Long lasting activity of nociceptive muscular afferents facilitates bilateral flexion reflex pattern in the feline spinal cord.

Chronic muscular limb pain requires the adoption of motor patterns distinct from the classic ipsilateral flexion, crossed extension and corresponding reciprocal inhibitions to acute exteroceptive stimulation. Using selective chemical activation of group III/IV afferents in gastrocnemius-soleus (GS) muscles we investigated bilaterally their reflex responses conditioned by (a) acute 'myositis' induced by intramuscular carrageenan; and (b) sub-acute 'myositis' induced by infusion of complete Freund's adjuvant (CFA). Reflex transmission was detected by monosynaptic testing and c-fos staining used to identify increased neuronal activity. In all control experiments with chemical stimulation of group III/IV afferents, ipsilateral responses conformed to the flexor reflex pattern. However, the expected contralateral facilitation of GS motoneurones occurred in fewer than 50% trials while only 9% of trials induced contralateral inhibition of flexor posterior-biceps-semitendinosus (PBSt) motoneurones. During carrageenan acute myositis contralateral PBSt was transiently facilitated by selective activation of group III/IV afferents. During CFA-induced myositis, contralateral only inhibition of GS motoneurones occurred instead of any facilitation, while bidirectionally a crossed facilitation of PBST dominated. These reflex changes were mirrored in an enhanced number of neurones with enhanced c-fos expression. Muscle pain, particularly if chronically persistent, requires another behavioural response pattern than acute exteroceptive pain.

Target-dependence of sensory neurons: an ultrastructural comparison of axotomised dorsal root ganglion neurons with allowed or denied reinnervation of peripheral targets.

Evidence is emerging for a role of rough endoplasmic reticulum (RER) in the form of stress granules, the unfolded protein response and protein bodies in the response of neurons to injury and in neurodegenerative diseases. Here, we have studied the role of the peripheral target in regulating the RER and polyribosomes of Nissl bodies in axotomised adult cat dorsal root ganglion (DRG) neurons where axonal regeneration and peripheral target reinnervation was either allowed or denied. Retrograde labelling with horseradish peroxidise was used as an independent marker to enable selection of only those DRG neuronal cell bodies with axons in the injured intercostal nerves. Indications of polyribosomal dispersal were seen by 6h following axotomy, and by 24h the normal orderly arrangement of lamellae of RER in Nissl bodies had become disorganised. These ultrastructural changes preceded light microscopical chromatolysis by 1-3d. The retrograde response was maximal 8-32 d after axotomy. Clusters of debris-laden satellite cells/macrophages were present at this time but no ultrastructural evidence of neuronal apoptosis or necrosis was seen and there were no differences in the initial retrograde response according to the type of injury. By 64 d following axotomy with reinnervation, approximately half the labelled DRG neurons showed restoration of the orderly arrangement of RER and polyribosomes in their Nissl bodies. This was not seen after axotomy with reinnervation denied. We propose that the target-dependent changes in Nissl body ultrastructure described here are part of a continuum that can modify neuronal protein synthesis directed towards growth, maintenance or death of the neuron. This represents a possible structural basis for mediating the varied effects of neurotrophic interactions.

Vagal afferent projections to lobule VIIa of the rabbit cerebellar vermis related to cardiovascular control.

In decerebrate rabbits we recorded simultaneously field potentials in lobule VIIa of the vermal cerebellar cortex and the vagal compound action potentials (vCAPs) proximally in the vagus nerve following electrical stimulation distally in the same nerve at different intensities. Four principal components of the vCAP were distinguished based on their peak conduction velocities. Their velocities were component I, 67-100 m/s; II, 28-50 m/s; III, 6-28 m/s, IV, 0.4-1.3 m/s. A collision test based on stimulating the recurrent laryngeal nerve identified component I and sub-component IIa of the vCAP as being due to the motor fibres of the descending limb of the nerve. The field potentials evoked in lobule VIIa by electrical stimulation of the cervical vagus nerve were climbing fibre responses as judged by the characteristics of their lamina profile and their response to high frequency stimulation. These field potentials in lobule VIIa correlated most closely with the component III of the vCAP; particularly with a sub-component IIIa of the vagus. Based on the investigations by Evans and Murray (1954) (Histological and functional studies on the fibre composition of the vagus nerve of the rabbit. J. Anat. (Lond.) 88, 320-337) in the rabbit, and by Paintal (1963) (Vagal afferent fibres. Ergeb. Physiol. 52, 74-156) and Mei (1970) (Cardiovascular and respiratory vagal mechanoreceptors in the cat. Exp. Brain Res. 11, 480-501) in the cat, component III is most likely to be due to receptors from the heart and a part of the pulmonary stretch receptors.

