Laminae III-VI Research Articles

Spinal Glycine Receptor Alpha 3 Cells Communicate Sensations of Chemical Itch in Hairy Skin.

Glycinergic neurons regulate nociceptive and pruriceptive signaling in the spinal cord, but the identity and role of the glycine-regulated neurons are not fully known. Herein, we have characterized spinal glycine receptor alpha 3 (Glra3) subunit-expressing neurons in Glra3-Cre female and male mice. Glra3-Cre(+) neurons express Glra3, are located mainly in laminae III-VI, and respond to glycine. Chemogenetic activation of spinal Glra3-Cre(+) neurons induced biting/licking, stomping, and guarding behaviors, indicative of both a nociceptive and pruriceptive role for this population. Chemogenetic inhibition did not affect mechanical or thermal responses but reduced behaviors evoked by compound 48/80 and chloroquine, revealing a pruriceptive role for these neurons. Spinal cells activated by compound 48/80 or chloroquine express Glra3, further supporting the phenotype. Retrograde tracing revealed that spinal Glra3-Cre(+) neurons receive input from afferents associated with pain and itch, and dorsal root stimulation validated the monosynaptic input. In conclusion, these results show that spinal Glra3(+) neurons contribute to acute communication of compound 48/80- and chloroquine-induced itch in hairy skin.

Temporal and spatial dynamics of peripheral afferent-evoked activity in the dorsal horn recorded in rat spinal cord slices.

In the present study, multi-electrode array recording was used to examine dorsal horn activity following stimulation of primary afferents in a rat dorsal root attached-spinal cord slice preparation. The multi-electrode array probe was placed under the dorsal horn slice and local field potentials evoked by stimulation on the dorsal root were analyzed. Three kinds of dorsal root-evoked responses were identified. In lamina IIo, local field potentials exhibited P1 (peak latency 1.46±0.08ms), N1 (2.77±0.18ms, n=12), N2 (7.31±0.48ms), N3 (12.12±0.73ms) and P2(18.30±0.80ms) waves. In lamina IIi local field potentials exhibited P (1.99±0.10ms), N1 (3.35±0.17ms) and N2 (8.58±0.44ms) waves. In laminae III-VI, local field potentials exhibited P1 (3.01±0.07ms), P2 (7.02±0.21ms) and N waves (22.57±0.79ms). Sweep spread was calculated by two dimensional current source density (2D-CSD) analysis. Both α-amino-3-hydroxy-5-methylisoxazole-4-propionic a/kainate and N-methyl-d-aspartate-type glutamate receptors participated in this neuronal circuitry. Morphine diminished local field potentials. Gabapentin diminished the negative components in lamina II and P2 component in lamina IIo, but increased the positive components in lamina IIi and laminae III-VI. The present study revealed that functional dorsal horn activity was preserved in the spinal cord slice preparation. Glutamatergic synapses were crucially involved in information processing. Opioid interneurons and gabapentin may play a modulatory role in regulating signal flows in the dorsal horn. Taken together, these results identify a spatio-temporal profile of dorsal horn activity evoked by dorsal root stimulation, and implicate glutamatergic and opioidergic receptors and gabapentin in this activity.

Dynamic variations of c‐Fos expression in the spinal cord exposed to intramuscular hypertonic saline‐induced muscle nociception

Numerous studies focus on intramuscular (i.m.) injection of hypertonic saline-induced muscle pain and nociception. The spatio-temporal characteristics and dynamic variation of spinal neuronal activities elicited by i.m. hypertonic saline remain unknown. The spatio-temporal variations of c-Fos expression in the lumbar spinal cord exposed to i.m. injection of 5.8% saline were investigated in male rats. After a unilateral i.m. 5.8% saline injection, c-Fos expression in dorsal horn of spinal L4-6 segments was significantly enhanced bilaterally (p < 0.05 and p < 0.001). These 5.8% saline-induced bilateral spinal Fos expression occurred rapidly 0.5 h following the i.m. injection, and reached the peak levels within 1 h, which declined gradually and returned to the control levels within 8 h. Compared with intact rats without i.m. insults, no significant influence of the spinalization on spinal c-Fos expression was found. However, the 5.8% saline-induced increases in Fos expression in intact and spinalized rats differed significantly. During muscle nociception, the c-Fos expression within the superficial layer (laminae I-II) and the deep layer (laminae V-VI) in spinalized rats were significantly lower and higher than that of in intact rats (p < 0.05 and p < 0.001). Fentanyl (20 μg/kg, intraperitoneal) completely attenuated the 5.8% saline intramuscularly induced increases in c-Fos expression in laminae III-VI (p < 0.001), but not laminae I-II. It is suggested that spinal nociceptive neuronal activities in superficial and deep layers may differently be modulated by endogenous descending facilitation and inhibition, respectively.

