Activation of NMDA receptors drives action potentials in superficial dorsal horn from neonatal rats.

We have investigated the role of NMDA receptors in mediating synaptic transmission in spinal cord lamina II over the first 2 weeks of postnatal development. High intensity root stimulation evoked D-APV-sensitive slow synaptic activity in lamina II neurons that drove action potential firing. This NMDA receptor-mediated activity was enhanced when bicuculline and strychnine were used to block synaptic inhibition. When activated by repetitive focal stimulation, synaptic activity mediated by NMDA receptors alone drove action potential firing. NMDA receptors were also able to drive action potential firing at synapses where AMPA receptors were present but blocked. Our data show that in lamina II of the dorsal horn, NMDA receptors significantly affect neuronal excitability even in the absence of co-activation of AMPA receptors.

Effects of the P2-purinoceptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid on glutamatergic synaptic transmission in rat dorsal horn neurons of the spinal cord.

The effects of suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) on glutamatergic synaptic transmission were studied on dorsal horn lamina II neurons of rat spinal cord slice preparation and cultured dorsal horn neurons. Suramin at 100 microM significantly suppressed the amplitude of the evoked excitatory postsynaptic currents (EPSCs) by 33%, miniature EPSC (mEPSC) amplitude was decreased by 46% and the mEPSC frequency also decreased by 41%. PPADS at 50 microM had little effect on either the evoked EPSCs or mEPSCs. The lack of effect of PPADS on glutamatergic synaptic transmission suggests that the effect of suramin is less likely to be mediated by P2x receptors. When whole-cell (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) currents evoked by glutamate were examined, both suramin and PPADS showed no inhibition of peak amplitude. However, the onset of glutamate-evoked whole-cell currents became significantly slowed by suramin but not by PPADS. The suppression of synaptic transmission by suramin may be due, in part, to the slowed onset of glutamate-evoked AMPA currents. These results suggest that the analgesic effects of suramin shown in cancer patients and animal pain models may not be solely due to its antagonism to purinoceptors. PPADS is probably a more suitable antagonist for the study of synaptic P2x receptor function at excitatory synapses mediated by AMPA receptors.

NMDA EPSCs at glutamatergic synapses in the spinal cord dorsal horn of the postnatal rat.

In rat dorsal horn, little is known about the properties of synaptic NMDA receptors during the first two postnatal weeks, a period of intense synaptogenesis. Using transverse spinal cord slices from postnatal day 0-15 rats, we show that 20% of glutamatergic synapses tested at low-stimulation intensity in spinal cord laminae I and II were mediated exclusively by NMDA receptors. Essentially all of the remaining glutamatergic EPSCs studied were attributable to the activation of both NMDA and AMPA receptors. Synaptic NMDA receptors at pure and mixed synapses showed similar sensitivity to membrane potential, independent of age, indicating similar Mg2+ sensitivity. Kinetic properties of NMDA EPSCs from pure and mixed synapses were measured at +50 mV. The 10-90% rise times of the pure NMDA EPSCs were slower (16 vs 10 msec), and the decay tau values were faster (tau1, 24 vs 42 msec; tau2, 267 vs 357 msec) than NMDA EPSCs at mixed synapses. Our results indicate that NMDA receptors are expressed at glutamatergic synapses at a high frequency, either alone or together with AMPA receptors, consistent with the prominent role of NMDA receptors in central sensitization (McMahon et al., 1993).

Electrical properties of periglomerular cells in the frog olfactory bulb.

Whole-cell patch clamp recording techniques were applied to periglomerular (PG) cells in slices of the frog olfactory bulb (OB) preparation to study the basic electrical properties of these inhibitory interneurons. The cells were intracellularly stained with Lucifer Yellow for precise identification. Under current-clamp conditions PG cells showed rich spontaneous excitatory synaptic activity at rest, usually leading to overshooting, TTX-sensitive action potentials. The passive cable properties of the cell membrane have been carefully characterised. Depolarisation of this neurone under voltage-clamp conditions activated a complex pattern of current flow, that has been dissected into its main components. The currents have been isolated resorting to their different kinetic and pharmacological properties. Four main voltage dependent ionic currents have been isolated, two inward currents, I(Na) and I(Ca), and two outward currents carried by potassium ions, one fast transient, I(A)-type and another similar to the delayed rectifier type. These currents have been characterised kinetically and pharmacologically. The functional implications of their properties are discussed.

Excitatory synapses in the glomerular triad of frog olfactory bulb in vitro.