Differences in the synaptic complement of thoracic motoneurons of adult and ageing cats after permanent or reversible axotomy.

We have compared the effects of intercostal nerve crush (reversible axotomy) or nerve transection with proximal ligation (permanent axotomy) on the somatic synaptic terminals of thoracic motoneurons of adult (1-2 years) and ageing (10-15 years) cats. Retrograde axonal transport of horseradish peroxidase (HRP) was used to identify axotomised motoneurons; control motoneurons were labelled by the intramuscular injection of HRP. Synaptic frequency and cover of control motoneurons in adult and ageing cats was similar. In adults, 8-16 days following both types of axotomy, synaptic cover was halved without any significant change in synaptic frequency. By 32-64 days following reversible axotomy, synaptic frequency and cover were not significantly different from controls. By contrast, 32-64 days following permanent axotomy synaptic frequency and cover were reduced to 30-50% of adult control values. In ageing cats 2 months following reversible axotomy, synaptic frequency and cover were reduced to 40% and 33% of ageing control values, respectively, while no significant change from controls was found 2 months following permanent axotomy. The long-term synaptic response of axotomised motoneurons in ageing cats is therefore opposite to that of adults.

Spontaneous synchronized neural activity in decerebrate gallamine-paralysed cats.

In decerebrate cats paralysed with gallamine, over a period of several days there develops a remarkable synchronization of discharge in widely different motor nerves throughout the body, including intercostal nerves and limb nerves. These discharges are also in synchrony with slow waves approximately 100 ms in duration in the inferior olive. The slow waves and discharges are at first irregular and only weakly synchronized, but become increasingly strongly synchronized and by about the fourth day exhibit a strong 6-8 Hz rhythm. The degree of synchronization is greater the lower the end-tidal CO2 concentration. Transection of the spinal cord at a high cervical level breaks the synchrony and may abolish the discharge in the nerves, but the slow waves in the inferior olive continue rhythmically. It was shown, however, that gallamine injected subdurally at cervical vertebra 7 or lumbar vertebra 7 has a direct excitatory action on the spinal cord. Slow waves in the inferior olive are elicited by gallamine in the decerebrate, spinalized and decerebellectomized cat, and therefore must originate in the brainstem. Gallamine is known to act directly on olivary neurons and the slow waves may originate in the inferior olive, but further experiments are needed to determine what other structures it affects. The condition of the cat a few days after decerebration and paralysis resembles the clinical condition of reticular reflex myoclonus and it is suggested that the genesis of the myoclonus may be similar in the two conditions.

Ultrastructure of gamma-motoneurons after temporary or permanent interruption of peripheral target contact.

The paradigm of nerve crush, vs. nerve transection and ligation, was used to examine the effects of temporary or permanent interruption of peripheral target contact on the ultrastructure of cat thoracic gamma-motoneurons. The normal, highly ordered ultrastructure of Nissl bodies was lost 8 days after axotomy. Nissl bodies remained disorganised up to 305 days after nerve transection and ligation. In contrast, normal ultrastructural orderliness was restored for many of the Nissl bodies of gamma-motoneurons 64 days following nerve crush. A decrease in the area of the Golgi apparatus was found 64 days following both nerve crush and nerve transection with ligation. Other organelles were unaltered.

Contralateral projections of thoracic respiratory interneurones in the cat.

1. Retrograde neuronal transport following small iontophoretic injections of horseradish peroxidase was used to investigate the location of neurones projecting to the thoracic ventral horn of the cat. 2. A concentration of labelled neurones was seen in the contralateral medial ventral horn at the immediately opposite rostrocaudal position. 3. The participation of respiratory interneurones in the projection was investigated by means of spike-triggered averaging, as follows. 4. Spike trains of single interneurones whose firing patterns were related to the central respiratory cycle were recorded extracellularly in the thoracic ventral horn of anaesthetized, paralysed cats. Firing patterns were defined by the construction of cycle-triggered histograms. 5. Spike-triggered averaging of the signal from an extracellular tungsten microelectrode in the opposite ventral horn was performed to test for the presence of axonal, terminal or focal synaptic potentials. 6. At least one of these types of potential was found for 34/55 units. Terminal potentials were found for thirty-one units, accompanied by focal synaptic potentials for twenty-seven units. Potentials were found for units with all types of firing patterns. Units whose activity elicited these potentials were generally located in the medial half of the ventral horn. 7. We conclude that at least 60% of the respiratory interneurones project to the immediately opposite ventral horn.

