Differential effects of muscimol upon the firing frequency of large and small amplitude antidromic dorsal root action potentials in rat spinal cord in vitro.

The effects of bath applied muscimol upon spontaneous and evoked antidromic activity recorded from lumbar dorsal roots was investigated in hemisected, isolated preparations of rat spinal cord. In magnesium free medium containing 0.1 microM 4-aminopyridine, bursts of high amplitude (up to 1 mV), dorsal root reflexes were recorded. These were blocked by low concentrations of muscimol (2-5 microM). Higher concentrations (5-20 microM) of muscimol caused a concentration-dependent increase in the frequency of small amplitude (<200 microV) spontaneous dorsal root action potentials. The possibility that the large and small amplitude extracellular action potentials reflect activity in large and small diameter dorsal root axons, and that these respond in different ways to the GABA(A) agonist muscimol, is discussed.

Cervicocephalic kinesthetic sensibility in patients with chronic, nontraumatic cervical spine pain.

OBJECTIVE: To investigate cervicocephalic kinesthetic sensibility (head repositioning accuracy to subjective straight ahead) in patients with chronic, nontraumatic cervical spine pain. DESIGN: A prospective, 2-group, observational design. SETTING: An outpatient chiropractic clinic in the United Kingdom. PARTICIPANTS: Eleven patients (6 men, 5 women; mean age +/- standard deviation, 41.1 +/- 13.3 yr; range, 18-55 yr) with chronic, nontraumatic cervical spine pain (mean duration, 24 +/- 18 mo), with no evidence of cervical radiculopathy and/or myelopathy or any other neurologic disorder. Eleven asymptomatic, unimpaired volunteers (5 men, 6 women; mean age, 39.3 +/- 10.3 yr; range, 28-54 yr) with no history of whiplash or other cervical spine injury or pain served as controls. MAIN OUTCOME MEASURES: Cervicocephalic kinesthetic sensibility was investigated by testing the ability of blindfolded participants to relocate accurately the head on the trunk, to a subjective straight-ahead position, after a near-maximal active movement of the head in the horizontal or vertical plane. The active cervical range of motion and the duration and intensity of neck pain were also recorded. RESULTS: Mann-Whitney U testing indicated that the patient (P) group was no less accurate in head repositioning than the control (C) group for all movement directions except flexion (median global positioning error [95% confidence interval], P = 5.7 degrees [5.03-9.10], C = 4.2 degrees [3.17-5.32]; p <.05). CONCLUSIONS: Nontraumatic neck pain patients show little evidence of impaired cervicocephalic kinesthetic sensibility. These results contrast with studies of chronic cervical pain patients in which the origin was not controlled or involved a cervical whiplash injury.

Effects of delta-aminolaevulinic acid upon electrical activity of rat spinal cord in vivo.

Bath applied delta-aminolaevulinic acid (ALA, 1-1000 microM), significantly depressed the frequency of spontaneous antidromic activity in the lumbar dorsal roots, and the amplitude of the monosynaptic component of the dorsal root-evoked ventral root reflex, in isolated rat spinal cord in a concentration-related manner. In contrast bath concentrations up to 1 mM ALA were found to produce no significant change in either the conducted afferent volley, nor field potentials recorded in the lumbar dorsal horn. These results indicate that the raised levels of ALA found in cerebrospinal fluid during porphyrias and lead poisoning may contribute to the neurological symptoms of these diseases.

The effect of FMRF-amide-like peptides on electrical activity in isolated mammalian spinal cord.

FMRFamide and seven FMRFamide-like peptides were manually synthesized on solid phase and their effects tested upon the amplitude of the dorsal root-ventral root monosynaptic reflex (MSR) and dorsal horn field potentials (FP) in an isolated preparation of rat spinal cord. FLFQPQRFamide (NPFF) and AGEGLSSPFWSLAAPQRFamide (NPAF) both depressed a fast component of the FP with similar potencies. FMRFamide also inhibited the FP but its potency was much lower. NPFF and NPAF potentiated the amplitude of the MSR while FMRFamide had no effect. PQRFamide, PFRFamide, FFRFamide, DPQRFamide and Fmoc-FLFQPQRFamide were also examined on the MSR. PQRFamide and PFRFamide potentiated the MSR whereas FFRFamide and DPQRFamide caused a small depression at high concentrations. The increase in amplitude of the MSR induced by NPFF was completely abolished when the N-terminal was left protected with an Fmoc-group. The results suggest that PFRFamide and PQRFamide may act as agonists of NPFF and NPAF whereas the other peptides did not show such activity.

c-fos expression in isolated rat spinal cord.

