Neuronal activity recorded from the spinal dorsal horn of physiologically intact, awake, drug-free, restrained cats: a preliminary report

A newly developed technique was used to record extracellular single unit activity in the dorsal horn of the spinal cord of physiologically intact, awake, drug-free, restrained cats. Activity of low threshold neurons in the intact animal differs from activity in spinal cord-transected animals in that there is little or no spontaneous activity present in the intact preparation. These data suggest important differences between the intact and spinal cord-transected preparations.     

Classification of dorsal horn neurons based on somatic receptive fields in cats with intact and transected spinal cords: neural plasticity

Classification of dorsal horn neurons based on cell activity responses to somatic receptive fields stimulation, was compared between anesthetized cats with transected or intact cords. Results showed a significant (P≤0.001) difference. In animals with transected cords, dorsal horn neurons responded with less specificity to noxious and innocuous stimulation. The results are consistent with the proposition that loss of supraspinal influences plays a significant role in determining response characteristics of dorsal horn neurons.     

Inhibition of rat spinal cord dorsal horn neurons by non-segmental, noxious cutaneous stimuli

Extracellular single unit recordings were obtained from spinal cord dorsal horn neurons in halothane-anesthetized rats. Inhibitory effects induced by noxious mechanical or electrical stimuli applied to a remote area of the body surface were assessed on the spontaneous or evoked activity of these cells. Noxious mechanical stimulation inhibited 59% of the cells receiving nociceptive inputs (wide dynamic range and nociceptive specific) but only 5% of the other cell types. Inhibition produced by mechanical stimulation lasted for the full duration of stimulus application (up to 30 s) whereas inhibition produced by electrical stimulation lasted less than 500 ms. Increasing the depth of anesthesia was found to depress or abolish the inhibition.     

A technique for chronic extracellular recording of neuronal activity in the dorsal horn of the lumbar spinal cord in drug-free, physiologically intact, cats

A technique has been developed which allows extracellular recording from single neurons in the spinal cord of physiologically intact, awake, drug-free, restrained cats. The technique involves the surgical placement, under general anesthesia, of a stainless steel recording chamber which has a rectangular opening in its center that is 6 mm wide by 12 mm long. The recording chamber is attached to the vertebral column so that the opening is positioned over a similar opening that has been made in the bone of the vertebral column overlying the lumbar enlargement. Following a two week recovery period, the cats, which have been trained to accept restraint, are placed in a plexiglass restraining box, and a microdrive assembly is attached to the recording chamber. Tungsten microelectrodes (Frederick Haer & Co.) are then advanced through the dura into the dorsal horn of the spinal cord while receptive fields are stimulated. The chamber, when properly positioned, provides access to neurons with receptive fields on both hindlimbs. The stability of the system makes it possible to maintain single cell recordings from neurons in the dorsal horn of the spinal cord in the presence of either experimenter-induced or spontaneous movement of hindlimbs, tail or back musculature or during and after intramuscular, intravenous, spinal and epidural drug injections. Of greatest importance is the fact that the animals remain healthy and normal throughout the period of neuronal recordings.     

Increased receptive field size of dorsal horn neurons following chronic spinal cord hemisections in cats

The somatotopic organization of the 17 dorsal horn was studied using extracellular recordings in normal cats, and in cats with acute or chronic spinal cord hemisection at T13, sparing the dorsal columns. Based on data concerning recovery of function and collateral sprouting of afferents following hemisections, we predicted that the lesion would result in increases in receptive field size and decreases in the specificity of the somatotopic map. In normal animals, the usual mediolateral, rostrocaudal and dorsoventral somatotopic sequences were found. Following acute hemisections (6 h–5 days), there were changes in spontaneous and evoked activity, but receptive field sizes and somatotopic organization remained unchanged. Following chronic hemisections (88–174 days), proximal hindlimb receptive fields in the lateral dorsal horn ipsilateral to the lesion increased dramatically in size and were significantly larger than similar receptive fields on the contralateral side. The largest of these fields extended from the dorsal midline to the middle of the foot. Receptive field sizes elsewhere in the dorsal horn remained unchanged, as did somatotopic organization in general. These findings indicate that hemisections result in a complex series of changes consisting of an early stage of anatomically generalized changes in excitability and a later stage of highly localized changes in receptive field size. Possible mechanisms for these changes, as well as their relationship to recovery of function, are discussed.     