Functional identities of thoracic respiratory interneurones in the cat.

1. Spike-triggered averaging was used to reveal focal synaptic potentials (FSPs) in the thoracic ventral horn resulting from impulses in individual respiratory interneurones situated in the contralateral ventral horn of the same segment (T6 or T7) in anaesthetized paralysed cats. 2. FSPs were of two types, negative-going or positive-going, and were of the same sign at each of several sites for each unit. These two types were interpreted as indicating excitatory or inhibitory actions respectively. 3. The positive-going FSPs were almost all the result of activity in the most strongly modulated, phasic respiratory interneurones, whereas most of the negative-going ones were derived from neurones with a continuous tonic component in their firing patterns. 4. Positive-going FSPs (particularly those from inspiratory neurones) were generally found at more ventral sites in the ventral horn than negative-going ones. 5. It is suggested that an important role for the phasically active interneurones is to provide phasic inhibition, which is combined with largely tonic excitation to produce strong respiratory modulation in the activity of other neurones in the thoracic spinal cord, particularly in motoneurones. However, some groups of respiratory interneurones, such as a group of tonic inspiratory ones which are located more dorsally than the phasic inspiratory ones, may not receive such phasic inhibition.

The effects of opiates on the respiratory activity of thoracic motoneurones in the anaesthetized and decerebrate rabbit.

1. Efferent discharges were recorded from inspiratory and expiratory intercostal nerve filaments (T2-T10) in artificially ventilated, anaesthetized or decerebrate rabbits with or without vagotomy. 2. Hypocapnic apnoea was used to study the fractional end-tidal CO2 (FET,CO2)-dependent tonic discharges of the expiratory motoneurones, the FET,CO2 threshold for rhythm generation and the FET,CO2 response curve of both inspiratory and expiratory burst activity. 3. Incremental doses of morphine (e.g. 1 mg kg-1 I.V.) produced slowing of the respiratory rhythm due to prolongation of the expiratory duration and an elevation of the FET,CO2 threshold for rhythm generation. Eventually apnoea supervened with associated tonic firing of the expiratory motoneurones. At the elevated levels of FET,CO2 bursts of inspiratory activity, with concomitant phasic inhibition of the tonic expiratory activity, could occur either spontaneously or following sensory stimulation. The peak integrated activities of these bursts were closely similar to the values obtained for corresponding levels of FET,CO2 before the administration of morphine. 4. Tonic expiratory activity responded to increased levels of FET,CO2, as it had during hypocapnic apnoea prior to morphine, by an increased discharge frequency of single units or recruitment of new units. 5. All of these effects of morphine were immediately reversed by naloxone (100 micrograms kg-1). 6. Naloxone (greater than 100 micrograms kg-1), without pre-treatment with morphine, led to an increase in respiratory frequency due to a shortening of the expiratory duration and a dose-dependent reduction in the FET,CO2 threshold for rhythm generation. There was little alteration either in the inspiratory response to FET,CO2 during rhythm or in the FET,CO2 response of the expiratory output whether expressed as tonic activity during hypocapnic apnoea or phasic activity following the onset of rhythm. 7. Thus opiates act upon the mechanisms of rhythm generation without depressing the FET,CO2 drive as expressed either as phasic or tonic activation of the motoneurones.

Focal blockade of single unit synaptic transmission by iontophoresis of antagonists.

We have assessed the use of iontophoresis for investigating the pharmacology of synaptic transmission from individual presynaptic neurones in-vivo, by modifying extracellular focal synaptic potentials (FSPs) recorded by spike-triggered averaging. FSPs from two types of excitatory neurone (muscle spindle primary afferents and expiratory bulbospinal neurones) and from one inhibitory interneurone were studied in the thoracic ventral horn of anaesthetized cats. The antagonist of excitatory amino acids at non-NMDA receptors, DNQX, blocked the FSPs from the first two, as did bicuculline for the third. Thus the FSPs were generated by excitatory amino acids acting via non-NMDA receptors and GABA, respectively. The postsynaptic neurones were probably motoneurones. Merits and limitations of the method are discussed.

Retrograde response to axotomy of motoneurons in the thoracic spinal cord of the aging cat.

