GABA-immunoreactive neurons in the dorsal horn of the rat spinal cord

An antiserum to GABA was used on semithin resin-embedded sections of rat dorsal horn. Immunoreactive neurons were evenly distributed throughout laminae I-III and constituted between 24 and 33% of the total neuronal population within three laminae. Fifty Golgi-stained cells in lamina II were tested with the antiserum. Most of the islet cells examined were immunoreactive, although some small islet cells were not. None of the 14 stalked cells tested was immunoreactive. These results provide further evidence that the stalked and islet cells of lamina II form two distinct functional classes and suggest that the islet cells function as inhibitory interneurons.     

Wnt signaling plays an essential role in neuronal specification of the dorsal spinal cord

In the developing spinal cord, signals from the roof plate are required for the development of three classes of dorsal interneuron: D1, D2, and D3, listed from dorsal to ventral. Here, we demonstrate that absence of Wnt1 and Wnt3a, normally expressed in the roof plate, leads to diminished development of D1 and D2 neurons and a compensatory increase in D3 neuron populations. This occurs without significantly altered expression of BMP and related genes in the roof plate. Moreover, Wnt3a protein induces expression of D1 and D2 markers in the isolated medial region of the chick neural plate, and Noggin does not interfere with this induction. Thus, Wnt signaling plays a critical role in the specification of cell types for dorsal interneurons.     

Response characteristics of spinal cord dorsal horn neurons in chronic allodynic rats after spinal cord injury

The physiological mechanisms of chronic pain in patients with spinal cord injury (SCI) are poorly understood. In the present study, we explored response characteristics of dorsal horn neurons of spinally injured rats exhibiting chronic pain (pain-like response to innocuous mechanical and cold stimulation). Several abnormalities were found in the distribution and response characteristics of dorsal horn neurons in chronic allodynic rats. First, 17% of the recorded neurons (vs. 0% in control animals) had no receptive field. Most of these units were located at or close to the lesioned spinal segment, and they discharged spontaneously at high frequencies. Allodynic rats also showed a significant decrease in the proportion of low-threshold (LT) neurons and an increase in the proportion of wide dynamic range (WDR) neurons. The rate of spontaneous activity of high-threshold (HT) neurons was significantly higher in allodynic compared with control rats. Moreover, HT neurons in allodynic animals showed increased neuronal responses to mechanical stimulation. WDR neurons responded with higher discharge rates to innocuous von Frey hair stimulation in allodynic compared with control rats. The percentage of WDR and HT neurons showing afterdischarges to noxious pinch was also significantly increased in the allodynic rats. The proportion of WDR and HT neurons responding to innocuous cold stimulation respectively increased from 53 and 25% in control rats to 91 and 75% in allodynic animals. These results suggest that the chronic pain-like behaviors in spinally injured rats may be generated and maintained by abnormalities in dorsal horn neurons.     

Synapses upon the axon origin of dorsal horn neurons in the rat spinal cord

Axon terminals synapsing with axon hillocks or origins of Golgi-impregnated and gold-toned neurons in the dorsal horn of the rat were shown in serial electron micrographs. Synapses occurred irrespective of the site (perikaryon or dendrite) and mode (with or without an axon hillock) of the axon origin. The synapsing axon terminals contained 3 populations of vesicles: pleomorphic and flattened synaptic vesicles and a combination of pleomorphic and dense-core vesicles. The membrane thickening in the axon-axon hillock synapses was of the symmetrical type.     

Intersegmental synchronization of spontaneous activity of dorsal horn neurons in the cat spinal cord