Expression of c-fos-immunoreactivity (c-fos-ir) has been demonstrated in the dorsal horn of lumbar segments of an isolated spinal cord preparation from 3 week old rats. The method of preparation generated a low level of c-fos-ir activity which was not significantly altered by low intensity (1.5 times threshold) dorsal root stimulation, but was significantly increased by high intensity (20 times threshold) stimulation. Replacement of the calcium in the bathing medium by 2 mM manganese suppressed all detectable c-fos-ir, whereas inclusion of 0.5 microM capsaicin caused intense c-fos-ir expression in the absence of stimulation. The number of dorsal horn cells exhibiting c-fos-ir increased between 0.5 and 1 h after stimulation, reaching a maximum at 2 h, with no further increase at longer intervals. Few positive cells were found when the incubation temperature was reduced from 27 to 20 degrees C. The strongest increase in c-fos-ir was found in the dorsal horn ipsilateral to the stimulated dorsal root and a smaller, but significant, increase was also seen in the contralateral dorsal horn. Cords obtained from animals treated at 1 day old with capsaicin to destroy afferent C fibres showed a reduction in the number of c-fos-ir positive cells induced by high intensity dorsal root stimulation. This preparation will aid detailed investigation of the pharmacology of nociceptive pathways.

Comparison of responses of spontaneously active cells in the cerebellar Purkinje layer to parallel fibre stimulation in slice preparations and urethane-anaesthetised rats: effects of benzodiazepine receptor ligands.

1. GABA-mediated inhibitory responses were induced in spontaneously active Purkinje cells by parallel fibre stimulation in cerebellar slices or in urethane-anaesthetised rats. Effects of agonist and inverse agonist benzodiazepine (BDZ) receptor ligands were compared in the preparations. 2. Purkinje cells fired simple spikes at higher rates in slice preparations while complex spikes were seldom (in vivo) or never observed (slice). Cells fired more regularly in vivo resulting in the occurrence of rhythmic postinhibitory responses in the PSTH analysis in some preparations. 3. Single pulse stimulation of parallel fibres at just suprathreshold intensity induced inhibition of Purkinje cell activity in both preparations. At lower firing rates there was a marked increase in the duration of this response, which was more evident in vivo where more slowly firing cells were encountered. 4. BDZ receptor ligands modified inhibitory responses in slice preparations with only weak effects on the firing rates of the cells. These compounds predominately induced changes in firing rate in the anaesthetised rat with little evidence of direct modification of GABA-mediated synaptic transmission. 5. In a few experiments, following injection of the partial inverse agonists beta-CCE and beta-CCM, block of the inhibitory response was observed independent of changes in firing rate. Bidirectional efficacy of BDZ receptor ligand (agonists decrease firing and increase inhibitory response, inverse agonists increase firing and decrease inhibitory response) was demonstrated for modulation of inhibitory responses in slices and for changes in firing rate in vivo. The increased firing rate response in vivo was biphasic the magnitude of the later phase being correlated with efficacy of inverse agonists. 6. It is concluded that cerebellar slice preparations are more appropriate for studying direct effects of BDZ receptor ligands on GABA-mediated synaptic inhibition than in vivo preparations.

Toxin I, but not 4-aminopyridine, blocks the late inhibitory component of the dorsal root reflex in an isolated preparation of rat spinal cord.

Bursts of spontaneous antidromic dorsal root action potentials, and evoked dorsal root reflexes (DRR), have been recorded from lumbar roots of isolated spinal cord preparations of rats weighing 70-90 g. The pattern of dorsal root activity was similar to that reported for isolated cord preparations from hamsters, but the frequency of spontaneous dorsal root activity was approximately 10 times slower in the rat. Toxin I and 4-aminopyridine (4-AP) both increased the frequency of spontaneous dorsal root activity. The threshold concentration of 4-AP was 1 microM, with EC50 at 20 microM. Insufficient Toxin I was available to reach a maximal response, but the threshold concentration for producing an increase in spontaneous activity was 0.1 microM, and the curve appeared to be parallel to that of 4-AP. The patterns of spontaneous dorsal root activity in the presence of 4-AP and Toxin I differed. In 4-AP bursts of large amplitude action potentials were followed by periods of depressed activity lasting up to 500 ms, whereas in Toxin I bursts of large amplitude action potentials caused no change in the continuously firing small amplitude action potentials. DRR evoked by stimulation of adjacent dorsal roots also showed differences in the presence of 4-AP and Toxin I. In 4-AP the excitatory phase of the reflex was followed by a period of depressed activity lasting up to 500 ms. This was was reduced or absent in the presence of Toxin I. Paired pulse stimulation confirmed the presence of inhibition in 4-AP, and its reduction in Toxin I. Examination of the pattern of spontaneous dorsal root activity following dorsal root stimulation showed strong oscillatory activity in the presence of 4-AP, but little such activity in the presence of Toxin I. It was concluded that the actions of 4-AP and Toxin I on the isolated preparation of rat spinal cord are similar in that both cause an increase in the spontaneous dorsal root firing rate, but that Toxin I also blocks the period of inhibition which follows bursts of large amplitude action potentials.