The retrograde response of HRP-labelled intercostal motoneurons was compared in adult (1-2 years) and aging (10-15 years) cats, 64-68 days following crush of intercostal nerves or following nerve transection with proximal ligation. A comparison of the neuroglial response to these two lesions was also made. In both adult and aging cats, 64-68 days following nerve crush, most labelled motoneurons had a normal appearance. In contrast, 64-68 days following nerve transection and ligation the Nissl bodies of labelled motoneurons lacked the highly ordered ultrastructure characteristic of normal and control motoneurons. No axotomy-induced neuronal loss was found in aging cats. A three-fold increase in numbers of microglial cells was quantified in the ipsilateral ventral horn of aging cats following nerve transection and ligation. This increase was not seen following nerve crush in aging cats, nor following either type of nerve injury in adult cats. Numbers of astroglia and oligodendroglia were unaffected by axotomy in adult and aging animals.

A threshold effect of the major isoforms of NCAM on neurite outgrowth.

Interactions between recognition molecules on the surface of neuronal growth cones and guidance cues present in the local cellular environment are thought to account for the growth of neurites in the highly stereospecific manner that contributes to correct target cell innervation. In vitro assays have been used to identify candidate molecular components of this system, either directly by demonstrating their ability to promote neurite outgrowth, or indirectly by the ability of specific antibodies to inhibit neurite outgrowth. The role of the neural cell adhesion molecule (NCAM) in pathway finding is not fully understood. Some immunological studies support a positive role; others do not, and it has been reported that purified NCAM does not support neurite outgrowth. We have previously shown that an arbitrary biochemical index of neurite outgrowth, the relative level of immunoreactive neurofilament protein, is increased when human and rat dorsal root ganglion neurons are cultured on monolayers of cells expressing transfected human NCAM. But, the complexity of growth precluded a simple morphological analysis and we did not determine the 'dose-response' relationship between NCAM expression and neuronal response. Here, we report on the morphology of rat cerebellar neurons cultured on monolayers of 3T3 cells transfected with complementary DNAs encoding all of the main NCAM isoforms found in cells such as astrocytes, Schwann cells and skeletal muscle. The data indicate that both transmembrane and glycosyl-phosphatidylinositol linked NCAM isoforms are potent substrates for neurite extension. A critical threshold value of NCAM expression is required for increased neurite outgrowth. Above this threshold, small increases in NCAM induce substantial increases in neurite outgrowth.

Organelle changes in cat thoracic alpha- and gamma-motoneurons following axotomy.

Horseradish peroxidase was used as a retrograde marker to identify the cell bodies of cat thoracic alpha- and gamma-motoneurons 8 days following axotomy. A quantitative ultrastructural comparison revealed changes in Nissl bodies, the Golgi apparatus, mitochondria and lysosomes for axotomised alpha-motoneurons, while axotomised gamma-motoneurons only showed changes in Nissl bodies and nuclear pores.

Ultrastructure of axotomized alpha and gamma motoneurons in the cat thoracic spinal cord.

Using horseradish peroxidase as a retrograde marker, the ultrastructural response of alpha and gamma motoneuronal cell bodies in the cat thoracic spinal cord has been compared 1-8 days following intercostal nerve transection and ligation. By light microscopy, reduction of Nissl body size, together with nuclear and nucleolar alterations were seen in alpha motoneurons 4-8 days following axotomy, but not at any stage in axotomized gamma motoneurons. In the electron microscope, disorganization of Nissl body ultrastructure was seen in both alpha and gamma motoneurons 2 days following axotomy. Only in alpha motoneurons, however, did these disorganized Nissl bodies subsequently fragment into smaller pieces. Both alpha and gamma motoneurons lost synapses following axotomy, but the proportional loss from gamma motoneurons was two-fold greater than that from alpha motoneurons. Loss of synaptic terminals with flattened synaptic vesicles was two-fold higher than that of synaptic terminals with round synaptic vesicles from axotomized gamma motoneurons, whereas axotomized alpha motoneurons lost both types of synaptic terminal equally.

Ultrastructure of interneurons within motor nuclei of the thoracic region of the spinal cord of the adult cat.

Alpha (greater than 40 microns) and gamma (less than 30 microns) motoneurons in inspiratory motor nuclei of the thoracic spinal cord of the adult cat were labelled retrogradely by the intramuscular injection of HRP. Small (less than 30 microns) unlabelled neurons within 200-300 microns of labelled motoneurons were analysed qualitatively and quantitatively with both the light and electron microscope. Most of these small unlabelled neurons had inconspicuous nucleoli, wrinkled nuclear membranes, low numbers of nuclear pores, and Nissl bodies which were either small or had the form of an amorphous perinuclear band. Such Nissl bodies were composed primarily of aggregates of polyribosomes within which short fragments of granular endoplasmic reticulum were distributed. Alpha and gamma motoneurons in contrast had prominent nucleoli, smooth-contoured nuclei, more nuclear pores and large, discrete Nissl bodies. Such Nissl bodies were composed primarily of several lamellae of granular endoplasmic reticulum with linear arrays of polyribosomes arranged between individual cisternae. Alpha motoneurons had most synaptic terminals on their cell bodies, gamma motoneurons had least and small unlabelled neurons had intermediate values. Synaptic terminals of the S-, F- T- and C-type were observed on alpha motoneurons, whereas only S- and F-types were observed on gamma motoneurons and small unlabelled neurons. Since they were unlabelled and differed morphologically from both alpha and gamma motoneurons, but were similar to small interneurons described elsewhere in the spinal cord and brain, it is suggested that the small unlabelled neurons located in the external intercostal and levator costae motor pools are interneurons. The functional significance of some of the morphological features which distinguish interneurons from motoneurons is discussed.

