The acute and chronic effects of capsaicin on slow excitatory transmission in rat dorsal horn

The acute and chronic effects of capsaicin on rat spinal dorsal horn neurons and the excitatory transmission in the dorsal horn were investigated by means of intracellular recording techniques in the spinal cord slice preparation. Bath application of capsaicin (1–2 × 10⁻⁵M) produced in a majority of cells a prolonged depolarization associated with an increase in synaptic activity and intense neuronal discharges. During and immediately following the capsaicin depolarization, repetitive stimulation of a dorsal root failed to elicit the slow depolarization.

After neonatal capsaicin treatment the proportion of dorsal horn neurons exhibiting the slow excitatory transmission was markedly reduced, however, the fast excitatory postsynaptic potentials were present in all examined cells. In addition, the proportion and sensitivity of the cells responding with a slow depolarization to substance P increased.     

Slow excitatory transmission in rat dorsal horn: possible mediation by peptides

Repetitive stimulation of a dorsal root elicited a slow depolarization in about half of the dorsal horn neurons examined in the rat spinal cord slice preparation. The response was markedly depressed or abolished in the presence of substance P, substance P antagonists and capsaicin. In some dorsal horn neurons a slow hyperpolarization was also observed.     

Interstitial potassium concentration, slow depolarization and focal potential responses in the dorsal horn of the rat spinal slice

Extracellular potassium concentration ([K⁺]_o) was measured, and intra- and extracellular recordings made, in the dorsal horn of rat spinal cord slices maintained in vitro during repetitive dorsal root stimulation. In about half of the dorsal horn neurons, the stimulation evoked a possibly substance P-mediated slow depolarization. [K⁺]_o increased during stimulation, reaching its highest values approximately 150 μm from the dorsal surface. The time course of Δ[K⁺]_o was different from that of the slow depolarization. Substance P itself evoked a much smaller Δ[K⁺]_o (0.4 mM) in the dorsal horn. It is concluded that the slow depolarization is not mediated by elevated [K⁺]_o.     

Cholecystokinin octapeptide excites dorsal horn neurons both in vivo and in vitro

Cholecystokinin octapeptide (CCK-8) applied microiontophoretically causes a moderate to strong excitation of about half of all tested dorsal horn neurons located in laminae I-VII of both the cat intact spinal cord and the rat in vitro spinal cord slice preparation. In the cat intact spinal cord the excitation is not limited to a single population of neurons but is observed in all categories of units recognized in spinal preparations of cats in this area on the basis of their excitability by different kinds of cutaneous afferent input. In the spinal cord slice preparation the excitatory action of CCK-8 persists even when the spinal cord slices are perfused with a Ca2+-free, Mg2+-high Krebs solution. The latter finding indicates that the action of CCK-8 might be a direct one exerted on the postsynaptic sites of dorsal horn units. These results are consistent with the possibility that CCK-8 acts on postsynaptic sites in the dorsal horn of the spinal cord as a neurotransmitter or modulator.     

The actions of neuropeptides on dorsal horn neurons in the rat spinal cord slice preparation: an intracellular study

Responses of dorsal horn neurons to bath application of substance P, somatostatin and enkephalin were studied by intracellular recording in the neonatal spinal cord slice preparation. Substance P depolarized dorsal horn neurons and increased their excitability. The depolarization was most commonly associated with an increase in neuronal input resistance. Somatostatin and enkephalin hyperpolarized dorsal horn neurons and caused reduction or abolition of spontaneous firing. While the hyperpolarization produced by enkephalin was always associated with a fall in neuronal input resistance, in the case of somatostatin the similar effect was less consistently observed.     