A human gene encoding morphine modulating peptides related to NPFF and FMRFamide.

FMRFamide-related peptides have been isolated from both invertebrates and vertebrates and exhibit a wide range of biological effects in rats. We show here that in humans 2 FMRFamide-related peptides are encoded by a single gene expressed as a spliced mRNA. The larger predicted peptide (AGEGLNSQFWSLAAPQRFamide) differs from the peptide isolated from bovines (AGEGLSSPFWSLAAPQRFamide) by the substitutions of 2 amino acids. The shorter predicted peptide (NPSF, SQAFLFQPQRFamide) is 3 amino acids longer than the bovine 8 amino-acid NPFF (FLFQPQRFamide) or the human NPFF peptide isolated from serum [5], suggesting that the encoded protein is subject to cleavage by a tripeptidyl peptidase or by a novel processing mechanism. On rat spinal cord, the larger peptide is indistinguishable in activity from the equivalent bovine peptide whereas the smaller extended peptide is inactive.

Analysis of neural bursting: nonrhythmic and rhythmic activity in isolated spinal cord.

Neuronal firing recorded from primary afferents and central spinal neurons in isolated mammalian spinal cord in vitro was analyzed using interspike interval histograms and Poincaré maps (Return maps). Interspike interval histograms described the time between two successive spikes. However, Poincaré maps represented the interspike interval preceding a spike against the interspike interval following the spike. The variability in firing of neurons occurring naturally in different preparations was explored. In a presumptive single central spinal neuron, the firing pattern provided a single cluster. During bursting in a presumptive single primary afferent, the Poincaré maps provided information on interburst intervals. Moreover, lengthening and or shortening of interspike intervals within bursts can be detected in the maps. The firing of a population of neurons was modulated using a pharmacological intervention with 4-aminopyridine and resulted in a complex Poincaré map.

The isolated mammalian spinal cord.

This review considers: spinal cord slices; isolated spinal cord sagitally or transversely hemisected; whole spinal cord; respiration control--[brain-stem spinal cord; brain-stem spinal cord with attached lungs]; nociception--[spinal cord with tail]; fictive locomotion--[spinal cord with one hind limb; spinal cord with two hind limbs]. Much of the functional circuitry of the CNS can be studied in the isolated spinal cord with the additional advantage that the isolated spinal cord can be perfused with known concentrations of ions, neurotransmitters, agonists, antagonists, and anaesthetics. These can be washed away, the circuitry allowed to recover and other drugs or different concentrations applied. Future preparations including the complete spinal cord, the two hind limbs, and a sagittal section of the complete brain will allow greater understanding of the multiple sensory and motor pathways and their interactions in the CNS.

The effects of RU 33965 and RU 34030, two new 3-cyclopropyl carbonyl imidazobenzodiazepines, on GABAA receptor-mediated synaptic transmission in cerebellar slices in the rat.

1. Two 3-cyclopropyl carbonyl imidazobenzodiazepines, RU 33965 and RU 34030, were tested for their ability to modulate GABAA synaptic transmission in rat cerebellar slices. The action of the full benzodiazepine agonist RU 32007 and the inverse agonist Ro19-4603 were tested for comparison. 2. Extracellular recordings were made from the Purkinje cell layer of the cerebellar slices and inhibition induced by just threshold electrical stimulation of the parallel fibres was bicuculline sensitive. 3. The major effect of RU 32007 when examined at 100 nM and 1 microM was to increase the GABAA mediated inhibition in the slice. 4. In contrast the major effect of the inverse agonist Ro19-4603 was to reduce the period of inhibition. 5. RU 33965 and RU 34030 at 10 and 1 microM respectively either had little effect on GABAA mediated inhibition or decreased it slightly. 6. RU 34030, 1 microM, abolished the agonist effect of RU 32007, 1 microM. 7. The effects of RU 32007 and Ro19-4603 were abolished by the benzodiazepine antagonist flumazenil. 8. It is concluded that both RU 33965 and RU 34030 have marginal inverse agonist properties.