Extracellular recordings of neuronal activity made in the lumbosacral spinal segments of the anesthetized cat have disclosed the existence of a set of neurons in Rexed's laminae III–VI that discharged in a highly synchronized manner during the occurrence of spontaneous negative cord dorsum potentials (nCDPs) and responded to stimulation of low-threshold cutaneous fibers (<1.5×T) with mono- and polysynaptic latencies. The cross-correlation between the spontaneous discharges of pairs of synchronic neurons was highest when they were close to each other, and decreased with increasing longitudinal separation. Simultaneous recordings of nCDPs from several segments in preparations with the peripheral nerves intact have disclosed the existence of synchronized spontaneous nCDPs in segments S1–L4. These potentials lasted between 25 and 70 ms and were usually larger in segments L7–L5, where they attained amplitudes between 50 and 150 μV. The transection of the intact ipsilateral hindlimb cutaneous and muscle nerves, or the section of the dorsal columns between the L5 and L6, or between the L6 and L7 segments in preparations with already transected nerves, had very small effects on the intersegmental synchronization of the spontaneous nCDPs and on the power spectra of the cord dorsum potentials recorded in the lumbosacral enlargement. In contrast, sectioning the ipsilateral dorsal horn and the dorsolateral funiculus at these segmental levels strongly decoupled the spontaneous nCDPs generated rostrally from those generated caudally to the lesion and reduced the magnitude of the power spectra throughout the whole frequency range. These results indicate that the lumbosacral intersegmental synchronization between the spontaneous nCDPs does not require sensory inputs and is most likely mediated by intra- and intersegmental connections. It is suggested that the occurrence of spontaneous synchronized nCDPs is due to the activation of tightly coupled arrays of neurons, each comprising one or several spinal segments. This system of neurons could be involved in the modulation of the information transmitted by cutaneous and muscle afferents to functionally related, but rostrocaudally distributed spinal interneurons and motoneurons, as well as in the selection of sensory inputs during the execution of voluntary movements or during locomotion. Electronic Publication     

Expression of nerve growth factor in spinal dorsal horn following crushed spinal cord injury

Expression of nerve growth factor in spinal dorsal horn following crushed spinal cord injury     

Acetylcholine system in the isolated ventral and dorsal horn neurons from bovine spinal cord

We have isolated the ventral and dorsal horn neurons from the bovine spinal cord using our isolation procedure and determined choline acetyltransferase, acetylcholinesterase and choline uptake activity in these neurons. Choline acetyltransferase and choline uptake activity were higher in the ventral than in the dorsal horn neurons, showing their cell specificity to some extent, but acetylcholinesterase activity did not show any differences between the two cell types.     

内脏伤害性刺激在脊髓突触后背柱神经元的信号转导机制

内脏痛是急慢性胃肠道、盆腔、泌尿道和其它实体器官疾病的最常见症状之一。许多疾病伴发的内脏性疼痛十分顽固,如肠道激惹综合征、间质性膀胱炎、胰腺炎、子宫内膜异位症和癌性内脏痛等,给临床医师带来了极大的挑战。内脏疼痛机制十分复杂。近年来,一系列的临床和基础研究发现起源于     

Modulation of AMPA excitatory postsynaptic currents in the spinal cord dorsal horn neurons by insulin

Glutamate AMPA receptors are critical for sensory transmission at the spinal cord dorsal horn (DH). Plasma membrane AMPA receptor endocytosis that can be induced by insulin may underlie long term modulation of synaptic transmission. Insulin receptors (IRs) are known to be expressed on spinal cord DH neurons, but their possible role in sensory transmission has not been studied. In this work the effect of insulin application on fast excitatory postsynaptic currents (EPSCs) mediated by AMPA receptors evoked in DH neurons was evaluated. Acute spinal cord slices from 6 to 10 day old mice were used to record EPSCs evoked in visually identified superficial DH neurons by dorsal root primary afferent stimulation. AMPA EPSCs could be evoked in all of the tested neurons. In 75% of the neurons the size of the AMPA EPSCs was reduced to 62.1% and to 68.9% of the control values when 0.5 or 10 μM insulin was applied. There was no significant change in the size of the AMPA EPSCs in the remaining 25% of DH neurons. The membrane permeable protein tyrosine kinase inhibitor, lavendustin A (10 μM), prevented the insulin induced AMPA EPSC depression. Our results suggest a possible role of the insulin pathway in modulation of sensory and nociceptive synaptic transmission in the spinal cord.     