Effects of polyclonal and monoclonal antibodies to substance P on slow excitatory transmission in rat spinal dorsal horn

The effects of bath perfusion of polyclonal and monoclonal antibodies to substance P (SP) on slow excitatory transmission in rat dorsal horn have been investigated by intracellular recording in the immature rat spinal cord slice preparation. Both polyclonal and monoclonal antibodies to SP produced a significant decrease in the amplitude and the duration of the slow depolarization generated in dorsal horn neurons by high intensity, repetitive dorsal root stimulation or exogenous SP application. The effect of endogenous SP, or SP-related peptide, released during dorsal root stimulation appears likely since bath perfusion of a slice with a normal rabbit serum, or affinity chromatography preadsorbed SP antiserum, or non-specific IgG, or 5-hydroxytryptamine antiserum had no similar depressant effect. These results, if taken together with other experimental evidence, suggest that SP, or SP-like peptide, is in some way involved in a generation of the dorsal root-evoked slow depolarization. In addition, a novel approach is presented for using polyclonal and monoclonal antibodies to SP as pharmacological antagonists. Use of a specific characterized monoclonal antibody for the detection of physiological and pharmacological effects of putative peptide transmitters in vitro opens new avenues for further investigations.     

Vasoactive intestinal polypeptide excites mammalian dorsal horn neurons both in vivo and in vitro

Vasoactive intestinal polypeptide (VIP) applied by iontophoresis and/or pressure microinjection causes a strong excitation of more than 75% of all tested spinal neurons in laminae I–VII of both the cat intact spinal cord and the rat spinal cord slice preparation. In the cat intact spinal cord the excitation is not limited to a single population of neurons but is observed in all cagegories of units recognized in spinal preparations of cats in this area on the basis of their excitability by different kinds of cutaneous afferent input. In the rat spinal cord slice preparation, VIP depolarized dorsal horn neurons and increased their excitability. The depolarization was associated wtih a decrease in neuronal input resistance. These results are consistent with the possibility that VIP may have a physiological role in synaptic function, either as a transmitter or as a modulator.     

Increase in substance P in the dorsal horn during a chemogenic nociceptive stimulus

Substance P has been implicated as a neuromediator of nociception in the dorsal horn of the spinal cord. This is the first report of quantitative increases in substance P following an acute nociceptive stimulus. Female Wistar rats were injected subcutaneously in the plantar aspect of the right hindpaw with 5% formalin. Substance P levels were determined by quantitative immunohistochemistry 1 h after the injection. The present results show that substance P levels in the upper two laminae of the ipsi-and contralateral dorsal horn were significantly increased 1 h after the formalin injection. These results suggest a direct functional relationship between substance P levels in the upper dorsal horn and an acute chemogenic nociceptive stimulus.     

Morphine does not antagonize the substance P mediated excitation of dorsal horn neurons

Multibarrelled microelectrodes were used to test the effects of iontophoretically released substance P (SP), morphine, glutamate, and naloxone on spinal cord dorsal horn neurons. Cells excited by SP were also excited by noxious stimuli, a finding consistent with the hypothesis that SP is the neurotransmitter released by primary nociceptor afferents to excite dorsal horn neurons. Iontophoretic morphine failed to depress the SP-induced discharges. Indeed, iontophoretic morphine frequently potentiated the SP responses. In addition to potentiating SP-induced discharges, iontophoretic morphine frequently increased both the spontaneous activity of dorsal horn neurons and the activity evoked in these cells by noxious cutaneous heat and iontophoretic glutamate. Naloxone did not antagonize these excitatory effects. Intravenous morphine only depressed spontaneous discharges. Nevertheless, iontophoretic morphine still produced excitatory effects in spinal animals pretreated with analgesic doses of intravenous morphine. It is concluded that such excitatory effects are toxic actions indicative of supratherapeutic morphine concentrations in the vicinity of the neuron being studied. Intravenously administered morphine depressed the spontaneous activity of dorsal horn neurons of spinal cats, but failed to depress their responses to SP. Morphine also failed to antagonize SP's biological effects in peripheral systems (contraction of isolated guinea pig ileum, rabbit hypotensive effect, rat sialogogic response). It is concluded that morphine is not a substance P receptor antagonist. The results are discussed with respect to the hypotheses that (1) the spinal analgesic effects of systemically administered morphine occur on presynaptic terminals of sensory neurons, and (2) an SP antagonist might be a unique analgesic agent.     

