Intrinsic optical signals in the dorsal horn of rat spinal cord slices elicited by brief repetitive stimulation

With repetitive electrical stimulation of the dorsal root (20 Hz for 1 s at C-fibre strength), intrinsic optical signals (IOSs), measured as changes in light transmittance, were recorded in the superficial dorsal horn of rat spinal cord slices using a photodiode array imaging device. The mechanism underlying the induction of IOSs was investigated. IOSs elicited by brief repetitive stimulation persisted for 1-2 min and were decreased by reducing external Cl- concentration or by cation-chloride cotransport inhibitors. Furosemide was most effective whilst bumetanide was least effective among the inhibitors tested. A 1-min elevation of external K+ concentration evoked IOSs in the dorsal horn in the absence of stimulation, and K+-induced IOSs were inhibited by furosemide. These results suggest that the uptake of excess K+ via the furosemide-sensitive, cation-chloride cotransporters underlies the induction of the IOSs. One-minute exposure to hypotonic solutions, which would cause cell swelling, induced IOSs in the superficial dorsal horn. Whilst osmotic-induced IOSs were not affected by furosemide, they were inhibited by HgCl2 in a 2-mercaptoethanol-sensitive manner. The stimulation-induced IOSs were similarly depressed by HgCl2. In contrast, voltage-sensitive dye signals and field potentials, evoked by single electrical stimuli, were significantly less affected by HgCl2. These results suggest that there is a specialized water transport pathway in the superficial dorsal horn, and that IOSs elicited by brief repetitive activation of C-fibres are attributable to cell swelling caused by water influx through this pathway, as an osmotic gradient is established by the uptake of K+ via the furosemide-sensitive cotransporters.     

Adenosine exerts multiple effects in dorsal horn neurones of the adult rat spinal cord

In the present study, we have examined the effects of adenosine and its analogues on the electrophysiological properties of dorsal horn neurones in the rat adult spinal cord. Adenosine and the A₁ receptor agonist R-phenylisopropyl adenosine (RPIA) reversibly hyperpolarised these neurones via the generation of an outward current at −60 mV that was inhibited by pre-application of barium or Rp-adenosine 3′, 5′-cyclic monophosphothioate triethylamine. In contrast, the A_2a receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680) had no effect on the resting membrane properties of these neurones. Stimulation of the dorsal root evoked non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) at −60 mV that were completely abolished by 2,3-dihydroxy-6-nitro-7-sulophamoyl-benzo(F)quinoxalone (NBQX). Bath application of adenosine or RPIA reversibly inhibited these EPSCs in a concentration-dependent manner via a presynaptic action. In contrast, CGS21680 increased the amplitude of the EPSC in 20% of neurones tested and decreased the EPSC amplitude in 30% of neurones tested. It is concluded that adenosine exerts multiple effects upon the electrophysiological properties of dorsal horn neurones in the adult spinal cord via interaction with multiple receptors. These findings have important implications in the understanding of adenosine action in preclinical models of pain.     

The selective effects of metorphamide on dorsal horn neurones of the cat spinal cord

In anaesthetized cats, the proenkephalin A derivative metorphamide was administered microelectrophoretically while recording the excitation of lumbar dorsal horn neurones by noxious and innocuous cutaneous stimuli. Administered in the substantia gelatinosa or more ventrally near the cell bodies, metorphamide reduced the nociceptive responses and spontaneous firing of laminae IV and V neurones. These effects were reversed by electrophoretic administration of naloxone at the same site. Responses to innocuous stimulation were partially but inconsistently reduced by metorphamide administration near cell bodies, and not reduced by administration in the substantia gelatinosa. These results suggest that metorphamide may be an endogenous ligand at opioid μ-receptors, and may also act at κ-receptors.     