Purine-induced depression of dorsal horn neurons in the cat spinal cord: enhancement by tachykinins

The neurokinins, physalaemin, substance P, neurokinin A and bradykinin, were tested on the responses of single spinal neurons to the purines, adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate and to GABA. Experiments were done on anaesthetized cats, recording extracellularly from functionally identified sensory neurons in the lumbar dorsal horn. All compounds were administered by iontophoresis. Neurokinins caused a slow, prolonged excitation which outlasted the period of application. Physalaemin was tested on responses to ATP in 24 units. In each case application of ATP caused either depression, excitation or a biphasic response when the application was not pre-conditioned by ejection of physalaemin. For 11 units, with ATP applications subthreshold to alter the on-going firing rate, such applications caused depression when they were preceded by administration of physalaemin. Three units were tested with ATP applications which caused the excitatory response; when the applications of ATP were preceded by ejection of physalaemin, there was then a depressant component in the response. In these 14 cases, the magnitude of the depression or of the depressant component of the response, was measured using currents which failed to produce depression in the absence of physalaemin ejection; the mean magnitude of this depression was 34.7 +/- 1.6% (+/- S.E.M.). With the 10 remaining units, responses to ATP were unaffected by application of physalaemin. The early components of the biphasic and excitatory responses were unaffected by physalaemin and hence it appeared to have a differential effect, enhancing only the depressant effects of ATP. The enhancement of depression was reversible, lasting up to 30 min following a single ejection. Neither control current nor glutamate mimicked the effect of physalaemin in the responses to application of ATP. The depressant response to adenosine 5'-monophosphate was also enhanced by physalaemin: ejections of adenosine 5'-monophosphate subthreshold to affect the on-going firing rate caused depression after physalaemin application in 3 of 8 units (average depression: 35.0 +/- 3.3%). On the other hand, depression induced by GABA was unaffected by physalaemin in every case (n = 8); in 4 of these cases GABA was tested on units for which purine-induced depression was enhanced by physalaemin. Thus, physalaemin preferentially affected depressant responses to ATP and to adenosine 5'-monophosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of neurons that express preprotachykinin B in the dorsal horn of the rat spinal cord

Although it is established that neurokinin B is expressed by some neurons in laminae I-III of the rat spinal dorsal horn, little is known about the proportions of cells in these laminae that express neurokinin B, or whether these are excitatory or inhibitory neurons. Neurokinin B is derived from preprotachykinin B, and we have used an antibody against preprotachykinin B to address these issues. We found that preprotachykinin B-immunoreactive neurons were present throughout laminae I-III, constituting 10-11% of the neuronal population in laminae I-II, and 4% of that in lamina III. They formed a prominent band in the ventral half of lamina II (where they made up 16% of the population) and the dorsalmost part of lamina III. The great majority (99%) of preprotachykinin B-immunoreactive axonal boutons contained the vesicular glutamate transporter 2, while none contained glutamic acid decarboxylase. Since most of these boutons are likely to be derived from local preprotachykinin B-expressing cells, these observations suggest that most of the latter are excitatory interneurons. Although 9% of preprotachykinin B-labeled axonal varicosities were substance P-immunoreactive, none contained calcitonin gene-related peptide, which is consistent with reports that neurokinin B is not expressed by primary afferent axons. Many of the preprotachykinin B-immunoreactive cells contained compounds that are present in putative excitatory neurons in laminae I-III: calbindin (84%), protein kinase Cgamma (76%) or somatostatin (31%). However, there was little or no overlap between preprotachykinin B and three other markers associated with excitatory neurons in these laminae: the mu opioid receptor MOR-1, the neurokinin 1 receptor and neurotensin. These results suggest that neurokinin B is expressed by specific populations of excitatory neurons in the superficial dorsal horn. By examining expression of Fos protein in response to intraplantar injection of formaldehyde we provide evidence that many of the preprotachykinin B cells in lamina I and the outer part of lamina II respond to noxious stimulation.     

Muscle stretch-induced modulation of noxiously activated dorsal horn neurons of feline spinal cord

The present work was designed to check for the possibility of interactions between mechanical innocuous and chemically induced noxious muscle afferent inputs on discharge behavior of nociceptive superficial dorsal horn neurons (SDHNs) of the spinal cord in decerebrated cats. The innocuous and noxious stimuli were applied separately and in combination, so that the effects of the innocuous stimulus on nociceptive processing could be evaluated. The innocuous stimulus consisted of ramp-and-hold stretches of the gastrocnemius muscles, whereas the noxious stimulus consisted of i.a. injections of bradykinin (BK; 0.5-1 ml, 50 microg/ml) into the arterial circulation of same muscles. Only neurons up to approximately 1mm depth and those that responded to noxious pinch of the gastrocnemius muscles were selected for further analysis. The activity of 16 dorsal horn neurons was recorded extracellularly with high-impedance glass microelectrodes, out of which seven responded to stretch, while 12 neurons responded to bradykinin injections. The bradykinin injections induced three types of responses: excitatory, inhibitory and mixed. The majority of the neurons that showed excitatory and mixed responses to bradykinin were also influenced by stretches applied directly after the bradykinin injection. In these neurons, the stretch usually counteracted the bradykinin-induced response, i.e. shortening and reducing bradykinin-induced excitation and re-exciting the cells after bradykinin-induced inhibition. The mechanism of the stretch modulation is proposed to reside in a segmental spinal control of the nociceptive transmission.     