Ultrastructure of chemically defined neuron systems in the dorsal horn of the monkey. I. Substance P immunoreactivity

The ultrastructural localization of substance P-like immunoreactivity (SPLI) in lamina I (marginal zone) and lamina II₀ (outer substantia gelatinosa) of the dorsal horn of the macaque monkey was examined by the indirect antibody peroxidase-antiperoxidase method. SPLI was found in small unmyelinated and finely myelinated axons and a variety of terminal types. The majority of SPLI terminals contained a few to many large granular vesicles (mean diameter 90 nm) in addition to a population of small clear round vesicles. A very few terminals contained mainly small round vesicles. SPLI terminals were presynaptic in axosomatic, axodendritic and axospinous contacts forming, in all but the axosomatic junctions, asymmetrical synapses. Some axosomatic junctions were symmetrical. SPLI terminals also formed the center of glomeruli with unlabeled dendrites and dendritic spines; some of the unlabeled dendrites contained a few small scattered vesicles and large dense-core vesicles. In more complex formations 2 to 4 SPLI terminals were associated with one another and linked by desmosomal contacts. The individual terminals in the complexes or ‘congregate terminals’ were simple large granular vesicle containing terminals (LGV), LGV-central terminals of associated glomeruli, or terminals containing mainly small round vesicles. In the apical region of lamina I an unlabeled terminal was found occasionally in contact with an SPLI terminal, which in turn synapsed onto a dendrite. These contacts have some synaptic characteristics and the SPLI terminal was possibly postsynaptic. Most of the types of SPLI terminals resemble closely terminal types shown to be of primary afferent origin. These terminals which make direct contact with dorsal horn dendrites may be the morphological substrate for postsynaptic excitation of dorsal horn neurons by substance P. The contacts of unlabeled terminals with SPLI terminals may represent a morphological substrate by which other neurochemical substances such as enkephalin or serotonin may modulate the substance P effects on dorsal horn neuronal activity.     

Evidence that substance P and somatostatin transmit separate information related to pain in the spinal dorsal horn

Effects of various types of natural skin stimuli on the in situ release of immunoreactive substance P and somatostatin from the rabbit dorsal horn were examined. Noxious mechanical or thermal stimuli specifically increased the release of immunoreactive substance P or somatostatin, respectively. Innocuous stimuli did not affect the release of these peptides. These results suggest that the nociceptive mechanical or thermal primary afferents contain substance P or somatostatin, respectively.     

Altered responsiveness to substance P and 5-hydroxytryptamine in cat dorsal horn neurons after 5-HT depletion with p-chlorophenylalanine

The responsiveness of functionally identified cat spinal dorsal horn neurons to iontophoretically applied substance P (SP) and 5-hydroxytryptamine (5-HT) has been investigated by means of extracellular recording after 5-HT depletion with p-chlorophenylalanine (p-CPA). In addition, the spinal levels of 5-HT, SP, cholecystokinin octapeptide, neurotensin, and vasoactive intestinal polypeptide have been measured in intact and p-CPA-pretreated cats. In the present study we have demonstrated an altered responsiveness of dorsal horn neurons to locally applied SP and 5-HT. We found in p-CPA-pretreated cats that the proportion of neurons responding with excitation to SP and 5-HT was significantly increased. At the same time, depression induced by 5-HT in the dorsal horn cells was virtually absent in p-CPA-pretreated animals. Our finding that spinal level of 5-HT was significantly decreased in p-CPA-treated animals is consistent with previous studies. No convincing alteration in the spinal levels of 4 analyzed peptides was found in p-CPA-treated animals. The present study has shown that pharmacological depletion of 5-HT has two major effects: (1) it increases significantly the proportion of dorsal horn neurons excited by SP and 5-HT; and (2) it is ineffective in inducing 5-HT supersensitivity. Further work is needed to explain mechanisms involved in these effects.     