Postnatal changes in the dorsal root reflex in the isolated spinal cord of the hamster, Mesocricetus auratus.

Spontaneous activity has been demonstrated in the lumbar dorsal roots of isolated spinal cord preparations taken from animals ranging in age from 2 to 65 days. Peaks of activity were recorded at 2 and 5 weeks of age, with mean firing frequencies of 33 Hz and 28 Hz respectively. The firing frequency in weeks 3 and 4 was lower (15 Hz) as was the frequency in cords taken from animals older than 6 weeks. The pattern of the spontaneous dorsal root activity changed during the first 5 weeks of life. In cords taken from animals less than 10 days old, the roots fired single action potentials, producing a single broad peak in Inter Spike Interval plots (ISI). Dorsal root recordings made from cords taken from animals in weeks 2 and 3 of life exhibited both single spikes and bursts of action potentials. By the end of the third week of life, individual spike activity had declined and the bursts of action potentials characteristic of the adult pattern had become dominant, producing a bimodal ISI plot. Cross correlation analysis of dorsal root and dorsal horn activity in lumbar segments up to five segments apart, revealed an increasing degree of correlation developing over the first 4 weeks of postnatal life. Dorsal horn responses to dorsal root stimulation in cords taken from young animals were prolonged, lasting in excess of 250 msec. In the third week of life, the duration of the excitatory component of the response was reduced to approximately 50 msec by the development of an inhibitory phase.

Postnatal changes in the role of NMDA in the isolated spinal cord of the hamster, Mesocricetus auratus.

The frequency of spontaneous activity recorded from lumbar dorsal roots and the lumbar dorsal horn of isolated spinal cord preparations taken from hamsters aged between 2 days to 8 weeks showed an age dependent sensitivity to 1 mM Mg2+ and 5 microM AP5. Spontaneous dorsal root and dorsal horn activity in cords from animals less than 3 weeks of age was depressed by 1 mM Mg2+ and 5 microM AP5. Cords taken from animals older than 3 weeks showed significantly less depression of spontaneous activity. The application of 10 microM NMDA to the cord produced a small (33%) depression in spontaneous dorsal root and dorsal horn activity in cords from 4 to 6 week old animals. Cords from younger animals exhibited a complex response to NMDA, with an initial increase in spontaneous activity followed by a profound (77%) depression of the firing rate. These results indicate that there are substantial changes taking place in the pharmacology of the dorsal horn during the early weeks of life, and care must be exercised when extrapolating results obtained from neonatal preparations to adult animals.

Spread of the dorsal root reflex in an isolated preparation of hamster spinal cord.

In an isolated preparation of hamster spinal cord maintained at 27 degrees C, stimulation of a single dorsal root has been shown to evoke dorsal root reflexes in ipsilateral roots throughout the lumbar and thoracic cord. The dorsal root reflex was evoked by activity in low threshold dorsal root fibres, and left the cord through dorsal root fibres conducting at 5.9 m/s. The ipsilateral dorsal root reflex travelled equally well in both directions along the cord at a calculated velocity of 0.5 m/s at 27 degrees C. Comparison of the stimuli required to evoke dorsal root reflexes in adjacent and distant ipsilateral dorsal roots suggests that once this system is activated it operates largely in an all-or-none manner. The delay in the generation of the dorsal root reflex within the cord was calculated to be 4.7 +/- 0.2 ms at 27 degrees C, corresponding to approximately 2 ms at body temperature, suggesting that a trisynaptic pathway is involved in the generation of the dorsal root reflex at the segmental level.

Correlation between spontaneous bursts of activity recorded from the dorsal roots in an isolated hamster spinal cord preparation.

At a temperature of 25-27 degrees C spontaneous antidromic activity has been demonstrated in lumbar and thoracic dorsal roots of an isolated spinal cord preparation taken from the golden hamster. A characteristic pattern of bursts of action potentials has been identified, which develops within 1-2 h following dissection and persists for more than 8 h. Simultaneous recordings made from pairs of dorsal roots have revealed correlations between the patterns of spontaneous activity in dorsal roots separated by up to sixteen segments longitudinally and across the cord. The strongest correlations were found between pairs of adjacent roots on the same side of the cord which produced a cross-correlation histogram having a single peak with a mode close to 0 ms. As the separation between the roots was increased the cross-correlation histogram became bimodal, with peaks equidistant on either side of 0 ms. Activity recorded in ipsilateral and contralateral pairs of roots supplying the same spinal segment also produced bimodal cross-correlation histograms. Transverse sectioning of the cord did not abolish spontaneous activity in any of the spinal roots examined, although there was a progressive reduction in the frequency of the bursts of spontaneous activity with shorter lengths of cord. These results suggest that each spinal segment is capable of generating spontaneous activity, and that there is a system by which adjacent segments are linked, allowing the activity to spread up and down the cord from the point of origin. The networks associated with the spread of dorsal root activity and primary afferent depolarization (PAD) in the spinal cord are discussed.