The development of voltage-dependent ionic conductances in murine spinal cord neurones in culture.

The development of voltage-dependent ionic conductances of foetal mouse spinal cord neurones was examined using the whole-cell patch-clamp technique on neurones cultured from embryos aged 10-12 days (E10-E12) which were studied between the first day in vitro (V1) to V10. A delayed rectifier potassium conductance (Ik) and a leak conductance were observed in neurones of E10, V1, E11, V1, and E12, V1 as well as in neurones cultured for longer periods. A rapidly activating and inactivating potassium conductance (IA) was seen in neurones from E11. V2 and E12, V1 and at longer times in vitro. A tetrodotoxin (TTX) sensitive sodium-dependent inward current was observed in neurones of E11 and E12 from V1 onwards. Calcium-dependent conductances were not detectable in these neurones unless the external calcium concentration was raised 10-to 20-fold and potassium conductances were blocked. Under these conditions calcium currents could be observed as early as E11. V3 and E12, V2 and at subsequent times in vitro. The pattern of development of voltage-dependent ionic conductances in murine spinal neurones is such that initially leak and potassium currents are present followed by sodium current and subsequently calcium current.

Respiratory interneurones in the thoracic spinal cord of the cat.

1. The discharges of spontaneously firing neurones, whose activity was modulated in phase with the central respiratory cycle, were recorded in the thoracic ventral horn (T3-T9) of anaesthetized, paralysed cats. 2. Out of 310 neurones, forty-six were positively identified as motoneurones by antidromic activation or spike-triggered averaging, fifty-four were positively identified as interneurones by antidromic activation from other spinal cord segments and ninety were indirectly identified as interneurones by virtue of their positions or firing rates as compared to the motoneurones. 3. Units were classified as inspiratory (64%), expiratory (25%) or post-inspiratory (7%) according to the times of their maximum firing rates. The remaining 4% consisted of units whose discharges were either strongly locked to the respiratory pump cycle or did not fit into the other categories. All but one of the motoneurones were classified as inspiratory or expiratory. 4. Inspiratory and expiratory units were further classified as early, late or tonic according to the starting times of their discharges in the respiratory cycle. The interneurones (both positively and indirectly identified) included more of the early and tonic categories and more fast-firing units than did the motoneurones in both the inspiratory and expiratory groups. 5. The locations of the motoneurones corresponded to the known positions of the intercostal and interchondral motor nuclei, including clear segregation of inspiratory and expiratory populations. The locations of neither the interneurones nor the unidentified units were segregated according to their firing patterns. These neurones were concentrated in the medial half of the ventral horn and were found generally more dorsally than the positions of the motoneurones, though their positions overlapped considerably with this group. 6. The axons of the positively identified interneurones were identified from one to five segments caudally and mostly contralaterally, but were not traced to their terminations. Some axons were located by microstimulation and found to run in the ventral or ventromedial white matter. Conduction velocities covered a wide range, 8 to around 100 m/s, mean 53 m/s. 7. Preliminary calculations indicate that there may be almost 10 times more respiratory thoracic interneurones as respiratory bulbospinal neurones.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and electrophysiological investigations of embryonic mammalian motoneurones in culture.

The retrograde transport of fluorochromes from muscle was used to label embryonic rat motoneurones in vivo. The fluorescent motoneurones were subsequently detected in cultures of dissociated spinal cord neurones using an image-intensification video camera, the images from which allowed identification of the motoneurones under phase-contrast microscopy. Such motoneurones were mostly phase dark, often with a multipolar appearance and were detectable for up to 4 days in culture. Whole-cell patch-clamp recordings from these motoneurones demonstrated mean input resistances of 635 M omega and resting membrane potentials of -65 mV; action potentials could be evoked and, under voltage-clamp, inward and outward currents were present.