Dynamic alterations in the cutaneous mechanoreceptive fields of dorsal horn neurons in the rat spinal cord

The effect of the application to the skin of the chemical irritant mustard oil on the size and responsiveness of the cutaneous mechanoreceptive fields of 32 lumbar dorsal horn neurons has been examined in the adult decerebrate, spinal rat. Mustard oil placed on a small region of skin outside the mechanoreceptive firing zone produced a brief (185 +/- 35 sec, SEM) discharge of action potentials in 17 neurons and, in 23 cells, a prolonged increase of the response to a standard low- or high-intensity mechanical stimulus applied to the firing zone of the receptive field. This increase was shown, in 6 intracellularly recorded cells, to be due to a significantly increased depolarization in response to the stimuli. An expansion of the mechanoreceptive firing zones that peaked at 26 +/- 3.7 min was seen in 21 cells. While 6 of 8 nociceptive-specific neurons and 11 of 18 multireceptive neurons showed such an expansion, it did not occur in the 6 cells with low-threshold-only receptive fields. The expansion of the firing zones in 4 intracellularly recorded cells was found to be due to an increased amplitude of the EPSPs evoked by stimuli applied to what had initially been low probability firing fringes (Woolf and King, 1989) outside the firing zones, so that subthreshold responses became suprathreshold after application of the mustard oil. In 4 of 8 nociceptive-specific cells, the mechanical threshold in the firing zone became reduced to innocuous levels after application of the mustard oil. The demonstration of the capacity of a relatively brief afferent barrage of chemosensitive nociceptors to produce an increase in the spatial extent of the cutaneous receptive fields of dorsal horn neurons, amplify their responsiveness, and reduce their thresholds has implications both for the pathogenesis of postinjury pain hypersensitivity phenomena and for receptive field plasticity in the somatosensory system.     

Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons

In decerebrate cats, electrical stimulation of nucleus raphe magnus (NRM) of the medulla produced marked inhibition of spinal neurons in lumbosacral dorsal horn. Only neurons with high threshold inputs were inhibited. These cells were located in lamina I and in or near laminae V and VI.The duration of inhibition produced was related to the stimulus train length. An ipsilateral lesion of the dorsolateral funiculus at L1 markedly reduced the inhibition of neurons caudal to the lesion.Although NRM stimulation was the most effective, inhibition from more lateral sites could be obtained at higher stimulus intensities.NRM induced inhibition is probably mediated by a direct projection via the dorsolateral funiculus to spinal dorsal horn laminae I, II, V and VI.The results are discussed in relation to proposed mechanisms underlying the analgesia produced by NRM stimulation.     

Dorsal root projections to dorsal horn neurons in the cat spinal cord

The projection of dorsal root fibers to the dorsal horn of the spinal cord of the cat has been studied by electron microscopy. Two distinct types of synaptic degeneration are seen with the electron microscope: small knobs which exhibit a marked increase in electron density, and large knobs which fill with neurofilaments. Variations in the distribution of degenerating knobs to the six laminae of the dorsal horn are observed: dense degenerating knobs are found throughout the horn, but are quite rare in laminae I and II. Lamina III exhibits the most dense knobs, and a great many are also present in laminae IV and VI with a somewhat lesser number seen in lamina V. Knobs undergoing neurofilamentous degeneration are found only in laminae V and VI, and are much Jess frequently seen than are dense knobs. The types of synaptic contacts made by degenerating dorsal root fibers also vary from one region of the dorsal horn to another. Dense degenerating knobs synapse primarily with small dendrites in laminae I, II and III, but not at all with large dendrites or nerve cell bodies. In lamina III, dense knobs are seen in axoaxonal synapses, and are always the pre-synaptic component of the synapse. In laminae IV, V and VI dark knobs are commonly seen to synapse upon cell bodies of large and medium sized neurons and their proximal dendrites, but not upon small cell bodies Dense knobs upon small dendrites are common. In degenerating axoaxonal synapses, the dense knob is always the post-synaptic component. Degenerating neurofibrillar knobs are only seen to synapse with cell bodies or larger dendrites in V and VI and not at all other synaptic knobs. The results are correlated with findings by light microscopy. Comparisons are made with some of the known physiological properties of dorsal horn neurons.     