Immunoreactivity of Somatostatin Receptor Subtype 2 in Dorsal Root Ganglia in the Rat

Purpose: Somatostatin has been suggested to play a role in the transmission of neurotransmitters and in the modulation of pain. Of the different subtypes of somatostatin receptors 2, sstr2A and sstr2B are important in the modulation and transmission of pain. The present study was carried out to investigate sstr2 immunoreactivity in rat. Materials and Methods: Dorsal root ganglia (DRG) cells at the levels of the spinal cord of 10 rats (Sprague-Dawley, 200-250 g) were examined for sstr2 by immunohistochemistry. Results: In the control group, sstr2A immunoreactivity was strongly positive in the dense network within laminae I and II of the dorsal horn at spinal levels (). In contrast to sstr2A, sstr2B immunoreactivity was observed throughout laminae III-VI. In the DRG, sstr2A and sstr2B immunoreactivities were mainly found in medium-sized neurons. Conclusion: The distribution of sstr2A immunoreactive cells among sstr2 in the dorsal root ganglia (L4-6) resembles that of somatostatin. Incontrast to sstr2A, sstr2B immunoreactivity showed a different distribution. The presence of sstr2A at laminae I and II, and sstr2B at laminae III-VI of the dorsal horn may modulate sensory functions at these different regions of the spinal cord. Considering different actions according to the receptors of the neurotransmitter, the functions of the isoforms of sstr2 appear variable in terms of modulating and transmitting pain.

The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn.

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid-lumbar spinal cord of the rat. The great majority (94-100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2-immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin- and enkephalin-containing boutons (including somatostatin-immunoreactive boutons that lacked calcitonin gene-related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present. We also investigated VGLUT expression in central terminals of primary afferents. Myelinated afferents were identified with cholera toxin B subunit; most of those in lamina I were VGLUT2-immunoreactive, whereas all those in deeper laminae were VGLUT1-immunoreactive, and some (in laminae III-VI) appeared to contain both transporters. However, peptidergic primary afferents that contained substance P or somatostatin (most of which are unmyelinated), as well as nonpeptidergic C fibres (identified with Bandeiraea simplicifolia isolectin B4) showed low levels of VGLUT2-immunoreactivity, or were not immunoreactive with either VGLUT antibody. As all primary afferents are thought to be glutamatergic, this raises the possibility that unmyelinated afferents, most of which are nociceptors, express a different vesicular glutamate transporter.

Selectivity of the central control of sensory information in the mammalian spinal cord.

Afferent feedback from muscle proprioceptors, as well as movement-induced activation of skin receptors plays an important role in the patterning of motor activity for stepping and postural control. An important component in this control is the presynaptic GABAergic modulation of the synaptic effectiveness of muscle and cutaneous afferents, known to change in phase with the locomotor cycle, during the execution of voluntary movements, or after a peripheral nerve injury. Recent electrophysiological studies, together with ultrastructural observations, indicate that the distribution of GABAa synapses in the intraspinal arborizations of muscle spindle and tendon organ afferents is not homogeneous. Namely, that some collaterals are the targets of one, or more, GABAergic interneurones, while other collaterals of the same fibre receive no GABAergic connections. In addition, both PAD and inhibition of PAD have a local character. This allows, at least in principle, decoupling the information arising from common sensory inputs. A spatially restricted modulation of PAD could play a significant role in the adjustment of the synaptic effectiveness of Ia afferents at the onset of voluntary contractions in humans, during movement-induced stimulation of the skin, or during the compensation of motor activity following partial denervation of muscles. Changes in the synchronization of the PAD-mediating interneurones can also have a profound effect on the information transmitted by a given set of afferent fibres. Data are presented that in the anesthetized cat, variation in the spontaneous activity of a population of dorsal horn neurones in laminae III-VI, that respond to stimulation of low-threshold cutaneous afferents, produce correlated fluctuations of monosynaptic reflexes by means of pre- and postsynaptic mechanisms. It is suggested that correlated changes in the level of PAD can also play a significant role in the presynaptic adjustment of the synaptic effectiveness of the afferent fibres during specific motor tasks.

