Excitatory synaptic transmission in the spinal substantia gelatinosa is under an inhibitory tone of endogenous adenosine

Exogenous adenosine produces potent synaptic inhibition in spinal substantia gelatinosa (SG), a region involved in nociceptive and thermoreceptive mechanisms. To examine the possibility that endogenous adenosine tonically modulates excitatory synaptic transmission in spinal SG, whole-cell, voltage-clamp recordings were made from SG neurons in adult rat spinal cord slices. In all SG neurons sensitive to exogenous adenosine, the adenosine uptake inhibitor, NBTI, mimics adenosine's inhibitory actions on dorsal root evoked EPSCs (eEPSCs) and miniature spontaneous EPSCs (mEPSCs). These inhibitory effects were antagonized by A1 adenosine receptor antagonist, DPCPX. DPCPX also potentates eEPSCs in those SG neurons in which adenosine or adenosine A1 receptor agonists (CHA, CCPA) suppressed eEPSCs. DPCPX often increases mEPSC frequency without altering mEPSC amplitude, suggesting presynaptic action on adenosine A1 receptors. Selective A2 (DMPX) and A2a (ZM 241385) adenosine receptor antagonists had no or minimal effects upon either eEPSCs or mEPSCs. The adenosine degrading enzyme, adenosine deaminase, mimicked the effects of DPCPX on the mEPSC frequency. We conclude that the excitatory synaptic transmission in the spinal SG is under an inhibitory tone of endogenous adenosine through the activation of A1 receptors. The present results suggested that the background activity of A1 receptors in the spinal SG might be contributed to setting the physiological “noceceptive thresholds”.     

Appearance of acid phosphatase in neonatal rat substantia gelatinosa

Summary The onset of acid phosphatase activity was observed in neonatal rat substantia gelatinosa using the Gomori method. Although acid phosphatase activity was not present at birth it appeared during the first day postnatally. By six to ten days postnatally enzyme activity appeared to reach its adult level. The activity was quantified using atomic absorption spectrophotometry which showed that acid phosphatase activity reached its adult level by 6 days postnatally. Acid phosphatase in the substantia gelatinosa is fluoride resistant from its first appearance.This project was supported by NIH Grant DA 02060     

Neurons with asymmetrical dendritic arbors in the substantia gelatinosa of the rat spinal cord

Summary Two types of neurons were observed in the substantia gelatinosa (SG) of the rat spinal cord which exhibit wide variations in dendritic symmetry. As demonstrated with the Golgi technique, islet cells with short dendritic arbors and type III stalk cells display dendritic patterns which vary from a bipolar type arrangement with two dendritic arbors of nearly equal dimensions to a unipolar arrangement with a dendritic arbor which extends in only one direction. Examination of the morphology and dendritic development of these neurons shows that they are unique compared with other SG neurons in that they have short, longitudinal dendritic arbors which undergo maturation relatively late in the postnatal period. As is discussed, variations in dendritic symmetry are probably dependent on the location of the terminal fields of primary and/or other types of afferents which are formed earlier in development.     

Absence of tonic supraspinal control of substance P release in the substantia gelatinosa of the anaesthetized cat

Summary Antibody microprobes were used to measure immunoreactive substance P (irSP) release in the substantia gelatinosa of the lower lumbar spinal cord of barbiturate-anaesthetized cats. Release of irSP was produced by noxious peripheral stimuli. Such release was not altered by blocking spinal conduction at the first lumbar segment by cooling or transecting the spinal cord. The results suggest that the release of irSP from the central terminals of nociceptors is not subject to tonic supraspinal inhibition.     

The median and lateral substantia gelatinosa in the cervical cord of the musk shrew (Suncus murinus) and its synaptic composition

Summary The extent and laminar arrangement of the substantia gelatinosa (SG) were examined in the cervical spinal cord of the shrew. Between C1 and C6 or C7 the three dorsal layers on either side of the gray matter were confluent at the commissura posterior grisea in shrews of both sexes. Lamina I was thin with no large marginal cells. Lamina II comprised the major part of the SG, consisting of outer cellular and inner neuropil layers. Lamina III was composed of a meshwork of axon terminals, dendritic profiles and myelinated fibers. Unlike the situation in other mammals, in shrew the incidence of axon terminals with round vesicles was similar in the three uppermost layers, but the occurrence of terminal profiles with flat vesicles was significantly greater in deeper laminae. Lamina IV was restricted to the dorsal horn and did not extend through the midline.     