Direct catecholaminergic innervation of spinal dorsal horn neurons with axons ascending the dorsal columns in cat

Previous ultrastructural studies have shown that catecholamine-containing nerve terminals in the spinal dorsal horn form synaptic junctions with dendrites and somata, but the identity of the neurons giving rise to these structures is largely unknown. In this study we have investigated the possibility that spinomedullary neurons, which project through the dorsal columns to the dorsal column nuclei, are synaptic targets for descending catecholaminergic axons. Neurons with axons ascending the dorsal columns were retrogradely labelled after uptake of horseradish peroxidase by their severed axons in the thoracic (T10–T12) or cervical (C2–C3) dorsal columns. After the retrogradely labelled neurons were visualized, the tissue was immunocytochemically stained with antisera raised against tyrosine hydroxylase or dopamine-β-hydroxylase. Three hundred forty-three retrogradely labelled neurons within laminae III–V of the lumbosacral dorsal horn were examined under high power with the light microscope. In Triton X-100 treated material, over 60% of cells were found to have dopamine-β-hydroxylase-immunoreactive varicosities closely apposed to their somata and proximal dendrites. The number of contacts per cell varied from 1 to 22, with a mean number of 4.5. Fewer cells (34%) received contacts from axons immunoreactive for tyrosine hydroxylase as a consequence of the weaker immunoreaction produced by this antiserum. Correlated light and electron microscopic analysis confirmed that many of these contacts were regions of synaptic specialization and that immunostained boutons contained pleomorphic (round to oval) agranular vesicles together with several dense core vesicles. These observations suggest that catecholamines regulate sensory transmission through this spinomedullary pathway by a direct postsynaptic action upon its cells of origin. Such an action would be predicted to suppress transmission generally through this pathway. © 1993 Wiley-Liss, Inc.     

Demonstration of thyrotropin-releasing hormone immunoreactivity in neurons of the mouse spinal dorsal horn

Thyrotropin-releasing hormone-like immunoreactivity was demonstrated in neurons within the superficial laminae (I, II, III) of the mouse spinal dorsal horn by light-microscopic peroxidase immunocytochemistry. The immunoreactivity was distributed in a narrow dorsoventral band that enclosed the substantia gelatinosa (lamina II), with a higher concentration along the lamina II/III border. At present, the functional significance of these neurons is unknown. Their existence within the substantia gelatinosa suggests a role in sensory information processing.     

Plasticity of some spinal dorsal horn neurons as revealed by pentobarbital-induced disinhibition

Extracellular activity was recorded from single spinal dorsal horn neurons in physiologically intact, awake, drug-free cats before and after the intravenous administration of 20 mg/kg pentobarbital (Pb). Pb produced a series of changes in response properties that reflect a significant moment-to-moment plasticity of some spinal dorsal horn neurons. Pb administration unmasked the ability of some low-threshold (LT) neurons to respond to noxious mechanical or thermal stimuli resulting in their being reclassified as wide dynamic range (WDR) neurons. Pb also appeared to unmask an afterdischarge in some neurons following noxious mechanical stimulation. In addition, some neurons appeared to be better able to signal changes in the intensity of mechanical stimulation after Pb. Neuronal receptive fields for low threshold stimulation were reduced in many instances but enlargement was also observed. The responses of some neurons to peripheral stimulation were unchanged by Pb. We hypothesize that the relatively low doses of Pb used in the study reduced tonic inhibition of some spinal dorsal neurons although the observed effects could have been produced by excitation.     

Quantitative study of NPY-expressing GABAergic neurons and axons in rat spinal dorsal horn