Calcium currents and transmitter output in cultured spinal cord and dorsal root ganglion neurons

The effects of repetitive activation upon voltage-dependent calcium currents (ICa) and transmitter release were studied in dissociated cell cultures of fetal mouse spinal cord and dorsal root ganglion. Sodium and potassium currents were suppressed with tetrodotoxin (TTX) and tetraethylammonium (TEA) ions, 4-aminopyridine (4-AP), and cesium sulfate. Calcium currents were compared under voltage clamp before and after a series of depolarizing clamp pulses in spinal cord (SC) and dorsal root ganglion (DRG) neurons. Repetitive activation resulted in an exponential decline in ICa, with the decrease in ICa being much more marked in DRG compared with SC neurons. Both voltage-dependent inactivation and inactivation related to the intracellular movement of Ca2+ appeared to be involved in the decrement in ICa with repetitive activation. A decrease in transmitter output occurred with repetitive activation in DRG neurons but not in SC neurons (either excitatory or inhibitory). DRG neuron synaptic boutons had fewer mitochondria than did the boutons of either excitatory or inhibitory of SC neurons. The decrement in both ICa and synaptic transmitter output in DRG neurons could last for prolonged periods (at least minutes) following repetitive activation. We hypothesize that this vulnerability of DRG neurons to repetitive activation may be related, at least in part, to a relative incapacity to maintain a low intracellular calcium ion concentration [Ca]i during periods of increased calcium ingress associated with excitation. Such an incapacity to buffer [Ca]i may be one mechanism leading to the inactive synapses seen in some studies in vitro and in vivo of synaptic transmission.     

脊髓半横断损伤后腹角神经元神经生长因子、脑源性神经营养因子、神经营养因子-3和神经营养因子-4表达的变化

目的:探讨大鼠脊髓半横断损伤(htSCI)后脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养因子(NT-3、NT-4)在脊髓腹角神经元表达的早期变化。方法:免疫组织化学ABC法分别染4种神经因子并作阳性细胞计数。结果:NGF主要分布于脊髓腹角神经元的胞核,BDNF、NT-4与NT-3主要分布于胞浆。htSCI前后它们在细胞内的分布范围没有变化。BDNF、NGF与NT-3的3 d在损伤尾侧段脊髓双侧腹角阳性神经元数与对照组相比显著减少。BDNF与NGF的14 d的双侧腹角阳性神经元数量均较正常组明显增多,NT-3与NT-4的14 d~21 d的双侧腹角阳性神经元数量均较正常组明显增多,BDNF7~21 d以及NGF14 d的健侧的阳性神经元数量均分别多于相应的伤侧。结论:内源性BDNF、NGF、NT-3、NT-4增加对脊髓损伤修复具有重要作用,BDNF和NGF在健侧表达的增加说明健侧代偿功能的活跃。     

Subthreshold components of the cutaneous mechanoreceptive fields of dorsal horn neurons in the rat lumbar spinal cord