The dorsal root distribution to the substantia gelatinosa of the rat with a note on the distribution in the cat

Summary The terminal degeneration in the substantia gelatinosa of the rat was studied with the Fink-Heimer silver technique following dorsal root section. Providing the survival time of the animal was in the range of 1–4 days, a massive degeneration was seen in lamina I, II and III of Rexed. The light microscope findings were corroborated by electronmicroscopic observations of degenerating boutons. Spinal cord material examined with silver methods one week after dorsal root section showed few signs of degeneration in the substantia gelatinosa. Although a significant dorsal root distribution to the substantia gelatinosa was found also in the cat, the terminal degeneration in lamina II showed considerable regional variations in this species.     

Functional type I vanilloid receptor expression by substantia gelatinosa neurons of trigeminal subnucleus caudalis in mice

The aim of this study was to investigate the existence of functional TRPV1 receptor by substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc), which is implicated in the processing of nociceptive information from orofacial regions. The direct membrane effects of a TRPV1 receptor agonist, capsaicin, were examined by gramicidin-perforated patch clamp recording using a trigeminal brainstem slice preparation containing Vc from immature mice. Capsaicin (1–2 μM) induced a membrane depolarization in 58 out of 71 SG neurons tested (82%). Capsaicin-induced depolarization was maintained in 20 out of 32 (63%) SG neurons in the presence of amino acid and voltage-dependent sodium channel blockers (10 μM CNQX, 20 μM AP-5, 0.5 μM TTX, 50 μM picrotoxin and 2 μM strychnine). In addition, capsaicin-induced depolarization was maintained in the presence of L-732,138 (1 μM), an NK1 receptor antagonist, in 14 out of 17 neurons (82%) tested. The capsaicin-induced depolarizing effects were blocked by a TRPV1 receptor antagonist, capsazepine (10 μM). These results indicate that a sub-population of SG neurons in the Vc express functional TRPV1 receptors, and that capsaicin can directly activate the TRPV1 receptor on the postsynaptic membrane of SG neurons.     

Noradrenaline inhibits substantia gelatinosa neurons in mice trigeminal subnucleus caudalis via alpha(2) and beta adrenoceptors

The actions of noradrenaline (NA) in the substantia gelatinosa (SG) are important for their antinociceptive effects. In order to identify the possible mechanisms underlying NA actions in the SG of trigeminal subnucleus caudalis (Vc), the direct membrane effects were examined by gramicidin-perforated patch clamp recording using brain slice preparation from immature mice brainstem. The majority (60/71, 85%) of neurons tested were hyperpolarized by NA application, and these hyperpolarizing effects were mimicked both by the alpha(2) adrenergic agonist, clonidine (18/28, 64%) and the beta adrenergic agonist, isoproterenol (9/24, 38%). NA-induced hyperpolarizing effect was also blocked by the alpha(2) adrenergic antagonist, yohimbine in five out of six neurons tested. However, a minority (5/71, 7%) of neurons tested were depolarized by NA, and these depolarizing effects were mimicked by the alpha(1) adrenergic agonist, phenylephrine (11/26, 42%). NA-induced hyperpolarizing effects were maintained in the presence of tetrodotoxin (TTX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), d,l-2-amino-5-phosphonopentanoic acid (AP5), picrotoxin and strychnine, a Na(+) channel, ionotropic glutamate receptor, GABA(A) and glycine receptor antagonists, respectively, indicating that the effects of NA are direct on the postsynaptic SG neurons. These results indicate that alpha(2) and beta adrenoceptor mediate inhibition, and alpha(1) adrenoceptor mediates facilitation of orofacial nociceptive processing in mouse trigeminal brainstem SG neurons by postsynaptic actions.     