Whole-cell patch clamp recording techniques were applied to periglomerular (PG) cells in slices of the frog olfactory bulb (OB) to study the properties of the excitatory synapses in the triad formed by the olfactory nerve (ON) and the dendrites of mitral/tufted (MT) cells and PG cells. The postsynaptic response evoked by ON stimulation was glutamatergic and could be dissected into NMDA and non-NMDA components of equivalent amplitudes. The dendro-dendritic synapse between MT and PG cells could be activated following antidromic stimulation of the lateral and medial olfactory tract (LOT and MOT). In this case the postsynaptic potentials had amplitudes and durations comparable to those obtained by ON stimulation, the neurotransmitter was glutamate, but the synapse was largely dominated by the slow NMDA component.

Potassium currents in periglomerular cells of frog olfactory bulb in vitro.

Voltage-activated currents have been recorded from periglomerular cells in thin slices of frog olfactory bulb. Cells were examined with whole-cell patch clamp methods. The voltage-dependent potassium currents were studied after pharmacological block of inward currents. Depolarising steps from -130 mV gave an early transient, A-type, outward current and a delayed rectifier K+ current (IKV). The two currents could be isolated on the basis of the differences in their kinetic properties. The A-current developed following a third-order kinetics when the membrane was depolarised to potentials more positive than -40 mV after preconditioning to potentials more negative than -60 mV. Once activated (tau a 2.5 ms at 0 mV), IA inactivated following a single exponential (tau ha about 60 ms). IKV activated with a second-order kinetics above -30 mV with a time constant of 4 ms at 0 mV. IA and IKV were sensitive, respectively, to 4-aminopyridine (4-AP) and tetraethylammonium (TEA).

Sodium current in periglomerular cells of frog olfactory bulb in vitro.

Kinetic properties of the sodium current in periglomerular (PG) cells were investigated by applying whole-cell patch-clamp techniques to thin slices of the frog olfactory bulb. Eight of the cells were intracellularly stained with Lucifer Yellow for precise identification. Under current-clamp conditions PG cells showed rich spontaneous activity at rest. Na current was isolated from other current contributions by equimolar substitution of K+ with Cs+ in the intracellular solution to prevent K-currents, and 100 microM Cd2+ in the external solution to block Ca-current. Depolarisations beyond -40 mV activated a fast transient TTX-sensitive inward current. Once activated, INa declined exponentially to zero following a single exponential. The underlying conductance showed a sigmoidal activation between -40 and +30 mV, with half activation at -17.4 mV and a maximal value of 9.7 nS per neurone. The steady-state inactivation was complete at -30 mV and completely removed at -90 mV, with a midpoint at -56 mV. The activation process could be adequately described by third order kinetics, with time constants ranging from 260 microseconds at -20 mV to 70 microseconds at +50 mV.

Electrophysiological effects of a neurotoxin extracted from the skin of the Australian frog Pseudophryne coriacea.

1. The electrophysiological effects of a pumiliotoxin-B-like alkaloid extracted from the skin of the Australian frog Pseudophryne coriacea (PsC) have been studied in rat superior cervical ganglia at 37 degrees C. 2. PsC (50 mg/ml) elicits a broadening of the evoked compound action potential and, at rest, the appearance of spontaneous spike discharge at 10-20 Hz. Action potentials presumably originate far away from the soma, which is invaded in a typical IS-SD sequence. 3. The toxin effect is not related to any direct action on the preganglionic fibers of the sympathetic trunk, and does not involve synaptic mechanisms. 4. Two-electrode voltage-clamp experiments showed that the main properties of the major voltage-dependent ionic currents are apparently unaffected by the toxin, while the cell input resistance is considerably reduced. 5. The data are consistent with the hypothesis that PsC elicits a cationic permeability increase generating a pacemaker current in a region close to the cell soma.

Effects of reinnervation with normal and tetrodotoxin-inactive nerves on resting membrane potential of rat skeletal muscle.

Resting membrane potentials (RMPs) have been recorded in vitro near the end-plate region of rat soleus muscles reinnervated with tetrodotoxin-inactive nerves and compared with those of denervated muscles whose reinnervation had been prevented. The two muscle groups exhibited the same low values of RMP typical of denervated muscles. In control muscles of rats in which impulse conduction was left unimpaired, reinnervation induced the expected increase in RMP values towards normal. It is suggested that, at least for this property, reinnervation restores to normal the muscle fibre membrane essentially through the return of activity.

Comparative neurophysiological assessments of nerve sutures performed by microsurgical methods and with fibrin glue: experimental study.