Between 25-40% of neurons in laminae I-III are GABAergic, and some of these express neuropeptide Y (NPY). We previously reported that NPY-immunoreactive axons form numerous synapses on lamina III projection neurons that possess the neurokinin 1 receptor (NK1r). The aims of this study were to determine the proportion of neurons and GABAergic boutons in this region that contain NPY, and to look for evidence that they selectively innervate different neuronal populations. We found that 4-6% of neurons in laminae I-III were NPY-immunoreactive and based on the proportions of neurons that are GABAergic, we estimate that NPY is expressed by 18% of inhibitory interneurons in laminae I-II and 9% of those in lamina III. GABAergic boutons were identified by the presence of the vesicular GABA transporter (VGAT) and NPY was found in 13-15% of VGAT-immunoreactive boutons in laminae I-II, and 5% of those in lamina III. For both the lamina III NK1r-immunoreactive projection neurons and protein kinase Cγ (PKCγ)-immunoreactive interneurons in lamina II, we found that around one-third of the VGAT boutons that contacted them were NPY-immunoreactive. However, based on differences in the sizes of these boutons and the strength of their NPY-immunoreactivity, we conclude that these originate from different populations of interneurons. Only 6% of VGAT boutons presynaptic to large lamina I projection neurons that lacked NK1rs contained NPY. These results show that NPY-containing neurons make up a considerable proportion of the inhibitory interneurons in laminae I-III, and that their axons preferentially target certain classes of dorsal horn neuron.     

Sodium salicylate reduces gamma aminobutyric acid-induced current in rat spinal dorsal horn neurons

Sodium salicylate is one of the nonsteroidal antiinflammatory drugs and is clinically used for antiinflammation and chronic pain relief. In the present study, we investigated the actions of sodium salicylate on gamma-aminobutyric acid type A receptor (GABA(A)) current in cultured rat spinal dorsal horn neurons. Sodium salicylate was found to reduce GABA(A) current in a reversible and concentration-dependent manner, but did not change its ion selectivity. Sodium salicylate was effective only when GABA and sodium salicylate were applied together. Application of sodium salicylate immediately before, but not during, the application of GABA did not result in a significant reduction of GABA(A) current. Our results demonstrate that sodium salicylate reversibly attenuates the GABA(A) response of dorsal horn neurons, suggesting that GABA(A) receptors in the region are pharmacological targets of sodium salicylate.     

Cholinergic effects on spinal dorsal horn neurons in vitro: an intracellular study

The cholinoceptive properties of dorsal horn neurons (lamina III–V) were investigated by means of intracellular recordings from the rat isolated spinal cord slice preparation. In half of the neurons investigated, acetycholine (ACh) evoked a dose-dependent slow depolarization and increase in excitability; hyperpolarization was observed in 10% of neurons. Acetyl-β-methylcholine (MCh) similarly depolarized 39% and hyperpolarized 25% of neurons tested; depolarization was also observed following bethanechol. Responses to the muscarinic agonists were abolished by atropine (10⁻⁵ M). Nicotine depolarized 84% of tested neurons; dihydro-β-erythroidine (5 × 10⁻⁵M) and (+)-tubocurarine (10⁻⁶ M) antagonized this depolarization. ACh-, MCh- and nicotine-induced depolarizations, associated with changes in input resistance, were maintained in the presence of tetrodotoxin (10⁻⁶ M). Substance P, as well as repetitive electrical stimulation of the dorsal root, also evoked depolarization in ACh-sensitive neurons. Atropine, but not (+)-tubocurarine, diminished responses to both substance P and dorsal root stimulation. These results indicate that dorsal horn neurons are ACh-sensitive and possess both muscarinic and nicotinic receptors. In addition, the parallel sensitivity of neurons to muscarinic agonists, substance P and dorsal root stimulation, as well as the parallel antagonistic effect of atropine, are supportive of a common ionic mechanism underlying the activation of muscarinic and substance P receptors.     

Spinal dorsal horn neurons in elevated extracellular calcium: cell properties and spontaneous discharges

Recordings were made from neurons in the dorsal horn (DH), and from dorsal and ventral roots (DRs and VRs) of isolated spinal cords of infant mice. Raising calcium concentration ([Ca2+]) in the organ bath from 1.2 to 2.4 mmol/l resulted in a slight hyperpolarization, elevation of threshold current (rheobase), and augmentation of excitatory postsynaptic potentials (EPSPs). In many cells EPSPs acquired a much prolonged late phase. Orthodromic stimulation evoked in some DH neurons an action potential that had the same threshold as, and coincided in time with, the 'dorsal horn response' (DHR) recorded from DR. In spinal cords bathed in elevated [Ca2+], DR recordings showed irregularly recurring spontaneous waves, and DH neurons generated spontaneous EPSPs, often with spikes. Some neurons fired irregularly timed spontaneous action potentials that did not appear triggered by EPSPs. In less than 50% of the neurons the spontaneous EPSPs coincided in time with the spontaneous DR waves. The action potentials that appeared without EPSP were fired independently from DR activity. These observations confirm that elevation of interstitial free calcium concentration results in strong enhancement of excitatory transmission, especially of an EPSP of much extended duration. Virtually all neurons showed increased spontaneous activity in high [Ca2+], but only a minority appeared recruited into the synchronized discharges that are detectable as spontaneous waves in DR and VR recordings.     