GABA decreases in the spinal cord dorsal horn after peripheral neurectomy

A significant fall in the number of GABA-immunoreactive cells in laminae I–III of the rat spinal cord occured in the somatotopic area of projection of the sciatic nerve after nerve transection. The decrease started at 2 weeks post-neurectomy, and at 4 weeks ipsilateral mean cell numbers were approximately 72% of contralateral control values. Similarly, the concentration of GABA in spinal homogenates was significantly reduced 4 weeks post-neurectomy. These data, together with our recent finding of an increase in spinal GABA during chronic inflammation of the hindlimb, suggest that the level of GABA in the dorsal horn is regulated by the amount of primary afferent input.     

Strychnine sensitive inhibition in the dorsal horn of mammalian spinal cord

Using an in-vitro mammalian spinal cord preparation 3 pharmacologically distinct phases of inhibition have been demonstrated in the dorsal horn. An early picrotoxin-sensitive inhibition was followed by an intermediate strychnine-sensitive phase and a late phase resistant to both strychnine and picrotoxin. The picrotoxin-sensitive inhibition was accompanied by an increase in excitability of the afferent fibres which was prolonged by strychnine, suggesting the presence of glycine mediated inhibition of GABA primary afferent depolarization.     

Microdialysis recovery of serotonin released in spinal cord dorsal horn

Methods for making and using hollow microdialysis fibers suitable for recovering extracellular substances from discrete regions of the spinal cord are described. After placement of the fiber, artificial cerebrospinal fluid was pushed through it at a low (4-5 microliters/min) rate. The perfusate was collected and samples analyzed on a high performance liquid chromatograph with an electrochemical detector. Serotonin, 5-hydroxyindole acetic acid and norepinephrine were recovered and identified. Single unit extracellular recordings were made during the perfusion and collection; thus simultaneous observation of neurotransmitter release and modulation of single cell activity is now possible.     

Optical responses evoked by single-pulse stimulation to the dorsal root in the rat spinal dorsal horn in slice

Neuronal excitation evoked after dorsal-root (DR) stimulation in the spinal dorsal horn (DH) of rats was visualized with a high-resolution optical-imaging method, and the propagation mechanism was studied. Transverse slices of the spinal cord were obtained from 2–4 week-old rats and stained with the voltage-sensitive dye RH-482. Single-pulse stimulation to the primary-afferent A fibers in the DR attached to the slice evoked a weak, brief (<10 ms) excitatory optical response in the laminae I and III–V. When the stimulus intensity and duration were increased to activate both A and C fibers, an additional, much greater, and longer-lasting (>100 ms) excitatory response was generated in the laminae I–III, most intensely in the lamina II. A treatment with excitatory amino acid (EAA) antagonists, -2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, significantly reduced the amplitude and duration of the response in the lamina II. The optical response in the antagonists-containing solution was quite similar to that recorded in a Ca²⁺-free solution that blocked afferent synaptic transmission. The late component (>10 ms) was, however, slightly greater than that in the Ca²⁺-free solution. Treatment with the ATP-receptor antagonist, suramin, had a minimal effect on the response in the presence of EAA antagonists. These results suggested that the propagation of the DR-stimulus-elicited excitation was contributed largely by EAA receptors, but also by other receptors to a much lesser extent.     

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

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

Nicotinic modulation of GABAergic synaptic transmission in the spinal cord dorsal horn