GABAA receptor-mediated tonic currents in substantia gelatinosa neurons of rat spinal trigeminal nucleus pars caudalis

In the present study, we describe GABAA receptor-mediated tonic inhibitory currents in the substantia gelatinosa (SG) region of rat spinal trigeminal nucleus pars caudalis (Vc). The GABA(A) receptor-mediated tonic currents were identified by bath-application of the GABAA receptor antagonists, picrotoxin (1mM), SR95531 (100microM) and bicuculline (100microM). All three antagonists completely blocked outward spontaneous (phasic) inhibitory postsynaptic currents, but only picrotoxin and bicuculline induced a significant (>5pA) inward shift of holding currents at a holding potential (Vh) of 0mV in 60-70% of SG neurons, revealing the existence of tonic outward currents. The tonic currents were resistant to further the blockades of glycine receptors or those in addition to glutamate receptors and voltage-dependent sodium channels. An acute bath-application of THDOC (0.1microM), the stress-related neurosteroid, did enhance tonic currents, but only in a small population of SG neurons. In addition, slices incubated with THDOC for 30min increased the probability of neurons with significant tonic currents. The GABAergic tonic inhibition demonstrated in this study may play a significant role in the sensory processing system of the Vc.     

Segmental and supraspinal control of synaptic effectiveness of functionally identified muscle afferents in the cat

The present investigation documents the patterns of primary afferent depolarization (PAD) of single, functionally identified muscle afferents from the medial gastrocnemius nerve in the intact, anesthetized cat. Classification of the impaled muscle afferents as from muscle spindles or from tendon organs was made according to several criteria, which comprised measurement of conduction velocity and electrical threshold of the peripheral axons, and the maximal frequency followed by the afferent fibers during vibration, as well as the changes in discharge frequency during longitudinal stretch, the projection of the afferent fiber to the motor pool, and, in unparalyzed preparations, the changes in afferent activity during a muscle twitch. In confirmation of a previous study, we found that most muscle spindle afferents (46.1–66.6%, depending on the combination of criteria utilized for receptor classification) had a type A PAD pattern. That is, they were depolarized by stimulation of group I fibers of the posterior biceps and semitendinosus (PBSt) nerve, but not by stimulation of cutaneous nerves (sural and superficial peroneus) or the bulbar reticular formation (RF), which in many cases inhibited the PBSt-induced PAD. In addition, we found a significant fraction of muscle spindle primaries that were depolarized by stimulation of group I PBSt fibers and also by stimulation of the bulbar RF. Stimulation of cutaneous nerves produced PAD in 9.1–31.2% of these fibers (type B PAD pattern) and no PAD in 8.2–15.4% (type C PAD pattern). In contrast to muscle spindle afferents, only the 7.7–15.4% of fibers from tendon organs had a type A PAD pattern, 23–46.1% had a type B and 50–61.5% a type C PAD pattern. These observations suggest that the neuronal circuitry involved in the control of the synaptic effectiveness of muscle spindles and tendon organs is subjected to excitatory as well as to inhibitory influences from cutaneous and reticulospinal fibers. As shown in the accompanying paper, the balance between excitation and inhibition is not fixed, but can be changed by crushing the afferent axons in the peripheral nerve and allowing subsequent reconnection of these afferent fibers with muscle receptors.     

大鼠桡神经感觉纤维在脊髓胶状质的纵横定位研究

目的　研究大鼠桡神经感觉纤维在脊髓胶状质的定位投射。方法　按照跨神经节溃变原理 ,采用酸性磷酸酶(ACP)法在 10只切断桡神经的昆明大鼠观察桡神经感觉纤维在颈髓 5～ 8和胸髓 1上、中、下段胶状质内ACP的变化。结果　大鼠桡神经感觉纤维纵向投射 ,主要至脊髓C6上段至T1中段胶状质。其横向投射 ,桡神经主要位于整个胶状质的中 1/ 3区域。结论　桡神经投射区居外 ,尺神经投射区居内     

Adenosine 5'-triphosphate inhibits slow depolarization induced by repetitive dorsal root stimulation via P2Y purinoceptors in substantia gelatinosa neurons of the adult rat spinal cord slices with the dorsal root attached

We previously reported that slow depolarization of substantia gelatinosa neurons is evoked by repetitive stimulation of C-fibers of dorsal root in adult rat spinal cord transverse slices with the dorsal root attached, which was considered to be an event relevant to spinal nociception. In the present study, we investigated the effects of adenosine 5′-triphosphate (ATP) and its analogs on the slow depolarization. ATP (10–100 μM) significantly inhibited the amplitude of slow depolarization in a concentration-dependent manner. The inhibitory effect of ATP was not reversed by suramin, an antagonist for some P2-purinoceptors, and was mimicked by a P2Y selective agonist uridine 5′-triphosphate, but not a P2X selective agonist ,β-methylene ATP. These results suggest that ATP inhibits the slow depolarization of substantia gelatinosa neurons relevant to nociceptive transmission in the spinal dorsal horn, via suramin-insensitive P2Y purinoceptors.     