The fine structure of neurons in the dorsal horn of the cat spinal cord

The fine structure of the dorsal horn of the cat cord has been compared with the light microscope cytoarchitectural studies. Several distinct laminae may be seen with the electron microscope. Lamina I contains marginal cells of Waldeyer and has its processes oriented in a horizontal direction. Lamina II contains small neurons and processes oriented vertically, and may be distinguished from lamina III by the rich content of myelinated axons in the latter. In laminae I–III there are numerous non-myelinated axons and many axodendritic and axoaxonal synapses but few axosomatic ones. Complex synaptic arrays made up of a central axon synapsing with other axons and dendrites are commonly seen. Laminae IV, V and VI are distinguished by their content of large neurons. They exhibit many axodendritic and axosomatic synapses but few axoaxonal ones and no complex synaptic arrays. Synaptic knobs containing rings of neurofilaments are present only in laminae IV–VI and synapse usually with somata or dendrites of neurons. There are differences in synaptic vesicle configuration and in the density of subsynaptic membranes associated with them. Differentiation of the dorsal horn into horizontal laminae is demonstrated by both light and electron microscope studies. The regional differences in neuronal organization may represent differences in functional organization as well.     

Membrane properties of deep dorsal horn neurons from neonatal rat spinal cord in vitro

Whole-cell patch-clamp recordings were undertaken to characterize and compare the membrane properties of deep dorsal horn neurons in transverse slices of rat lumbar spinal cord in two age groups, postnatal days (P) 3–6 and 9–16. In both age groups, significant correlations were observed between membrane time constant and cell resistance and between action potential height and its duration at half-maximal amplitude. Cell resistance and action potential half-width values were lower in the P9–16 age group. Neurons were divided into four categories based on their firing properties in response to intracellular current injection: single spike, phasic firing, repetitive firing, and delayed firing. The distribution of neurons within these categories was similar in both age groups which suggests that the firing properties of deep dorsal horn neurons are functionally differentiated at an early postnatal age.     

Modulation of visceral nociceptive responses of rat spinal dorsal horn neurons by sympathectomy

We determined whether sympathectomy modulates visceral nociception under physiological or inflammatory conditions. Recordings of sacral spinal dorsal horn neurons with sustained responses were performed in pentobarbitone-anesthetized rats. Graded colorectal distension (CRD, 20-100 mmHg) was used as a visceral nociceptive stimulus. Inflammation was induced by intracolonic instillation of turpentine (25%). Sympathectomy was produced by administering 6-hydroxydopamine. Inflammation produced an increase in the CRD-evoked responses. The CRD-evoked responses were attenuated following sympathectomy both under control and inflammatory conditions. These changes in the CRD-evoked responses were associated with corresponding changes in spontaneous discharge rate. The convergent input evoked by noxious pinch of the skin was not changed by any of the experimental conditions. The results indicate that sympathectomy attenuates visceral nociceptive responses and spontaneous activity of sacral spinal cord neurons, without effect on convergent cutaneous inputs, both under physiological and inflammatory conditions.     

Effects of morphine on the activity of various dorsal horn neurons of the spinal cord involved in nociception]

In spinal preparation, morphine exerts a specific direct inhibitory action on the activities of dorsal horn neurones induced by noxious stimuli. The effect of morphine is preferential for the responses evoked by A delta and C fibre afferents and its specificity of action has been demonstrated pharmacologically in terms of isomerism, dose dependency and reversal of the inhibitions by opiate antagonists. These results are in good agreement with recent data related to the localization at the spinal level of opiate receptors, and terminal rich in Enkephalin and substance P. Numerous behavioural and pharmacological investigations suggest that morphine is also acting at the level of the brainstem by reinforcing the activity of descending control systems which modulate the transmission of noxious inputs at the spinal level. However this second modality of action remains extremely difficult to demonstrate from an electrophysiological point of view.