The authors carried out an experimental study to compare the regeneration of nerves repaired with human fibrin glue and those sutured with nylon. The experiments were performed on rat peroneal nerves; the degree of regeneration was assessed by the neurophysiological strain gauge method, which involved recording the intensity of isometric contraction of the extensor digitorum longus muscle, which is innervated by the peroneal nerve. The results showed that the nerves "glued" with fibrin exhibited better regeneration than those repaired by suture.

Interaction of inactivity and nerve breakdown products in the origin of acute denervation changes in rat skeletal muscle.

The action of nerve breakdown products on innervated fibres of soleus and extensor digitorum longus muscles was investigated with the following procedures: partial denervation, sensory or sympathetic denervation, section of a previously transplanted foreign nerve. Each procedure was performed either in isolation or combined with chronic muscle inactivity obtained by blocking impulse conduction along the sciatic nerve. Silastic cuffs containing tetrodotoxin (TTX) and sodium chloride were utilized for the block. Partial denervation induced extrajunctional sensitivity to acetylcholine (ACh) and resistance to tetrodotoxin not only in the denervated but also in the innervated fibres. The effects in the innervated fibres were equal in magnitude to those in the denervated fibres, provided they were paralysed. The onset of the membrane changes was synchronous in the two classes of fibres and their amount correlated with the extent of partial denervation. If the innervated fibres were normally active, the membrane changes were still detectable, but considerably smaller than in the denervated fibres. Sensory denervation (removal of dorsal root ganglia L4 and L5) was followed by the development of moderate ACh supersensitivity and TTX resistance in chronically paralysed muscles. Furthermore, section of radicular nerves (total denervation, i.e. efferent plus afferent) induced larger membrane changes than those observed following section of ventral roots alone (efferent denervation). Sympathetic denervation was ineffective even when associated with chronic muscle paralysis. Section of a previously transplanted mixed nerve (superficial fibular) was ineffective if the soleus muscle was normally active, while it induced marked extrajunctional ACh sensitivity and TTX resistance when combined with chronic paralysis of the muscle. Section of a transplanted sensory nerve (sural) also induced extrajunctional membrane changes in paralysed soleus muscles, but their magnitude was much smaller than after section of mixed nerves. We conclude that products of nerve destruction, especially those of motor axons, induce membrane changes of striking magnitude when potentiated by muscle inactivity. Such an action may also explain the greater efficacy of denervation vs. pure inactivity, at least at early times after their onset.

Effects of reinnervation upon electrical membrane properties of normal and paralyzed muscles.

Resting membrane potentials (RMP) and resistance to tetrodotoxin (TTX) have been compared in denervated rat soleus muscles and muscles reinnervated with tetrodotoxin-inactive nerves for periods of 15-18 days. RMP's of the two muscle groups exhibited the same low values typical of denervated muscles. Similarly, comparable values of TTX-resistance were found in the two muscle groups, although exceptions with slightly lower values in the innervated-paralyzed muscles were noted. It is concluded that muscle reinnervation restores to normal the membrane properties altered by denervation essentially through the return of muscle activity.

[Role of NGF in the development of primary sensory neurons in the rat]. / Ruolo dell 'NGF nello sviluppo dei neuroni sensitivi primari nel ratto.

Two populations of neurons are present in the dorsal root ganglia: i) the first which is connected with the neuromuscular spindles is localized in the mediodorsal (MD) part of the ganglion; ii) the second which is connected with the cutaneous receptors is localized in the ventrolateral (VL) part of the ganglion. The growth of MD but not of VL neurons in vitro is dependent upon the presence of the NGF. In order to study the presence in vivo of such differential effect of the NGF on the two neuronal populations, we have injected rat embryos with NGF antiserum (AS-NGF) and recorded the compound action potentials of different hindlimb nerves. We found that the sensory component, in the studied muscular nerves of the hindlimb, is seriously depressed in treated animals. These results indicate that the animals treated with AS-NGF suffer of a depression of activity in the afferents from the muscle spindles. This is probably due to a decrease in the number of these fibers in their muscle nerves.

Responses of lateral reticular neurons to sinusoidal stimulation of labyrinth receptors in decerebrate cat.