A study of the cutaneous afferent input to substantia gelatinosa

The primary afferent input to substantia gelatinosa of the spinal cord was studied in the decerebrate cat. The responses of cells in the substantia gelatinosa to natural stimulation of the skin and to graded electrical stimulation of the sural nerve were compared. The results show that the responses of these neurones are not predictable from the known properties of the primary afferent input. Interactions between A-fibre and C-fibre input to substantia gelatinosa cells were studied using A-fibre polarization block and conditioning A-fibre stimulation during C-fibre activation of these cells. Whereas A-fibres have strong inhibitory effects on C-fibre input to large projection cells in the spinal cord, this is not true for substantia gelatinosa cells.The results do not support the idea of substantia geletinosa as a simple relay between primary afferent inputs and spinal cord projection systems.     

神经降压素对大鼠脊髓背角胶状质内突触前神经递质释放的影响

目的:研究内源性神经肽神经降压素(neurotensin,NT)在脊髓背角胶状质(substantia gelatinosa,SG)内对突触前神经递质释放的影响。方法:采用全细胞电压膜片钳记录方法,在脊髓薄片上观察NT对SG内微小兴奋性突触后电流(mEPSCs)和微小抑制性突触后电流(mIPSCs)的频率和幅值的影响。结果:(1)灌流NT(2μmol/L)对SG内神经元mEPSCs的频率和幅值均无明显影响,说明NT不影响SG内兴奋性神经递质的释放;(2)灌流NT(2μmol/L)能增加SG内神经元mIPSCs的频率,但对幅值无明显影响,即NT可引起突触前抑制性神经递质的释放增加,但对突触后神经元无明显影响。结论:NT可通过增加SG内抑制性神经递质释放的途径抑制伤害性信息的传递,从而实现镇痛效应。     

Influences of contralateral nerve and skin stimulation on neurones in the substantia gelatinosa of the rat spinal cord

Stimulation of high threshold A delta and C fibre peripheral afferents inhibits dorsal horn cells on the other side of the spinal cord. The substantia gelatinosa (SG) is an area full of interneurones known to have commissural connections across the spinal cord. The role of SG in this contralateral inhibitory pathway is investigated here. Forty-three SG cells were recorded in the lumbar dorsal horn of decerebrate spinal rats. Their ipsilateral excitatory receptive fields and responses to sciatic nerve stimulation were recorded. Repetitive electrical stimulation was then applied to the contralateral sciatic nerve. Eight (19%) units were excited by such stimulation. A brief tetanus was followed by an increase of ongoing activity lasting 30 s to 10 min. These cells did not, however, have excitatory contralateral fields. A small separate group of 4 cells (9%) were mildly inhibited by heating or pinching the contralateral limb. The significance of contralateral excitation of some SG cells is discussed in the light of the predominantly inhibitory contralateral effect on dorsal horn cells in laminae 4 and 5. It is suggested that some SG cells may be inhibitory interneurones in their effect on deeper cells.     

Excitability changes of superior laryngeal,vagal and depressor afferent terminals produced by stimulation of the solitary tract nucleus

Summary Single and repetitive stimulation of the solitary tract nucleus produced a long lasting hyperexcitability (LLH) of the superior laryngeal (SL) fibres terminating within the same nucleus but not of the terminals of afferent fibres from the cervical vagus and depressor nerve. LLH was independent of the number of SL fibres activated by the conditioning stimulus and was depressed by preceding stimulation of peripheral afferents. It is suggested that LLH results from activation of interneurones making axo-axonic synapses with the SL terminals.     