Serotonin 5-HT(2) receptor activation induces a long-lasting amplification of spinal reflex actions in the rat.

1. C-fibre activation induces a long-term potentiation (LTP) in the spinal flexion reflex in mammals, presumably to provide enhanced reflexive protection of damaged tissue from further injury. Descending monoaminergic pathways are thought to depress sensory input but may also amplify spinal reflexes; the mechanisms of this modulation within the spinal cord remain to be elucidated. 2. We used electrical stimulation of primary afferents and recordings of motor output, in the rat lumbar spinal cord maintained in vitro, to demonstrate that serotonin is capable of inducing a long-lasting increase in reflex strength at all ages examined (postnatal days 2-12). 3. Pharmacological analyses indicated an essential requirement for activation of 5-HT(2C) receptors while 5-HT(1A/1B), 5-HT(7) and 5-HT(2A) receptor activation was not required. In addition, primary afferent-evoked synaptic potentials recorded in a subpopulation of laminae III-VI spinal neurons were similarly facilitated by 5-HT. Thus, serotonin receptor-evoked facilitatory actions are complex, and may involve alterations in neuronal properties at both motoneuronal and pre-motoneuronal levels. 4. This study provides the first demonstration of a descending transmitter producing a long-lasting amplification in reflex strength, accomplished by activating a specific serotonin receptor subtype. It is suggested that brain modulatory systems regulate reflex pathways to function within an appropriate range of sensori-motor gain, facilitating reflexes in behavioural situations requiring increased sensory responsiveness.

Modulation of synaptic transmission from segmental afferents by spontaneous activity of dorsal horn spinal neurones in the cat.

We examined, in the anaesthetised cat, the influence of the neuronal ensembles producing spontaneous negative cord dorsum potentials (nCDPs) on segmental pathways mediating primary afferent depolarisation (PAD) of cutaneous and group I muscle afferents and on Ia monosynaptic activation of spinal motoneurones. The intraspinal distribution of the field potentials associated with the spontaneous nCDPs indicated that the neuronal ensembles involved in the generation of these potentials were located in the dorsal horn of lumbar segments, in the same region of termination of low-threshold cutaneous afferents. During the occurrence of spontaneous nCDPs, transmission from low-threshold cutaneous afferents to second order neurones in laminae III-VI, as well as transmission along pathways mediating PAD of cutaneous and Ib afferents, was facilitated. PAD of Ia afferents was instead inhibited. Monosynaptic reflexes of flexors and extensors were facilitated during the spontaneous nCDPs. The magnitude of the facilitation was proportional to the amplitude of the 'conditioning' spontaneous nCDPs. This led to a high positive correlation between amplitude fluctuations of spontaneous nCDPs and fluctuations of monosynaptic reflexes. Stimulation of low-threshold cutaneous afferents transiently reduced the probability of occurrence of spontaneous nCDPs as well as the fluctuations of monosynaptic reflexes. It is concluded that the spontaneous nCDPs were produced by the activation of a population of dorsal horn neurones that shared the same functional pathways and involved the same set of neurones as those responding monosynaptically to stimulation of large cutaneous afferents. The spontaneous activity of these neurones was probably the main cause of the fluctuations of the monosynaptic reflexes observed under anaesthesia and could provide a dynamic linkage between segmental sensory and motor pathways.

