Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord

ABSTRACT: BACKGROUND: Pain-related (nociceptive) information is carried from the periphery to the dorsal horn of the spinal cord mostly by two populations of small diameter primary afferents, the peptidergic and the non-peptidergic. The peptidergic population expresses neuropeptides, such as substance P and calcitonin gene-related peptide, while the non-peptidergic fibers are devoid of neuropeptides, express the purinergic receptor P2X3, and bind the isolectin B4 (IB4). Although it has been known for some time that in rat the peptidergic afferents terminate mostly in lamina I and outer lamina II and non-peptidergic afferents in inner lamina II, the extent of the termination of the latter population in lamina I was never investigated as it was considered as very minor. Because our preliminary evidence suggested otherwise, we decided to re-examine the termination of non-peptidergic afferents in lamina I, in particular with regards to their innervation of projection neurons expressing substance P receptors (NK-1r). We used retrograde labeling of neurons from the parabrachial nucleus combined with lectin IB4 binding and immunocytochemistry. Samples were examined by confocal and electron microscopy. RESULTS: By confocal microscopy, we studied the termination of non-peptidergic afferents in lamina I using IB4 binding and P2X3 immunoreactivity as markers, in relation to CGRP immunoreactivy, a marker of peptidergic afferents. The number of IB4 or P2X3-labeled fibers in lamina I was higher than previously thought, although they were less abundant than CGRP-labeled afferents. There were very few fibers double-labeled for CGRP and either P2X3 or IB4. We found a considerable number of IB4-positive fiber varicosities in close apposition to NK-1r-positive lamina I projection neurons, which were distinct from peptidergic varicosities. Furthermore, we confirmed at the ultrastructural level that there were bona fide synapses between P2X3-immunoreactive non-peptidergic boutons and neurokinin-1 receptor-positive lamina I dendrites. CONCLUSIONS: These results indicate the presence of direct innervation by non-peptidergic nociceptive afferents of lamina I projection neurons expressing NK-1r. Further investigations are needed to better understand the role of these connections in physiological conditions and chronic pain states.     

Effects of morphine, L-DOPA and tetrabenazine on the lamina V cells of spinal dorsal horn.

Experiments were performed to clarify the adrenergic mechanism of morphine action in the spinal cord. Unit activity of lamina V cell of spinal dorsal horn (L₆−L₇) of the rabbit was recorded using microelectrode. An administration of bradykinin into the femoral artery markedly increased in frequency of the unit activity. Morphine or L-Dopa inhibited the bradykinin-induced response of lumina V cell. In contrast, tetrabenazine facilitated the bradykinin-induced response. Electrical stimuli of the bulbar reticular formation, particularly the nucleus reticularis gigantocellularis markedly inhibited the dorsal horn response induced by bradykinin injection. Effect of stimulation of the bulbar reticular formation was blocked by tetrabenazine and reversed by L-Dopa. These results support the hypothesis that the bulbospinal catecholaminergic neuron inhibits the pain transmission at the dorsal horn of the spinal cord and analgesic action of morphine is mainly mediated by activation of this neuron.     

P2X receptor-mediated purinergic sensory pathways to the spinal cord dorsal horn

P2X receptors are expressed on different functional groups of primary afferent fibers. P2X receptor-mediated sensory inputs can be either innocuous or nociceptive, depending on which dorsal horn regions receive these inputs. We provide a brief review of P2X receptor-mediated purinergic sensory pathways to different regions in the dorsal horn. These P2X purinergic pathways are identified in normal animals, which provides insights into their physiological functions. Future studies on P2X purinergic pathways in animal models of pathological conditions may provide insights on how P2X receptors play a role in pathological pain states.     

Characteristics of dorsal horn neurons expressing subliminal responses to sural nerve stimulation

The present study was designed (1) to characterize the subliminal responses of dorsal horn neurons to stimulation of the sural nerve, and (2) to correlate the type of response to this stimulus with the responses to natural mechanical stimulation of the skin. To accomplish this, intracellular and extracellular recordings were carried out in L6 and L7 dorsal horn neurons in the cat. The excitatory responses of each cell to electrical stimulation of the sural nerve and to mechanical stimulation of the skin were noted. Of 35 dorsal horn cells recorded intracellularly, 11 responded with impulses to sural nerve stimulation, 9 responded with excitatory postsynaptic potentials (EPSPs) but not impulses, and 15 had no excitatory responses to this stimulus. The type of response to sural nerve stimulation was strongly correlated with receptive field modality. Most cells receiving an input from high-threshold cutaneous mechanoreceptors responded with impulses or gave no excitatory response to sural nerve stimulation, whereas most cells that had only low-threshold mechanoreceptor input responded with EPSPs only or gave no response. In cells with only low-threshold (LT) mechanoreceptive input, response to sural nerve stimulation was highly correlated with receptive field locus. Those LT cells with no excitatory responses to sural nerve stimulation had receptive fields confined to the foot and/or toes, whereas those that gave EPSPs had more proximal receptive fields. The possible significance of these data with reference to changes observed after lesions, such as increased response to sural nerve stimulation, increased receptive field size, and somatotopic reorganization, is discussed.     