While the mechanisms underlying nicotinic acetylcholine receptor (nAChR)-mediated analgesia remain unresolved, one process that is almost certainly involved is the recently-described nicotinic enhancement of inhibitory synaptic transmission in the spinal cord dorsal horn. Despite these observations, the prototypical nicotinic analgesic (epibatidine) has not yet been shown to modulate inhibitory transmission in the spinal cord. Furthermore, while nAChRs have been implicated in short-term modulation, no studies have investigated the role of nAChRs in the modulation of long-term synaptic plasticity of inhibitory transmission in dorsal horn. Whole-cell patch clamp recordings from dorsal horn neurons of neonatal rat spinal cord slices were therefore conducted to investigate the short- and long-term effects of nicotinic agonists on GABAergic transmission. GABAergic synaptic transmission was enhanced in 86% of neurons during applications of 1 microM nicotine (mean increased spontaneous GABAergic inhibitory postsynaptic current (sIPSC) frequency was approximately 500% of baseline). Epibatidine (100 nM) induced an increase to an average of approximately 3000% of baseline, and this effect was concentration dependent (EC50=43 nM). Nicotinic enhancement was inhibited by mecamylamine and DHbetaE, suggesting an important role for non-alpha7 nAChRs. Tetrodotoxin (TTX) did not alter the prevalence or magnitude of the effect of nicotine, but the responses had a shorter duration. Nicotine did not alter evoked GABAergic IPSC amplitude, yet the long-term depression (LTD) induced by strong stimulation of inhibitory inputs was reduced when paired with nicotine. These results provide support for a mechanism of nicotinic analgesia dependent on both short and long-term modulation of GABAergic synaptic transmission in the spinal cord dorsal horn.     

Paraventricular hypothalamic oxytocinergic cells responding to noxious stimulation and projecting to the spinal dorsal horn represent a homeostatic analgesic mechanism

The participation of the hypothalamic paraventricular nucleus (PVN) in an endogenous central mechanism of analgesia has been observed using rats in various experimental procedures including electrophysiological and behavioral tests. However, little is known about the PVN neuronal responses to noxious stimulation. The only data available indicate a c-fos increase after noxious visceral stimulations. Our electrophysiological recordings of single PVN cells showed that, out of 223 cells, 79 responded to noxious mechanical and/or thermal stimuli, and another 10 responsive cells were found in the Reuniers thalamic nucleus. These cells responded only to noxious stimuli mainly in the ipsilateral hind limb but we also observed cells responding to stimulation of both hind limbs and also the tail. Mechanical stimulation was most effective but some cells could respond to both mechanical and thermal noxious stimuli. Some of the responding PVN cells were identified by antidromic stimulation in the ipsilateral lumbar dorsal horn spinal cord. Finally, in order to document the nature of the neurotransmitter and the projection to the spinal cord of the PVN cells that responded to noxious stimulation, we used a juxtacellular approach to record and stain some neurons and found them to be oxytocinergic by immunofluorescence procedures. The PVN cells activated by noxious stimuli may suppress the peripheral incoming afferent A-delta and C fibers, completing a circuit involved in diffuse endogenous analgesia. This mechanism strongly suggests that the PVN participates in a homeostatic mechanism involved in pain and analgesia.     

Pulsed electrical stimulation protects neurons in the dorsal root and anterior horn of the spinal cord after peripheral nerve injury

Most studies on peripheral nerve injury have focused on repair at the site of injury, but very few have examined the effects of repair strategies on the more proximal neuronal cell bodies. In this study, an approximately 10-mm-long nerve segment from the ischial tuberosity in the rat was transected and its proximal and distal ends were inverted and sutured. The spinal cord was subjected to pulsed electrical stimulation at T10 and L3, at a current of 6.5 m A and a stimulation frequency of 15 Hz, 15 minutes per session, twice a day for 56 days. After pulsed electrical stimulation, the number of neurons in the dorsal root ganglion and anterior horn was increased in rats with sciatic nerve injury. The number of myelinated nerve fibers was increased in the sciatic nerve. The ultrastructure of neurons in the dorsal root ganglion and spinal cord was noticeably improved. Conduction velocity of the sciatic nerve was also increased. These results show that pulsed electrical stimulation protects sensory neurons in the dorsal root ganglia as well as motor neurons in the anterior horn of the spinal cord after peripheral nerve injury, and that it promotes the regeneration of peripheral nerve fibers.     