ラット坐骨神経絞扼後の痛覚過敏と脊髄細胞のアポトーシス発現

Peripheral nerve injury often leads to pathological pain processes including hyperalgesia and allodynia in animals and humans. Although peripheral neuronal mechanisms contribute to these pathological pain state, recent investigation shows that changes in central neuronal function, such as glutamatergic nerve activation and disinhibition of inhibitory neurons in the spinal cord, may play a pivotal role. Therefore, it is reasonable to determine whether apoptosis develops resulting from glutamatergic activation via acceleration of intracellular and nuclear signalling processes. The aim of the present study was to investigate the histopathological changes in relation to response to thermal stimulation with time in rats with chronic constriction injury (CCI). Male Sprague-Dawley rats were anesthetized with pentobarbital (50mg/kg, I.P.) and left sciatic nerve were ligated. Sham rats were performed skin incision only. Paw withdrawal latency times (PWL) of response to thermal stimulation were measured in these rats by using the plantar test equipment at the 6 th to 10 th postoperative days (POD). Immunohistochemical analysis for apoptosis (TUNEL stain) and neuronal degeneration (NAUTA stain) in the laminae I-II, III-VI and VII-IX in spinal cord (L3-5) in rats was performed with light microscope (40 and 400 times) at the 6 th, 7 th and 10 th POD, respectively. TUNEL stain score was obtained as 0 : no positive neuron to 3 : more than 10% neurons show positive, and NAUTA stain score was also obtained as 0 : normal neuron to 3 : more than 50% somato-dendritic argyrophyllic neurons. In the sham group, any remarkable change in the pain behaviour throughout period measured. While in the CCI rat, PWL decreased at the ipsilateral side after operation. In TUNEL stain, positive neurons developed in the ipsilateral laminae I-II at the 6th and 7th day and then became to less at the 10th day after CCI. In contrast, positive neurons of NAUTA stain were developed in the ipsilateral laminae III-VI after CCI (7th<10th day). The present results demonstrate that apoptosis development was firstly found in the superficial layer of spinal cord in rats with early state of hyperalgesia evoked by sciatic nerve ligation. This may suggest that sciatic nerve injury leads to increased ascending inputs or glutamatergic nerve activation followed by derangement of intracellular signalling-intranuclear process (c-fos expression) in the spinal cord which causes apoptosis leading to pain behaviour. It is also suggested that the delayed appearance of neuronal degeneration in the laminae III-VI causes dysfunction of inhibitory interneurons located in this region and this neuronal damage is sufficient to develop the sustained pain behaviour.

The medullary dorsal reticular nucleus facilitates pain behaviour induced by formalin in the rat.

The influence of the dorsal reticular nucleus (DRt) on pain behaviour during the formalin test was studied in the rat by lesioning the nucleus through local application of electrical current or quinolinic acid. Animals in which the DRt was lesioned ipsilaterally to the paw injected with formalin spent less time in focused (licking, biting or scratching the injected paw) and total (focused pain behaviour plus protection of the injected paw during movements) pain behaviour, and showed paw-jerks less frequently than non-lesioned animals in both phases 1 and 2 of the test. Animals in which the DRt was lesioned contralaterally to the injected paw presented a decrease in total pain behaviour and number of paw-jerks only during phase 2. The number of superficial (laminae I-II) and deep (laminae III-VI) spinal dorsal horn cells expressing the c-fos proto-oncogene 2 h after subcutaneous injection of formalin was reduced by 34% and 50%, respectively, in animals with an ipsilateral DRt lesion as compared to non-lesioned rats. No differences in c-fos expression were observed after lesioning the DRt contralateral to the formalin injection. The results indicate that the DRt is involved in the facilitation of nociception during the formalin test by enhancing the response capacity of dorsal horn neurons to noxious stimulation. It is suggested that the pronociceptive action of the DRt is mediated by the reciprocal connections it establishes with the spinal dorsal horn.

Modulated expression of c-Fos in the spinal cord following noxious thermal stimulation of monoarthritic rats.