Inhibitory Kcnip2 neurons of the spinal dorsal horn control behavioral sensitivity to environmental cold

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去甲肾上腺素在脊髓背角的镇痛机制

疹髓背角浅层是传递和调制外周伤害性信息的关键部位。起源于脑干的去甲肾上腺素（NA）能纤维终止脊髓背角,它们释放的NA具有抑制初级传入末梢释放谷氨酸和P物质、增加Ⅱ层（胶状质）抑制性神经活性物质释放的作用。此外,形态学研究提示NA可能直接抑制Ⅰ/Ⅲ层向丘脑传递伤害性信息的投射神经元。NA可能通过以上途径,实现对外周伤害性信息传递的调制而发挥镇痛作用。     

Short-term plasticity at primary afferent synapse in rat spinal dorsal horn and its biological function

Short-term plasticity (STP) is an important element of information processing in neuronal networks. As the first synaptic relay between primary afferent fibers (PAFs) and central neurons, primary afferent synapses in spinal dorsal horn (DH) are essential to the initial processing of somatosensory information. In this research, we examined the STP between Adelta-PAFs and spinal DH neurons by patch-clamp recording. Our results showed that depression dominated the STP at primary afferent synapses. The curves of STP had no significant changes in the presence of bicuculline, CTZ or AP-5. Lowering extracellular Ca(2+) concentration ([Ca(2+)](o)) from 2.4 to 0.8 mM reduced the depression of synaptic responses at all stimulus rates, while raising [Ca(2+)](o) from 2.4 to 4.0 mM increased the synaptic depression. Increasing the bath temperature from 24 to 32 degrees C clearly reduced the depression of all responses. These results indicate that the observed STP is of presynaptic origin and depends on transmitter release. By fitting the experimental data recorded under different conditions, a model of STP was used to quantitatively characterize the observed STP and to analyze the possible mechanisms underlying the effects of [Ca(2+)](o) and temperature. Furthermore, using a model neuron receiving synaptic inputs, we found that with this form of STP, postsynaptic DH neurons could detect rate changes in both rapidly- and slowly-firing afferents with equal sensitivity. The present study links the intrinsic STP properties of primary afferent synapses with their role in processing neural information, and provides a basis for further research on the STP in spinal DH and its biological function under in vivo conditions.     

可视膜片钳法记录初级传入纤维介导的脊髓背角神经元突触后电流

本文描述了用明胶半包埋法制备带背根脊髓薄片的实验步骤,和在脊髓背角记录由初级传入纤维介导的突触后电流的可视膜片钳法。手术制备一段带背根的脊髓标本,并用20％的明胶包埋在琼脂块上,再用振动切片机切片获得带背根的脊髓薄片。通过红外线可视的引导,在脊髓背角神经元上建立全细胞封接模式。在钳制电压为-70mV条件下,记录自发的和背根刺激引起的兴奋性突触后电流。以传入纤维的传导速度与刺激阈值为指标,可以区分A样纤维与C样纤维兴奋性突触后电流。在钳制电压为0mV条件下,记录自发的和背根刺激引起的抑制性突触后电流。用5μmol／L的士宁或20μmol／L的荷包牡丹碱分离出γ-氨基丁酸能或甘氨酸能的抑制性突触后电流。用可视膜片钳方法可以准确测量脊髓背角神经元的突触后电流,从而研究初级传入突触的传递过程。更重要的是,在红外线可视观察的帮助下,建立膜片钳封接的成功率显著提高,同时也使记录研究脊髓背角深层神经元变得更加容易。本研究为探索初级传入突触传递过程提供了一个有效的方法。     

Characterization of a thy1.2 GFP transgenic mouse reveals a tissue-specific organization of the spinal dorsal horn

In this study, transgenic mice in which membrane-linked enhanced green fluorescent protein (mGFP) is expressed from the Thy1.2 promoter were used. In these mice, a subpopulation of small to medium sized DRG neurons double stained for IB4 but not for CGRP. Most of the peripheral terminals traversed the dermis and ramify within the epidermis and form superficial terminals. Within the spinal cord, these afferents terminated exclusively within the substantia gelatinosa (SG). A second fibre type in the skin also expressed mGFP, and formed club-shaped endings towards the bases of hairs. Injury to the sciatic nerve resulted in mGFP loss from the SG ipsilateral to the nerve injury, but also in the corresponding region contralaterally. Together, these findings reveal the specificity of connectivity of a defined subpopulation of DRG sensory neurons innervating the epidermis and this will facilitate analysis of their physiological functions.     