Responses to 5-HT in morphologically identified neurons in the rat substantia gelatinosa in vitro

Bath application of 5-HT (1-1000 μM) induced a tetrodotoxin (TTX)-resistant outward current at the holding membrane potential (V_H) of −50 mV in 104/162 (64.2%) of substantia gelatinosa (SG) neurons from the rat spinal cord in vitro. The 5-HT-induced outward current was suppressed by an external solution containing Ba²⁺, or a pipette solution containing Cs₂SO₄ and tetraethylammonium. It was reversed near the equilibrium potential of the K⁺ channel. The response to 5-HT was abolished 30 min after patch formation with a pipette solution containing guanosine-5-O-(2-thiodiphosphate)-S. The 5-HT-induced outward current was mimicked by a 5-HT_1A agonist, (±)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide, and suppressed by a 5-HT_1A antagonist, WAY100635, suggesting the 5HT_1A receptor-mediated activation of K⁺ channels in the outward current. In 11/162 (6.8%) SG neurons, 5-HT produced an inward current, which was mimicked by a 5-HT₃ agonist, 1-(m-chlorophenyl)-biguanide (mCPBG). The 5-HT-induced outward currents were observed in vertical cells (21/34) and small islet cells (11/34), while inward currents were induced in islet cells (1/5) and small islet (4/5) cells, but not in vertical cells. It is known that most vertical cells and islet cells in the SG are excitatory (glutamatergic) and inhibitory interneurons, respectively, while small islet cells consist of both excitatory and inhibitory neurons. Bath application of 5-HT or mCPBG increased the amplitude and the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs), but no neurons showed a decrease in sIPSC. Furthermore, frequency, but not amplitude, of miniature IPSCs increased with perfusion with 5-HT in the presence of TTX. These findings, taken together, suggest that 5-HT induces outward currents through 5-HT_1A receptors in excitatory SG neurons. These findings also suggest that the inward currents are post- and presynaptically evoked through 5-HT₃ receptors, probably in inhibitory neurons.     

Effects of prolonged stretch of flexor muscles upon excitability of an extensor reflex arc

Summary Prolonged steady stretch of flexor muscles inhibits the monosynaptic response evoked by maximal stimulation of Group I extensor afferent fibers. However, the amplitude of the monosynaptic response tends to recover control values as a function of time. No temporal correlation between adaptation of inhibiting receptors and adaptation of inhibition has been found. As a consequence, it seems that the disinhibiting mechanism must be located mainly within the spinal cord. Usually, enhanced excitability of extensor primary afferent endings in monosynaptic contact with homonymous motoneurons occurs as long as the stretch of flexor muscles is maintained, but it dwindles to a relatively steady value, and so paralleling the changes of the monosynaptic responses, with opposite sign.This research was supported by USPHS NB 01143.     

内源性大麻素类似物NAGly对大鼠脊髓胶状质神经元的突触传递的抑制作用

目的:探讨内源性大麻素类似物N-花生四烯酰甘氨酸(N-arachidonylglycine,NAGly)对脊髓后角II层胶状质(SG)神经元突触兴奋性的影响。方法:选用生后4~6周雄性SD大鼠,深麻醉后蔗糖人工脑脊液快速心脏灌注处死,取脊髓腰膨大段,制备保留脊髓腰膨大处一侧后根的脊髓矢状切片。利用膜片钳技术,对脊髓后角II层SG神经元进行全细胞记录,通过分析后根刺激诱发的兴奋性突触后电流(eEPSC)的变化情况,观察NAGly(20μmol/L)对SG神经元突触传递兴奋性的影响,以及对自发性兴奋性突触后电流(sEPSC)的发放频率及幅度的影响。结果:通过诱发刺激的强度、潜伏期以及纤维传导速度我们将记录到的SG神经元分为Aδ纤维/C纤维投射神经元,NAGly对Aδ纤维和C纤维介导的eEPSC的幅度都有明显的抑制作用(P0.001),并且这种作用可以被洗脱。NAGly对SG神经元的sEPSC的频率有明显的抑制,但不明显改变其幅度,提示其作用部位在突触前。结论:内源性大麻素类似物NAGly可以抑制脊髓后角浅层Aδ纤维及C纤维介导的突触传递,并通过突触前机制抑制SG神经元的兴奋性。提示内源性大麻素类似物NAGly可通过抑制伤害性C和A纤维介导的突触传递发挥镇痛作用。