Peripheral noxious stimulation evokes functional and biochemical changes in the spinal cord which results in central sensitization and hyperalgesia, but at the same time also induces the activation of inhibitory control systems. The purpose of the present study was to investigate whether the adaptive changes induced by ongoing peripheral inflammation influence the spinal cord expression of c-Fos (a commonly used marker of neuronal activity) following an additional acute noxious stimulus. Therefore, the spinal expression of c-Fos was immunohistochemically investigated following noxious thermal stimulation of a rat monoarthritic hindpaw at various time points (1, 4, 8, 21 days) after induction of monoarthritis. Compared to normal rats, c-Fos expression following ipsilateral noxious thermal stimulation of monoarthritic rats was strongly modified in the deep laminae of the dorsal horn depending on the time course of inflammation. At 1 day of monoarthritis, an enhanced ipsilateral expression (135% and 208% of normal rats in laminae III-VI and VII, respectively) and at 3 weeks a reduced expression (38% and 23% of normal rats in laminae III-VI and VII, respectively) was detected. The amount of c-Fos-positive neurons in the ipsilateral superficial laminae I and II was unchanged at all time points investigated. To assess excitability changes on the contralateral side at an early stage of inflammation, a group of monoarthritic rats received a contralateral noxious stimulus at day 1 of monoarthritis. This resulted in a potentiated expression of c-Fos ipsilateral to the acute noxious stimulus (i.e., contralateral to the monoarthritic hindpaw) restricted to lamina II (137% of normal rats) of the dorsal horn. The data showed that changes in c-Fos expression depended on the time point of noxious heat stimulation (NHS) of monoarthritic rats, and differed in the ipsi- and contralateral side of the spinal cord. In addition to a possible habituation of c-Fos expression, it may be speculated that the time course-dependent changes reflect laminae-specific modulations of excitatory and inhibitory mechanisms during monoarthritis. Further studies are necessary in order to provide more insights into the contribution of these mechanisms on noxious stimulus-evoked c-Fos expression.

Cells of origin, course, and termination patterns of the ventral, uncrossed component of the mature rat corticospinal tract.

The cells of origin, the course, and termination patterns of the ventral, uncrossed component of the rat corticospinal tract (CST) was investigated by using retrograde and anterograde tracing methods. Anterograde tracing with biotin dextran-amine (BDA) revealed the position and detailed morphology of CST fibers in the spinal cord. Cross sections on spinal levels C4, T8, and L4 showed labeled fibers in the ipsilateral ventral funiculus on all levels. Although ipsilateral ventral CST fibers run close to the midline in the cervical cord, they were found to disperse more in the ventromedial funiculus at lower spinal levels. To study the termination patterns of the ipsilateral ventral projection, a dorsal spinal cord hemisection was performed at level T8, severing the crossed dorsomedial and dorsolateral components but leaving ipsilateral ventral running fibers intact. These fibers were observed to have sometimes several collaterals with terminal arbors extending into different spinal segments, innervating mostly laminae III-VI. Structures closely resembling synaptic boutons were identified in these arbors. By retrograde tracing in animals with dorsal spinal cord hemisection, we found labeled cells equally distributed throughout the spinally projecting cortical areas corresponding to the level of tracer injection. Labeled cells were found in layer V. The diameter of the labeled cells was not significantly different from other spinally projecting cortical neurons. In summary, a neuroanatomically complete ipsilateral, ventral corticospinal projection down to low spinal levels was found. The large extension of the terminal arborizations in intermediate laminae of the spinal cord suggests a modulatory role of this CST component.

Pre- and post-synaptic actions of 5-hydroxytryptamine in the rat lumbar dorsal horn in vitro: implications for somatosensory transmission.

Since the relative contribution of pre- versus post-synaptic actions of 5-hydroxytryptamine (5-HT) to modulation of somatosensory processing in the dorsal hom is not known, recordings from primary afferents and dorsal hom neurons from in vitro rat spinal cord were used to address this issue. 5-HT produced a depression of spontaneous dorsal root potentials and a slow primary afferent depolarization (PAD): the PAD versus 5-HT concentration-response curve was bell shaped (maximum at 5 microM; 250 +/- 41.5 microV). In 28/40 dorsal horn neurons, 5-HT elicited a slow depolarization not clearly associated with a specific input resistance change. Excitatory synaptic transmission from primary afferents to dorsal horn neurons was depressed by 5-HT in 40/45 neurons. 5-HT > or = 5 microM significantly (P < or = 0.05) decreased the amplitude, shortened the total duration and half-decay time of the excitatory post-synaptic potential (EPSP). A dominant effect of 5-HT on longer latency EPSP components was evident. There was not direct relationship between the magnitude of PAD and the reduction of the EPSP by 5-HT. 5-Carboxamidotryptamine, an agonist for 5-HT1 receptors, mimicked the depression of neurotransmission in the dorsal horn without producing PAD. A sample of dorsal horn neurons (n = 8) was injected with biocytin and their morphology described. All had somata within laminae III-VI. In five of these neurons 5-HT depressed the EPSP but in one interneuron-like and one unclassed neuron the EPSP was potentiated. These data suggest that whilst depression of synaptic transmission is the predominant effect of 5-HT in the deep dorsal horn, this is not easily related to PAD or cellular actions of 5-HT on dorsal horn neurons.