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.     

Experimental morphological study of primary afferent terminals at the base of the dorsal horn of the cat spinal cord

The distribution and ultrastructure of primary afferent terminals in the gray matter of the cervical and lumbar regions of the cat spinal cord were studied by the experimental degeneration method of Fink and Heimer. Most preterminals of primary afferents were shown to be concentrated in the region of the intermediate nucleus of Cajal (central part of Rexed's laminae VI–VII), in the substantial gelatinosa (laminae II–III), and in the nucleus proprius of the dorsal horn (central and medial parts of lamina IV). Fewer are found in the region of the motor nuclei. The number of degenerating axon terminals in the lateral parts of laminae IV and V differed: 31.5 and 0.4% respectively of all axon terminals. Many terminals of primary afferents in lamina IV contribute to the formation of glomerular structures in which they exist as terminals of S-type forming axo-axonal connections with other terminals. These results are in agreement with electrophysiological data to show that interneurons in different parts of the base of the dorsal horn differ significantly in the relative numbers of synaptic inputs formed by peripheral afferents and descending systems.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 5, No. 4, pp. 406–414, July–August, 1973.     

Changes in the efficacy of transmission at synapses of the dorsal horn of the cat spinal cord observed during repetitive activation of cutaneous afferents

Inhibitory patterns in monosynaptic components of field potentials and potentials in the dorsal spinal cord surface were investigated during acute experiments on cats involving low-frequency stimulation of cutaneous peripheral nerves. Approximation of experimental data obtained from a theoretical plot was performed adopting a standard model of transmitter storage and release. Parameters of fractional release and replenishment of transmitter depleted from synaptic junctions were determined. Processes of replenishing supplies of transmitter for release were seen to intensify under rhythmic stimulation. A comparison between these experimental findings on the character of synapses of the monosynaptic reflex arc and others found in the literature indicated a similarity between the parameters of the mechanism underlying transmitter mobilization and release at different synaptic junctions formed by primary afferent fibers.Dnepropetrovsk State University Commemorating the 300th Aniversary of Ukraine-Russian Reunion, Dnepropetrovsk. Translated from Neirofiziologiya, Vol. 19, No. 4, pp. 491–497, July–August, 1987.     

Locally released small (non-protein) ninhydrin-reacting molecules underlie developmental differences of cultured medullary versus spinal dorsal horn neurons

Neurons located in the trigeminal subnucleus caudalis (Vc) play crucial roles in pain and sensorimotor functions in the orofacial region. Because of many anatomical and functional similarities with the spinal dorsal horn (SDH), Vc has been termed the medullary dorsal horn--analogous to the SDH. Here, we report that when compared with embryonic SDH neurons in culture, neurons isolated from the Vc region showed significantly slower growth, lower glutamate receptor activity, and more cells undergoing cell death. SDH neuron development was inhibited in co-cultures of SDH and Vc tissues while Vc neuron development was promoted by co-culture with SDH tissues. Furthermore, we identified that small (non-protein) ninhydrin-reacting molecules purified from either embryonic or post-natal Vc-conditioned medium inhibited neuronal growth whereas ninhydrin-reacting molecules from SDH-conditioned medium promoted neuronal growth. These findings suggest the involvement of locally released factors in the region-specific regulation of neuronal development in Vc and SDH, central nervous system regions playing critical roles in pain, and point to novel avenues for investigating central nervous system regionalization and for designing therapeutic approaches to manage neurodegenerative diseases and pain.     

Ultrastructure of orexin-1 receptor immunoreactivities in the spinal cord dorsal horn

The ultrastructural properties of orexin 1-receptor-like immunoreactive (OX1R-LI) neurons in the dorsal horn of the rat spinal cord were examined using light and electron microscopy techniques. At the light microscopy level, the most heavily immunostained OX1R-LI neurons were found in the ventral horn of the spinal cord, while some immunostained profiles, including nerve fibers and small neurons, were also found in the dorsal horn. At the electron microscopy level, OX1R-LI perikarya were identified containing numerous dense-cored vesicles which were more heavily immunostained than any other organelles. Similar vesicles were also found within the axon terminals of the OX1R-LI neurons. The perikarya and dendrites of some of the OX1R-LI neurons could be seen receiving synapses from immunonegative axon terminals. These synapses were found mostly asymmetric in shape. Occasionally, some OX1R-LI axon terminals were found making synapses on dendrites that were OX1R-LI in some cases and immunonegative in others. The synapses made by OX1R-LI axon terminals were found both asymmetric and symmetric in appearance. The results provide solid morphological evidence that OX1R is transported in the dense-cored vesicles from the perikarya to axon terminals and that OX1R-LI neurons in the dorsal horn of the spinal cord have complex synaptic relationships both with other OX1R-LI neurons as well as other neuron types.     

