation of the ear. Depolarization of primary afferents was shown to be simulated by the central scratching generator. Antidromic spike discharges appeared at the peak of the primary afferent depolarization waves in certain afferent fibers. Similar discharges arise in the resting state in response to stimulation of limb mechanoreceptors. It is suggested that during real scratching primary afferent depolarization and antidromic spikes evoked by it may effectively modulate the level of the afferent flow to spinal neurons.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 2, pp. 173–176, March–April, 1978.     

Effect of serotonin on isolated cells with the various functionality from the lamprey spinal cord

The differential actions of 5-hydroxytryptamine (5-HT) (100 microM) were investigated on isolated motoneurons, interneurons, and primary sensory neurons from the lamprey spinal cord using patch-clamp techniques. Application of 5-HT did not evoke membrane currents in any of the spinal neurons tested (n = 62). However, in most motoneurons and interneurons (15 of 18), 5-HT produced a small depolarization (2-6 mV), which was not accompanied by a change in input resistance. In the remaining motoneurons and interneurons (3 of 18), 5-HT induced a large depolarization (up to 10-20 mV) and a decrease in input resistance of 20-60%. In most sensory neurons (dorsal sensory cells, DSCs), 5-HT evoked a short-lasting, low-amplitude depolarization, followed by a long-lasting hyperpolarization of 2-7 mV. The DSCs showed no significant change in input resistance to 5-HT application (n = 8). Spike afterpolarization were also differentially modulated by 5-HT. In motoneurons and interneurons, 5-HT decreased the amplitude of the afterhyperpolarization following the action potential while increasing the amplitude of the after depolarization. In the DSCs, no significant effect of 5-HT on spike afterpolarization was observed. 5-HT differentially modulated the current induced by application of N-methyl-D-aspartate (NMDA). In motoneurons and interneurons, 5-HT enhanced NMDA-evoked current, while in DSCs, 5-HT decreased this current. These results demonstrate that 5-HT differentially modulates the activity of functionally different groups of spinal neurons. In motoneurons and interneurons, 5-HT enhances excitation by inducing depolarization and decreasing the afterhyperpolatization, while NMDA currents are enhanced. These effects facilitate the appearance of rhythmic discharges in these cells in the presence of NMDA. In primary dorsal sensory cells, 5-HT enhances inhibition by hyperpolarizing the cells and depressing NMDA currents. These differential effects are presumably mediated by different types of 5-HT receptors on these classes of spinal neurons.     

Action of ammonium acetate on central primary afferent depolarization

Experiments on anesthetized spinal cats showed that ammonium acetate, injected intravenously (2–4 mmoles/kg) inhibits the depolarization of the central endings of primary afferent fibers activated by stimulation of afferent nerves. Inhibition of primary afferent depolarization is transient in character and develops parallel with depression of postsynaptic inhibition of monosynaptic reflexes. The depression produced by the action of ammonium was not due to blocking of negative postsynaptic potentials of the dorsal surface of the spinal cord or blocking of reflex electrical discharges in the ventral spinal roots. It is suggested that depression of primary afferent depolarization is due to a decrease in the emf for synaptic ion currents producing depolarization.Allergologic Research Laboratory, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 1, pp. 52–60, January–February, 1977.     

Microiontophoresis of 5-hydroxytryptamine, epinephrine, and prostaglandin E1 on spinal neurons in the frog.

5-Hydroxytryptamine (5-HT) and epinephrine were applied by microiontophoresis to single neurons in the isolated spinal cord of the frog. 5-HT depressed all but two of the responsive cells, whereas the response to epinephrine consisted exclusively of depression. 5-HT action was more marked than that of epinephrine on most cells. With either compound, responseve units were diffusely distributed throughout the tissue. While it was proven that prostaglandin E1 (PGE1) exerts a direct excitatory action on spinal neurons, no evidence of an antagonism between PGE1 and the monoamines was obtained. These findings provide additional support to the hypothesis that 5-HT and epinephrine are transmitters in the frog spinal cord. The possibility that PGE1 may 'modulate' the responsiveness of spinal neurons to the monoamines was not confirmed.