Distribution of Fos-like immunoreactivity in the caudal brainstem of the rat following noxious chemical stimulation of the temporomandibular joint.

Central expression of the protooncogene c-fos was used to examine areas receiving noxious sensory input from the rat temporomandibular joint (TMJ). Fos-like immunoreactivity (Fos-LI) in the caudal brainstem was visualized 2 hours after unilateral injection of the small-fiber-specific excitant/inflammatory irritant mustard oil into the TMJ region. Control animals received injection of either mustard oil into the subcutaneous fascia overlying the masseter muscle or mineral oil vehicle into the TMJ region. In all groups, Fos-LI was consistently observed ipsilaterally in the spinal trigeminal nucleus and cervical dorsal horn and, bilaterally, in the nucleus of the solitary tract and the ventrolateral medulla. The expression of Fos-LI ipsilaterally in the paratrigeminal nucleus was variable. Within the trigeminal sensory complex, Fos-LI was restricted to subnucleus caudalis and the caudal portions of subnucleus interpolaris near the level of the obex. Approximately 12% of Fos-LI cells in subnucleus caudalis and in the cervical dorsal horn were found in laminae III-VI. Compared to TMJ mustard oil injection, mineral oil injection produced less Fos-LI at all rostrocaudal levels, whereas subcutaneous mustard oil injection produced less Fos-LI in caudal subnucleus caudalis but similar amounts in the cervical dorsal horn. Neither of these injections yielded significant ipsilateral responses in subnucleus caudalis, indicating that Fos-LI in this region following TMJ mustard oil injection could be ascribed solely to small-fiber stimulation in the deep TMJ region. The wide rostrocaudal distribution of Fos-LI within the caudal brainstem reflects the distribution of TMJ-responsive nociceptive neurons that may underlie the spread and referral of pain from the TMJ region.

Field potential mapping of neurons in the lumbar spinal cord activated following stimulation of the mesencephalic locomotor region

The spinal neurons involved in the control of locomotion in mammals have not been identified, and a major step that is necessary for this purpose is to determine where these cells are likely to be located. The principal objective of this study was to localize lumbar spinal interneurons activated by stimulation of the mesencephalic locomotor region (MLR) of the cat. For this purpose, extracellular recordings of MLR-evoked cord dorsum and intraspinal field potentials were obtained from the lumbosacral enlargement during fictive locomotion in the precollicular-postmammillary decerebrate cat preparation. Potentials recorded from the dorsal surface of the cord between the third lumbar (L3) and first sacral (S1) segments typically showed four short-latency positive waves (P1-P4). These P-waves were largest between the L4-L6 segments. The amplitude of the P2-4 waves increased with the appearance of locomotion and displayed rhythmic modulation during the locomotor step cycle. Microelectrode recordings from the L4-L7 spinal segments during fictive locomotion revealed the presence of both positive and negative short-latency MLR-evoked intraspinal field potentials, and were used to construct isopotential maps of the evoked potentials. Positive field potentials were observed throughout the dorsal horn of the L4-L7 spinal segments with the largest amplitude potentials occurring in laminae III-VI. Negative field potentials were found in laminae VI-X of the lumbar cord. The shortest latency negative field potentials were observed in lamina VII and at the border between laminae VI and VII and were considered to be evoked monosynaptically from the arrival of the descending volley. Short-latency mono- and disynaptic negative field potentials were also observed in lamina VIII. Longer latency, tri- and polysynaptic field potentials were observed in laminae VII and VIII. Many of the longer latency negative waves observed in laminae VII and VIII followed shorter latency negative potentials recorded from the same location. Laminae VII and VIII negative field potentials were largest in the L5-6 and L4-5 spinal segments, respectively. Negative field potentials were also evoked in the motor nuclei of the L4-7 spinal segments. The segmental latencies for these potentials indicate that they were evoked di- and trisynaptically.(ABSTRACT TRUNCATED AT 400 WORDS)