Effects of RU33368, a low affinity ligand for neuronal benzodiazepine receptors, on rodent behaviours and GABA-mediated synaptic transmission in rat cerebellar slices.

1. The effects of the low affinity benzodiazepine receptor ligand RU33368 were studied on rodent behaviours and on GABA-mediated synaptic transmission in rat cerebellar slices. 2. RU33368 inhibited stress induced ultrasounds in rat pups without inducing marked muscle relaxation. RU33368 also enhanced operant responding in rats that had been suppressed by mild footshock. These effects of RU33368 in these two models of anxiety were both blocked by the benzodiazepine antagonist Ro15-1788 (flumazonil). 3. In cerebellar slices RU33368 enhanced stimulus-induced synaptic inhibition of Purkinje layer cells with a minimal effective concentration in the order of 1 microM. The classical benzodiazepine agonist RU32007 was approx. 10 times more potent. This action of RU33368 was blocked by Ro15-1788. 4. The minimal effective concentration of RU33368 fully blocked the effect of RU32007 in 2 of 4 cells tested and partially antagonized it in a third cell. 5. These data suggest that RU33368 is a partial agonist at benzodiazepine receptors and this, at least in part, explains its non-sedative anxiolytic behavioural profile.

Receptor sub-types involved in responses of Purkinje cell to exogenous excitatory amino acids and local electrical stimulation in cerebellar slices in the rat.

The effects of the NMDA receptor antagonist, 2-amino-5-phosphonovalerate (APV) and non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on responses of Purkinje cells to exogenous excitatory amino acids and to electrical stimulation of the parallel fibres, were investigated in slices of the cerebellum of the rat. Glutamate, aspartate, kainate and quisqualate all induced excitation of Purkinje cells. Responses to kainate and quisqualate were blocked by CNQX (10 microM) but not by APV (10 microM). N-Methyl-D-aspartate induced biphasic excitatory-inhibitory responses, both components of which were blocked by APV but not by CNQX. The inhibitory component was less sensitive to blockade by APV but was totally blocked by bicuculline, the GABAA receptor antagonist. Parallel fibre stimulation most commonly induced inhibition of Purkinje cells, with or without preceding excitation. This inhibition was blocked by APV and excitatory responses were often revealed. A less commonly-observed predominantly excitatory response was blocked by CNQX but not by APV and inhibition tended to be revealed. These data suggest that parallel fibre-Purkinje cell synapses possess non-NMDA postsynaptic receptors, while the parallel fibre-inhibitory interneuron synapses possess functional NMDA receptors.

The effect of bicuculline and baclofen on the dorsal root-ventral root reflex in the isolated spinal cord.

1. In the lumbar spinal cord, 10(-6) M bicuculline reduces the inhibition of the dorsal root-ventral root (DR-VR) reflex produced by lateral stimulation of the spinal cord. The inhibition returns on washing in control artificial cerebrospinal fluid (ACSF). 2. Baclofen (10(-6) M) inhibits the monosynaptic DR-VR reflex which returns on washing in control ACSF. 3. This suggests an involvement of GABAA receptors in the lateral inhibition and GABAB receptors in the DR-VR reflex system. 4. The increased activity seen following bicuculline or tubocurarine probably resets the "set point" of neural activity to a lower level.

Modulation of GABA-mediated inhibition in rat cerebellar slices by benzodiazepine receptor ligands.

1. Extracellular recordings were made from the Purkinje cell layer of rat cerebellar slices. Compounds were perfused over the slice and bipolar stimulating electrodes placed in the external layer of the slice close to the recording electrode. 2. Stimulus-evoked inhibition of Purkinje layer cell activity was sensitive to bicuculline methiodide and picrotoxin, suggesting it was gamma-aminobutyric acid (GABA) mediated. The benzodiazepine ligands RU 32007 and Ro 19-0528 reversibly increased the period of inhibition, as did pentobarbital. This benzodiazepine effect was antagonised by Ro 15-1788. 3. Five inverse agonists all reduced the period of stimulated inhibition and this effect was reversed by Ro 15-1788, suggesting the involvement of benzodiazepine receptors. 4. It is concluded that this system provides a convenient physiological and possibly quantitative model for studying the action of benzodiazepine receptor ligands.