A-fiber sprouting in spinal cord dorsal horn is attenuated by proximal nerve stump encapsulation

Peripheral nerve transection in the rat alters the spinal cord dorsal horn central projections from both small and large DRG neurons. Injured neurons with C-fibers exhibit transganglionic degeneration of their terminations within lamina II of the spinal cord dorsal horn, while peripheral nerve injury of medium to large neurons induces collateral sprouting of myelinated A-fibers from lamina I and III/IV into lamina II in rats, cats, and primates. To date, it is not known what sequelae are responsible for the collateral sprouting of A-fibers after peripheral nerve injury, although target-derived factors are thought to play an important role. To determine whether target-derived factors are necessary for changes in A-fiber laminar terminations in rat spinal cord dorsal horn, we unilaterally transected the sciatic nerve and ensheathed the proximal nerve stump in a silicone cap. Three days before sacrifice of rat, the injured sciatic nerve was injected with cholera toxin beta-subunit conjugated to horseradish peroxidase (betaHRP) that effectively labels both peripheral and central A-fiber axons. The effect of the ligature, axotomy, and silicone cap treatment was evaluated by analyzing the extent of betaHRP-, Substance P-(SP-), and isolectin B4- (IB4-) immunoreactive (ir) fibers in the somatotopically appropriate spinal cord dorsal horn regions. In all animals, 2-5 weeks after nerve transection (treated or otherwise), IB4- and SP-ir is absent from lamina II. Animals without nerve cap treatment exhibited robust fiber sprouting into lamina II at 2 weeks. In sharp contrast, animals treated with silicone caps did not exhibit betaHRP-ir fibers in lamina II at 2 weeks. This observation was extended up to 5 weeks postinjury. These results suggest that axotomy-induced expansion of betaHRP-ir primary afferent central terminations in the spinal cord dorsal horn is dependent on factors produced in the injury site milieu. While our understanding of local repair mechanisms of injured peripheral nerves is incomplete, it is clear that the time-dependent production of growth factors in the nerve injury microenvironment favor nerve fiber outgrowth, both peripherally and centrally.     

The contralateral input to the dorsal horn of the spinal cord in the decerebrate spinal rat

Input from the contralateral limb and tail was examined in the lumbar dorsal horn of decerebrate spinal rats. Fifty-three cells were recorded from laminae 4, 5 and 6 and classified according to their ipsilateral response to natural and electrical stimulation. Twenty-nine (54%) of these cells were found to have inhibitory contralateral fields. This inhibition was evoked by noxious pinching or heating of the skin. In most cases the inhibitory field was a mirror image of the excitatory ipsilateral field although it also often included the tail. Activity evoked by natural and electrical stimulation as well as spontaneous activity was inhibited by contralateral skin stimulation. Noxious specific and wide dynamic range cells displayed these fields but low threshold mechanoreceptive cells did not. Twenty-six cells (49%) received direct short-latency excitatory input from the contralateral sciatic nerve; this correlated well with the presence of contralateral fields. Trains of stimuli applied to the contralateral sciatic nerve at Aδ- and C-fibre strength resulted in inhibition of the cell whereas trains of Aβ strength had no effect. The results demonstrate the existence of segmental contralateral control over dorsal horn cell activity, not involving supraspinal pathways.     

关节炎大鼠脊髓背角内脑源性神经营养因子和促肾上腺皮质激素释放因子表达增加(英文)

研究表明 ,外周感染可导致脑源性神经营养因子 (BDNF)的过度表达 ,BDNF可影响其他神经递质的合成。采用免疫组化和原位杂交的方法观察了完全福氏佐剂所致的关节炎大鼠脊髓内 BDNF及其功能性受体酪氨酸蛋白激酶 B(trk B)的表达和促肾上腺皮质激素释放因子 (CRF) m RNA的水平。实验发现 ,在皮下注射完全福氏佐剂后 4h,脊髓腰膨大同侧背角中的 BDNF免疫活性神经元和 CRF m RNA阳性神经元数升高 ,在 2 4 h时达到峰值 ,在 7d时仍维持在较高水平。实验提示 ,关节炎大鼠脊髓内的 BDNF和 CRF可能参与了慢性痛反应。     