1. The electrical activity of 106 individual neurons located in the precerebellar lateral reticular nucleus (NRL) and the surrounding medullary reticular formation (RF) has been recorded in precollicular decerebrate cats during sinusoidal tilt around the longitudinal axis of the whole animal leading to stimulation of labyrinth receptors. 2. Among these lateral reticular neurons tested, 48 of 712 (67.6%) NRL neurons and 11 of 35 (31.4%) RF neurons responded to slow rotation of the animal at the standard frequency of 0.026 Hz and at the peak amplitude of displacement of 5-10 degrees. 3. All the responsive units showed a periodic modulation of firing rate during the sinusoidal stimulus. In particular, 35 of 57 units (i.e., 61.4%) were excited during side-up and depressed during side-down tilt of the whole animal; on the other hand, 14 of 57 units (i.e., 24.6%) showed the opposite behavior. In both instances, the peak of the responses occurred with an average phase lead of about 16 degrees with respect to the extreme side-up or side-down position of the animal. The remaining eight units (i.e., 14%) showed a phase shift of the peak of their response of about 90 degrees with respect to the animal position. 4. The sensitivity of the responses, expressed in percentage change of the average firing rate per degree of displacement, did not change by increasing the peak amplitude of tilt from 5 to 15 degrees at the frequency of 0.026 Hz. This finding indicates that the system was relatively linear with respect to the amplitude of stimulation. The sensitivity of the units, however, slightly increased but the phase angle of the responses did not change by increasing the frequency of tilting from 0.015 to 0.15 Hz at the peak amplitude of 5 or 10 degrees. These findings indicate that the responses depended on stimulation of macular labyrinth receptors. 5. Most of the lateral reticular units affected by tilt received also a bilateral convergent input from the hindlimbs. 6. These observations are related to the results of previous studies in which the responses of macular afferents, vestibular nuclei neurons, and corticocerebellar Purkinje (P) cells to sinusoidal tilt of the whole animal have been investigated. A possible role of lateral reticular neurons in the labyrinth control of posture in decerebrate cat is also discussed.

Responses of Purkinje cells of cerebellar vermis to sinusoidal rotation of neck.

1. The response of Purkinje (P) cells located in the vermal cortex of the cerebellar anterior lobe to sinusoidal rotation of the neck was investigated in precollicular decerebrate cats. The head of the animal was fixed in a sterotaxic frame while the spinous process of the second cervical vertebra was held by a clamp rigidly fixed to the tilting table. It was then possible to elicit a selective neck input by rotating the neck and the body simultaneously along the longitudinal axis of the animal while maintaining the head in horizontal position. 2. Among the 95 P-cells tested for neck stimulation, 35 units showed a mossy fiber (MF) or a climbing fiber (CF) response to sinusoidal rotation of the axis vertebra at the frequency of 0.026 Hz and at the peak amplitude of displacement of 5--10 degrees. The response consisted in a periodic modulation of the discharge frequency during sinusoidal rotation of the neck. Most of these units were excited during side-down rotation of the neck, but were inhibited during side-up rotation. 3. The threshold amplitude of neck rotation responsible for the MF-induced responses varied in different units from 1 to 3 degrees at the frequency of 0.026 Hz. The sensitivity of the units, expressed in percentage change of the average firing rate per degree of displacement, either did not change or very slightly decreased as a result of increasing amplitude of stimulation from 1--3 degrees to 10--15 degrees at the frequency of 0.026 Hz or by increasing frequency of neck rotation from 0.015 to 0.15 Hz at the amplitude of neck displacement of 5--10 degrees. 4. Changes in amplitude or frequency of stimulation at the parameters reported above did not greatly modify the phase of the unit responses relative to the side-down position of the neck. These findings indicate that the MF and CF responses of P-cells to sinusoidal rotation of the neck depended on changes in neck position and not on changes in velocity of neck rotation. 5. The observation that the majority of responding P-cells located in the vermal cortex of the cerebellar anterior lobe increased their firing rate during side-down rotation of the neck is discussed in relation to the results of stimulation and lesion experiments, indicating that postural changes can be elicited either during asymmetric stimulation of neck receptors or by unilateral interruption of the neck afferents.

Responses of Purkinje cells of the cerebellar vermis to neck and macular vestibular inputs.