Two forms of inhibition of spinothalamic tract neurons produced by stimulation of the periaqueductal gray and the cerebral cortex

1. Recordings were made from the lumbosacral spinal cord in anesthetized macaque monkeys. The inhibitory effects of electrical stimulation of the periaqueductal gray (PAG) and the cerebral cortex or cerebral peduncle (CP) were tested and compared by recording 1) cord dorsum potentials evoked by stimulation of the sural nerve, 2) discharges recorded extracellularly, and 3) membrane potentials recorded intracellularly from spinothalamic tract (STT) neurons at rest (background activity) or in response to stimulation of the sural nerve. 2. Stimulation of the cortex or in the CP preferentially reduced the amplitude of the N1 and N2 waves of the cord dorsum potential evoked by stimulation of the sural nerve, without affecting the N3 wave. Stimulation of the PAG, on the other hand, reduced the amplitude of the N3 wave with little effect on the N1 and N2 waves. 3. The activity of 62 STT neurons was recorded extracellularly. Stimulation of the PAG or the cortex/CP inhibited nonpreferentially the responses of the neurons in the superficial laminae to all afferent inputs. On the other hand, stimulation of the PAG or the cortex/CP inhibited preferentially the responses of most STT neurons in deep layers of the dorsal horn to the small or large afferent input, respectively. 4. Thirty-five neurons were recorded intracellularly. The membrane potential of the neurons averaged -45.5 +/- 10.1 (SD) mV. All neurons were recorded in laminae III-VI; the neurons were of the wide-dynamic-range (WDR) type and had background activity. 5. The inhibitory effects of stimulation of the PAG were tested on all 35 neurons. In 32 of the neurons, stimulation of the PAG evoked a hyperpolarization. The background activity of the neurons was reduced (generally it completely ceased) by the hyperpolarization. In three neurons stimulation of the PAG did not evoke a hyperpolarization and the background activity of the neurons did not change. Nevertheless, the responses of these three neurons to afferent input were inhibited by stimulation in the PAG. 6. The inhibitory effects of stimulating the cortex and/or the CP were tested in 26 of the 35 neurons. Stimulation of the cortex and/or the CP evoked a hyperpolarization in all the neurons, although, in 10 of the 26 neurons, stimulation of the CP also evoked a depolarization. The hyperpolarization generally blocked the background activity of the neurons. 7. The effective stimuli in the PAG and the cortex/CP to evoke a hyperpolarization in STT neurons were short, high-frequency trains of pulses.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of intravenous mu and kappa opioid receptor agonists on sensory responses of convergent neurones in the dorsal horn of spinalized rats.

1. Electrophysiological experiments have been performed to assess the effects of intravenously administered mu and kappa opioid agonists on the responses to noxious thermal and mechanical and non-noxious tactile stimuli of single convergent neurones in laminae III-VI of the dorsal horn of spinalized rats anaesthetized with alpha-chloralose. 2. The mu receptor agonists tested were fentanyl (1-16 micrograms kg-1) and morphine (0.5-16 mg kg-1) and the kappa-receptor agonists U-50,488 (1-16 mg kg-1) and tifluadom (0.1-1.6 mg kg-1). Multiple drug tests were made on each cell so that compounds could be compared under closely comparable conditions. 3. In one protocol, thermal and mechanical nociceptive responses of matched amplitudes were elicited alternately. Both mu and kappa agonists dose-dependently reduce the neuronal responses. Thermal nociceptive responses were as sensitive to the kappa agents as were the mechanical nociceptive responses; the mu agonists similarly reduced both types of response in parallel. 4. In another protocol, nociceptive and non-nociceptive responses were elicited alternately to permit the degree of selective antinociception to be assessed. The mu agonists were scarcely selective, fentanyl reducing nociceptive only slightly (but significantly at 4-16 micrograms kg-1) more than non-nociceptive responses. The kappa-opioid agonist U50,488 reduced tactile responses somewhat more than nociceptive responses. 5. The spontaneous discharge of these cells with ongoing activity was reduced to a significantly greater degree than the evoked responses; this is likely to have contributed to the non-selectivity of the reduction of the evoked responses. 6. The results are discussed with respect firstly to previous reports that K opioids are ineffective in tests of thermal nociception, and secondly to the likely spinal mechanisms by which opioid receptor agonists mediate antinociception.