Peripheral noxious stimulation induces phosphorylation of the NMDA receptor NR1 subunit at the PKC-dependent site, serine-896, in spinal cord dorsal horn neurons

The N‐methyl‐D ‐aspartate receptor (NMDAR) contributes to central sensitization in the spinal cord and the generation of pain hypersensitivity. NMDAR function is modulated by post‐translational modifications including phosphorylation, and this is proposed to underlie its involvement in the production of pain hypersensitivity in the spinal cord. We now show that a noxious heat stimulus applied to the rat hindpaw induces phosphorylation of the NMDAR NR1 subunit at a protein kinase C (PKC)‐dependent site, serine‐896, in superficial dorsal horn neurons. Phosphorylation of NR1 serine‐896 is essentially absent in the superficial dorsal horn laminae of naïve rats, but there is rapid (< 2 min) induction following a noxious but not innocuous heat stimulus. The number of pNR1‐immunoreactive neuronal profiles in the superficial dorsal horn peaks 30 min after noxious heat stimulation and persists for up to 1 h. pNR1serine896 induction occurs in the endoplasmic reticulum, suggesting that it contributes to trafficking of the receptor from intracellular stores to the membrane. The phosphorylation of the subunit is attenuated by intrathecal injection of the NMDAR antagonist, MK801, suggesting that the NMDAR is involved via a feed‐forward mechanism in its own phosphorylation. The pNR1serine896‐positive neurons are highly co‐localized with PKCdelta and only rarely with PKCgamma. These data provide evidence for an activity‐dependent NMDAR phosphorylation at the PKC‐dependent site, serine‐896, in spinal cord dorsal horn neurons initiated by peripheral noxious stimuli.     

Selective excitation of neurons in the mammalian spinal dorsal horn by aspartate and glutamate in vitro: correlation with location and excitatory input

The electrical activity of mammalian dorsal horn neurons was recorded with pipette microelectrodes in an in vitro spinal cord slice preparation with dorsal roots intact. Addition of relatively low concentrations of aspartate or glutamate to the superfusion solution or through the recording pipette with small iontophoretic currents excited only a subset of neurons. The majority of these excited neurons were located in the superficial dorsal horn (Rexed's laminae I and II) and a preponderance were excited by the C-fiber components of dorsal root volleys. These findings are consistent with the idea that aspartate or glutamate may function as a synaptic mediator for some neurons terminating in the superficial dorsal horn.     

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.     

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.     

Inhibition of nociceptive evoked activity in spinal neurons through a dorsal column-brainstem-spinal loop

In decerebrate-decerebellate cats, dorsal column stimulation (DCst), rostral to bilateral dorsal column cuts, inhibited dorsal horn neurons discharging to various types of nociceptive stimuli. Similar inhibitory effects were observed from conditioning nucleus raphe magnus stimulation. Activation of this dorsal column-brainstem-spinal loop could be part of an important supraspinal "gating' system to account for the alleviation of pain both by DCst and peripheral nerve stimulation in man.     

PKC在角叉菜胶致炎引起的脊髓背角神经元敏感化中的作用

组织损伤或炎症引起的脊髓背角神经元兴奋性的变化同痛觉过敏、痛觉超敏、自发痛等病理过程密切相关。本实验大鼠足底注射角叉菜胶后 ,脊髓背角神经元发生敏感化 ,自发放电及对伤害性刺激的诱发反应明显增强。背角局部经微透析给予蛋白激酶 C(PKC)非特异性抑制剂氯丙嗪 (CPZ)或特异性抑制剂 H- 7后 ,自发及诱发反应均明显回降 ,提示 PKC激活参与中枢敏感化的形成与维持