关节炎大鼠脊髓背角内脑源性神经营养因子和促肾上腺皮质激素释放因子表达增加

研究表明,外周感染可导致脑源性神经营养因子(BDNF)的过度表达,BDNF可影响其他神经递质的合成.采用免疫组化和原位杂交的方法观察了完全福氏佐剂所致的关节炎大鼠脊髓内BDNF及其功能性受体酪氨酸蛋白激酶B(trkB)的表达和促肾上腺皮质激素释放因子(CRF)mRNA的水平.实验发现,在皮下注射完全福氏佐剂后4 h,脊髓腰膨大同侧背角中的BDNF免疫活性神经元和CRF mRNA阳性神经元数升高,在24 h时达到峰值,在7 d时仍维持在较高水平.实验提示,关节炎大鼠脊髓内的BDNF和CRF可能参与了慢性痛反应.     

Neonatal rat spinal cord slice preparation: postsynaptic effects of neuropeptides on dorsal horn neurons

In the neonatal rat spinal cord slice preparation responses of the dorsal horn interneurons to iontophoretic or bath application of methionine-enkephalin (ME), substance P (SP) and somatostatin (SS) were qualitatively similar to those obtained in intact spinal cord. Thus, SP powerfully excited almost all neurons tested (15/16), while ME and SS depressed neuronal discharges in 13/14 and 4/6 units respectively. In some dorsal horn neurons the iontophoretic application of ME caused a marked depression of the SP-induced excitation. Angiotensin II (AgII) had no effect on dorsal horn units (n = 8). In the slices perfused with a Ca2+-free, Mg2+-high Krebs solution the extracellularly recorded effects of ME, SP and SS were not significantly modified, suggesting that the peptides were acting directly on postsynaptic sites. The results also indicate that the in vitro rat spinal cord slice preparation can be successfully utilized for further studies on the cellular mechanisms of actions of neuropeptides, particularly in relation to synaptic transmission processes in the dorsal horn.     

Regulation of flunitrazepam binding in the dorsal horn of the spinal cord by adrenalectomy and corticosteroids.

Adrenal corticosteroids and adrenalectomy (ADX) have opposing effects on benzodiazepine binding sites in brain regions. These treatments were employed to study [3H]flunitrazepam (FLU) binding in regions punched out from the rat spinal cord. We found that binding was higher in dorsal horn than in ventral horn, and minimal in white matter. Clonazepam and RO 15-1788 largely displaced [3H]FLU binding, whereas RO 5-4864 was weakly active. Four days post-ADX, binding increased exclusively in the dorsal horn, and this effect was reversed by administration of corticosterone (CORT), but not dexamethasone (DEX) or aldosterone (ALDO) given over 4 days. When endogenous CORT was increased by administration of cold stress to adrenal-intact rats, reduced benzodiazepine (BDZ) binding was also observed in the dorsal horn. When added in vitro, only ALDO and not CORT or DEX, inhibited [3H]FLU binding. It is suggested that steroids with affinity for the type I corticosteroid receptor (CORT, ALDO) decrease [3H]FLU binding to a neural-type BDZ receptor in the dorsal horn. Reduction of the inhibitory BDZ system may be physiologically important, and can partly explain the enhancement of excitatory synaptic transmission produced by corticosteroids at the level of the spinal cord.     

Synaptic and non-synaptic components of the dorsal horn potential in isolated hamster spinal cord

Slow negative potentials, evoked by stimulation of the lumbar dorsal roots, have been demonstrated in the dorsal horn of an isolated, hemisected spinal cord preparation from golden hamsters. Paired stimuli revealed a period of partial suppression of this slow potential persisting for up to 2 s following the conditioning stimulus, but with high stimulation frequencies this effect was masked and above 20 Hz a tetanic train of stimuli produced a smoothly rising potential. The response evoked by tetanic stimulation was shown to consist of two components, a manganese-sensitive, synaptically generated component, and a manganese-resistant, frequency-dependent element. Treatment with 10⁻⁴ M 4-aminopyridine blocked the manganese-resistant tetanic response but did not reduce the manganese-sensitive component. Bicuculline, picrotoxin and tubocurare had little effect upon the tetanic response, but 10⁻³ M procaine blocked it completely. The possibility that the manganese-resistant response was due to the release of potassium ions is considered.     