1. The dynamic analysis of the control exerted by neck and macular vestibular receptors on the cerebellar cortex has been investigated in precollicular decerebrate cats submitted to sinusoidal rotation along the longitudinal axis of the animal at the frequency of 0.026 Hz and at peak amplitudes up to 10 degrees for the neck input and 15 degrees for the macular input. 2. Purkinje (P) cells located in the vermal cortex of the cerebellar anterior lobe, particularly in the longitudinal parasagittal zone which projects to the ipsilateral lateral vestibular nucleus (LVN), showed a sinusoidal modulation of the firing rate in response to sinusoidal stimulation of the neck receptors or the vestibular receptors, the phase of the responses being in most units related to the extreme neck or head position. Mossy fiber (MF) and/or climbing fiber (CF) responses of the same or different P-cells to the two inputs were observed. 3. The sensitivity of the MF-response of the P-cells to the neck input, elicited by sinusoidal rotation of the neck and expressed in per cent of the average firing rate per degree of neck rotation, corresponded on the average to 2.71 +/- 1.67, S.D. This value was significantly higher than that of the MF-response of the P-cells to the macular input elicited by sinusoidal tilt along the longitudinal axis of the whole animal, which correspond to 1.71 +/- 1.01, S.D. 4. Most of the MF-responses of the P-cells to the neck input were characterized by an excitation during side-down rotation of the neck and by an inhibition during side-up rotation, whereas most of the MF-responses of the P-cells to the macular input showed just the opposite behavior, being inhibited by side-down tilt of the animal and excited by side-up tilt. 5. Units which received a convergent input from both neck and macular receptors and showed an antagonistic pattern of response to the two inputs were tested during rotation of the head alone, in order to excite simultaneously the two kinds of receptors. Due to the higher sensitivity of the neck over the macular response, the magnitude of the combined response tended to be similar to the difference between the individual ones. Moreover, the phase of the resulting response was always modified with respect to that of the response to the neck input alone, and became in some instances related to velocity of neck rotation rather than to neck position. 6. These findings indicate that opposite responses to neck and macular inputs occur at corticocerebellar level. However, a final integration of the two inputs, leading to suppression of the conflicting responses, may occur either at medullary (LVN) or at spinal cord level.

Sensitivity of lateral cerebellar nucleus to macular stimulation in the rabbit.

Experiments were performed in decerebrate rabbits to determine the sensitivity of the lateral cerebellar nucleus (LCN) to macular stimulation. Twenty-three percent of the neurons recorded extracellularly in the LCN showed steady changes in their discharge rate during 20 degrees tilt in both directions of the medial plane. Most of these neurons exhibited an alpha- and beta-type of response. A few gamma-types, but no delta-types were observed. The units sensitive to tilt were restricted to the caudal half of the LCN. Some of these positionally sensitive neurons responded monosynaptically to ipsilateral labyrinthine stimulation, but many received a polysynaptic input. These units could be activated antidromically by stimulation of the oculomotor nucleus but at a very high intensity suggesting current spread to the nearby brachium conjunctivum fibers. These results exclude a role of the LCN in the disynaptic otolith-ocular reflex.

Functional organization of vestibular and visual inputs to neck and forelimb motoneurons in the frog.

1. Intracellular responses in neck and forelimb motoneurons to electrical stimulation of the vestibular nerve, the optic tectum, and the optic nerve were studied in frog. 2. Stimulation of the anterior branch of the vestibular nerve typically produced EPSPs, bilaterally, in neck, shoulder (DOR), and forelimb extensor (TRI, RAD) motoneurons, and bilateral IPSPs in forelimb adductor (PED) and flexor (ULN, COR) motoneurons. 3. Latencies of PSPs recorded in neck, shoulder, and proximal extensor motoneurons (TRI) were mostly in the disynaptic range, whereas many of those recorded in distal extensor (RAD) and in adductor and flexor motoneurons involved three synapses. 4. Lesion of the vestibulospinal fibers greatly reduced the vestibular nerve-evoked field potentials in the spinal cord and the occurrence of PSPs in forelimb motoneurons. These results as well as the latency measurements suggest that the pathway linking vestibular nerve and forelimb motoneurons mainly consists of vestibulospinal fibers, though involvement of other structures for production of later PSPs could not be completely ruled out. Hemisection of the brain stem at its most caudal level showed that the pathway to the contralateral motoneurons crosses at the level of brain stem as well as in the spinal cord. 5. Stimulation of the optic tectum produced EPSPs, IPSPs, and a mixture of EPSPs and IPSPs in neck, shoulder, and forelimb motoneurons, bilaterally. Most frequently, a combination of an excitation and inhibition was observed. The pathway from the optic tectum to neck and limb motoneurons is at least dysnaptic in nature. 6. Stimulation of the optic nerve produced IPSPs and a mixture of EPSPs and IPSPs in neck and forelimb motoneurons. Impulses originating from the optic nerve descend as far as to lumbar motoneurons producing EPSP-IPSP sequences bilaterally. 7. Interaction studies suggested that the vestibular and optic pathways to neck and forelimb motoneurons are separate from each other so that the site of integration of vestibular and visual input occurs at the level of motoneurons. 8. Evidence for electronic coupling among forelimb motoneurons and electrical synaptic transmission in th pathway linking vestibular nerve and forelimb motoneurons is presented.