1. Intracellular recordings have been made from 76 neurons in the dorsal horn of the fourth and fifth lumbar segments of the spinal cord in decerebrate-spinal rats. The locations of the neurons were identified after horseradish peroxidase (HRP) ionophoresis (n = 18) or calculated from depth readings (n = 58). Sixty-nine of the neurons were found or estimated to lie within the deep dorsal horn (laminae III-V), with the remaining 7 in laminae I and II. 2. Background excitatory activity was present in all the neurons in the absence of peripheral mechanical stimuli. In 22 neurons, this consisted only of subthreshold excitatory postsynaptic potentials (EPSPs), but in 54, a proportion of the EPSPs reached threshold, producing a spontaneous spike discharge (frequency 0.2-50 Hz) that had a rhythmic component in six cells. Spontaneous hyperpolarizations occurred but were uncommon (n = 10). 3. All the neurons had excitatory cutaneous mechanoreceptive fields on the ipsilateral hindlimb. The receptive fields, defined in terms of action-potential discharge, could be subdivided into two areas: a high-probability "firing zone," where skin stimulation elicited an action-potential discharge above the mean + 1 SD of the background activity; and a low-probability firing fringe, where the stimulus elicited a distinct subthreshold depolarization, but the action-potential response fell within the variability of the background discharge. 4. Mechanical stimulation in the middle of the firing zone in all cells generated both supra- and subthreshold excitatory responses, with the former predominating. As the stimuli were applied progressively farther away from the center of the firing zone, the subthreshold component became relatively more prominent. 5. Fifty percent of the 15 neurons that were recorded from for sufficient time (greater than 30 min) to enable the presence, extent, and characteristics of subthreshold responses to be examined in detail were found to have a low-probability firing fringe to their receptive fields. The response to stimulation within this fringe typically consisted of high-frequency, low-amplitude PSPs riding on a sustained depolarization, with an action-potential discharge that could not readily be distinguished from the spontaneous activity. The size of the fringe ranged from a small area adjacent to the firing zone to almost the entire hindlimb. 6. The firing zones of 20 neurons were low-threshold only and in 5 cells were high-threshold only. The majority of neurons were multireceptive, responding both to low- and high-intensity stimuli (n = 51).(ABSTRACT TRUNCATED AT 400 WORDS)     

Upregulation of persistent and ramp sodium current in dorsal horn neurons after spinal cord injury

Traumatic spinal cord injury (SCI) results not only in motor impairment, but also in chronic central neuropathic pain, which often is refractory to conventional treatment approaches. Upregulated expression of sodium channel Nav1.3 has been observed within the spinal dorsal horn neurons after SCI, and appears to contribute to neuronal hyperresponsiveness and pain-related behaviors. In this study we characterized the changes in sodium current properties within dorsal horn neurons after contusive SCI. Four weeks after adult male Sprague-Dawley rats underwent T9 spinal cord contusion injury, when behavioral nociceptive thresholds were decreased to both mechanical and thermal stimuli, whole-cell patch-clamp recordings were performed on acutely dissociated lumbar dorsal horn neurons. The cells demonstrated characteristic fast-activating and fast-inactivating sodium currents. SCI led to a shift of the steady-state activation and inactivation of the sodium current towards more depolarized potentials. The shifted steady-state inactivation shows similarities to that obtained from axotomized dorsal root ganglions, which were shown to upregulate Nav1.3. Small slow depolarizations below action potential threshold produced ramp currents, which were markedly enhanced by SCI (from 182 ± 41 to 338 ± 55 pA). The density of the noninactivating persistent sodium current was also significantly enhanced in neurons from SCI animals (from 17.4 ± 3.2 to 27.7 ± 4.4 pA/pF at 50–70 ms of depolarization). The increased persistent sodium current and ramp current, which are consistent with upregulation of Nav1.3 within dorsal horn neurons, suggest a basis for the hyperresponsiveness of these neurons following SCI.     

Immunocytochemical evidence for calcitonin gene-related peptide-like neurons in the dorsal horn and lateral spinal nucleus of the rat cervical spinal cord

Calcitonin gene-related peptide (CGRP) in the dorsal horn of the rat spinal cord was assumed until now to be principally of primary afferent origin. It is shown here, on the basis of both light and electron microscopic immunocytochemical evidence, that some cell bodies of the dorsal horn and lateral spinal nucleus (LSn) of the rat cervical spinal cord contain a CGRP-like immunoreactivity. At the light microscopic level, immunoreactive cell bodies were observed in animals pretreated with colchicine injected intraventricularly, CGRP-like cell bodies were morphologically heterogeneous and distributed in the three superficial layers of the dorsal horn. They were very rare in lamina I and more numerous in laminae II and III. A group of immunoreactive cell bodies was also observed in the LSn. Using electron microscopic techniques, a few immunoreactive cell bodies were observed even in control animals. In addition, relatively numerous immunoreactive dendrites were observed in lamina II. The specificity of the reaction and the physiological implications of the results are discussed.