Multiple effects of phorbol esters in the rat spinal dorsal horn

Spinal cord slice preparation and intracellular recording techniques were used to examine the effects of phorbol esters on the sodium- and calcium-dependent action potentials, the excitatory synaptic transmission, the basal (resting) and the dorsal root stimulation-evoked release of 9 endogenous amino acids, including glutamate and aspartate, and the responsiveness of the rat dorsal horn neurons to excitatory amino acids (glutamic, kainic, quisqualic, and N-methyl-D-aspartic). 4-beta-Phorbol-12, 13-dibutyrate and 4-beta-phorbol-12, 13-diacetate produced minor alterations in membrane potential and resistance, but they broadened the sodium-dependent action potential and reduced the duration of the calcium-dependent action potential. In addition, phorbol esters caused a marked and long-lasting increase in the amplitude and the duration of excitatory postsynaptic potentials (EPSPs) evoked in dorsal horn neurons by orthodromic stimulation of a lumbar dorsal root. Phorbol esters produced a brief increase in the basal and electrically evoked release of endogenous excitatory (glutamic, aspartic) and inhibitory amino acids (glycine, GABA). In addition, the rates of release of alanine, serine, and threonine were also elevated. In the presence of TTX, phorbol esters selectively enhanced, in a reversible manner, the depolarizing responses of dorsal horn neurons to N-methyl-D-aspartic acid and L-glutamate but not the responses to kainic or quisqualic acids. The potentiation of the NMDA response was blocked by APV, a specific NMDA receptor antagonist. Thus, phorbol esters appear to enhance excitatory synaptic transmission in the rat spinal dorsal horn slice preparation by acting both at pre- and postsynaptic sites. Phorbol esters could potentiate excitatory synaptic transmission by acting predominantly at a postsynaptic site (NMDA receptor), since the duration of the increased responsiveness of dorsal horn neurons to glutamate and NMDA correlates better with the enhancement of EPSPs than with the increased release of the stimulation-evoked glutamate and aspartate. The increased release of endogenous amino acids is consistent with a presynaptic (terminal) site of action, but it could also be explained by enhanced interneuronal activity. Although our results suggest that in the rat spinal dorsal horn protein kinase C may have a role in controlling the release of putative excitatory and inhibitory neurotransmitters and may also be involved in the regulation of postsynaptic NMDA receptors, the identity of endogenous substance(s) participating in these effects is presently unknown.     

The effects of sandostatin and somatostatin on nociceptive transmission in the dorsal horn of the rat spinal cord.

The role of somatostatin and a stable analogue, sandostatin (Octreotide), on the responses of spinal cord neurones in vivo was investigated in the rat. Electrical C-fibre stimulation was used as a model of acute nociception and the response to subcutaneous formalin was used as a model of longer term events. Intrathecal pre-treatment with sandostatin and somatostatin did not alter the C-fibre response, wind up or A beta responses of the cells. However, intrathecal pre-treatment with sandostatin and somatostatin inhibited both the first and second phases of the formalin response dose dependently. Thus, sandostatin (20 micrograms) and somatostatin (150 micrograms) inhibited the first phase (66 +/- 12% inhibition and 52 +/- 13% respectively) and second phase (91 +/- 2% inhibition and 39 +/- 16% inhibition respectively). The second phase of the formalin response was more sensitive to somatostatin and sandostatin than the first. Sandostatin was approximately 400 times more potent than somatostatin on the second phase of the response. Subcutaneous sandostatin (100 mg/kg) significantly inhibited both the first and second phase of the formalin response whereas the local peripheral administration of sandostatin (20 micrograms) only inhibited the second phase of the formalin response.