异丙酚对正常大鼠脊髓背角感觉神经元反应性的抑制作用

脊髓背角感觉神经元不仅在感觉信息的传递和调节中起到重要作用,也是各种内源性和外源性药物的作用靶位.为了解静脉麻醉剂异丙酚是否对背角感觉神经元的反应性具有调节作用,本实验采用在体单细胞胞外记录技术,观察了脊髓背表面直接滴注0.5 μmol异丙酚对戊巴比妥钠麻醉大鼠脊髓背角广动力域(WDR)神经元和低阈值机械感受型(LTM)神经元反应性的影响.实验发现,异丙酚能抑制背角WDR神经元由施加于外周感受野伤害性热刺激(45、47、49和53℃,15 s)和夹捏机械刺激(10 s)诱发的反应性,与DMSO对照组比较具有显著性统计学差异(P＜0.05);同样,异丙酚对非伤害性机械刺激诱发的WDR或LTM神经元的反应性也具有显著的抑制作用(P＜0.05).本结果提示,异丙酚可直接作用于正常大鼠脊髓背角神经元,对由非伤害性和伤害性纤维介导的神经元反应性均产生抑制作用,因此异丙酚的脊髓抗伤害作用可能不是特异性的.     

High-resolution detection of ATP release from single cultured mouse dorsal horn spinal cord glial cells and its modulation by noradrenaline

Purinergic Signalling - Human embryonic kidney 293 (HEK293) cells stably transfected with the rat P2X2 receptor subunit were preincubated with 200 nM progesterone (HEK293-P2X2-PROG), a...     

Immunohistochemical studies of peptidergic neurons in the dorsal horn of the spinal cord

The indirect immunofluorescence technique was used to localize substance P, somatostatin, methionine--enkephalin, neurotensin, and oxytocin in the dorsal horn of the rat spinal cord. The unique distribution of each peptide is described and the relative amount of each peptide in laminae I--III of the dorsal horn and the dorsal part of the lateral funniculus qualitatively assessed. Colchicine treatment and dorsal rhizotomy were used to determine, in part, the origin of immunoreactive fibers and terminals observed in the dorsal horn.     

MK-801抑制福尔马林实验引起的大鼠脊髓背角环氧合酶-2的表达

应用免疫组织化学方法,观察鞘内注射N-methyl—D—aspartate(NMDA)受体拮抗剂MK-801对福尔马林实验引起的大鼠脊髓背角环氧合酶-2(cyclooxygenase-2,COX-2)表达的影响。结果表明：MK-801对福尔马林实验引起的第1相缩足反射仅有一定抑制作用,但对第2相缩足反射有显著的抑制作用,且呈剂量依赖性。与这种行为学的变化相对应,MK-801可显著抑制福尔马林实验引起的脊髓背角COX-2表达的增加,并且这种抑制作用与MK-801的剂量呈正相关。这些结果表明,在福尔马林实验中,NMDA受体的活动是引起脊髓背角COX-2表达增加的原因之一。     

Cyclic AMP-mediated long-term facilitation of glycinergic transmission in developing spinal dorsal horn neurons

cAMP is known to regulate neurotransmitter release via protein kinase A (PKA)-dependent and/or PKA-independent signal transduction pathways at a variety of central synapses. Here we report the cAMP-mediated long-lasting enhancement of glycinergic transmission in developing rat spinal substantia gelatinosa neurons. Forskolin, an adenylyl cyclase activator, elicited a long-lasting increase in the amplitude of nerve-evoked glycinergic inhibitory postsynaptic currents (IPSCs), accompanied by a long-lasting decrease in the paired-pulse ratio in immature substantia gelatinosa neurons, and this forskolin-induced increase in glycinergic IPSCs decreased with postnatal development. Forskolin also decreased the failure rate of glycinergic IPSCs evoked by minimal stimulation, and increased the frequency of glycinergic miniature IPSCs. All of these data suggest that forskolin induces the long-lasting enhancement of glycinergic transmission by increasing in the presynaptic release probability. This pre-synaptic action of forskolin was mediated by hyperpolarization and cyclic nucleotide-activated cation channels and an increase in intraterminal Ca²⁺ concentration but independent of PKA. The present results suggest that cAMP-dependent signal transduction pathways represent a dynamic mechanism by which glycinergic IPSCs could potentially be modulated during postnatal development.