Topographical localisation of endothelin mRNA and peptide immunoreactivity in neurones of the human brain

The distribution of endothelin mRNA and immunoreactivity in the human brain was investigated using the technique of in situ hybridization and immunocytochemistry. Cryostat sections from 22 cases of neurologically normal adult human brain, collected 3-7 h post-mortem were hybridized with 35S-labelled complementary (c)RNA probes prepared from the 3' non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization with all three cRNA probes revealed labelled neuronal cell bodies in laminae III-VI of the parietal, temporal and frontal cortices. Labelled cells were also seen, scattered throughout the para- and periventricular, supraoptic and lateral hypothalamic nuclei, the caudate nucleus, amygdala, hippocampus, basal nucleus of Meynert, substantia nigra, raphe nuclei, Purkinje cell layer of the cerebellum and in the dorsal motor nuclei of the vagus of the medulla oblongata. The distribution of neurones immunoreactive to endothelin was similar to that of endothelin mRNA, although fewer immunoreactive cells throughout the brain, were noted. Immunoreactive fibres were present mainly in the cortex and hypothalamus, and to a lesser extent in the brain stem. Combined in situ hybridization and immunocytochemistry on the same section revealed the presence of endothelin-1 mRNA and immunoreactivity in the same cortical neuronal cell. Colocalisation studies in the cortex revealed endothelin-1 mRNA and immunoreactivity in a number of cells which also expressed neuropeptide Y mRNA and immunoreactivity. In the hypothalamus and basal nucleus of Meynert endothelin immunoreactivity was colocalised to a subset of neurophysin- and galanin-immunoreactive cell bodies respectively. Endothelin mRNA and immunoreactivity was also seen in some blood vessel endothelial cells. The findings of endothelin mRNAs and immunoreactivity in heterogenous neuronal populations further emphasises the potential role of endothelin as a neuropeptide, probably having diverse actions in the nervous system of man.

An intracellular analysis of amino acid induced excitations of deep dorsal horn neurones in the rat spinal cord slice

The rat spinal cord slice preparation has been used to investigate the sensitivity of deep dorsal horn neurones to the excitatory amino acids N-methyl-D-aspartate (NMDA), quisqualate and L-glutamate. Intracellular recordings were made from 44 neurones in laminae III-VI of 14- to 16-day rats. Superfusion of quisqualate (30 microM) excited all neurones, NMDA (50 microM) excited 72% and L-glutamate (0.5-1 mM) 63% of the neurones. Depolarizations were retained after tetrodotoxin but with a reduced amplitude. The NMDA antagonist D-aminophosphonovalerate (D-APV, 10 microM) reduced NMDA and L-glutamate depolarizations by 66% and by 40%, respectively, while the quisqualate responses were enhanced by 27%. Dorsal root stimulation elicited two main patterns of activity; short-latency single/double spikes followed by subthreshold excitatory postsynaptic potentials (EPSPs) or a burst of spikes rising from a long duration composite EPSP. D-APV reduced the long-latency components of the first type and reduced the amplitude and duration of the composite EPSP of the second. These results support a specialized role for NMDA receptors in synaptic transmission in the dorsal horn.

Normal anatomy and physiology of the spinal cord dorsal horn.

The dorsal horn of the spinal cord receives afferent input from innocuous primary afferent neurons via collaterals from the dorsal columns. This input is integrated and relayed primarily by neurons in laminae III-VI. Dorsal horn neurons which encode innocuous inputs project to the medulla and the cervical spinal cord via the dorsal columns and the dorsolateral funiculus. Nociceptive primary afferent neurons enter the spinal dorsal horn via collaterals from Lissauer's tract. Nociceptive input is integrated and relayed by neurons in laminae I, II and V which project to the reticular formation and thalamus via the anterolateral tract.