In-Depth Characterization of Layer 5 Output Neurons of the Primary Somatosensory Cortex Innervating the Mouse Dorsal Spinal Cord.

Neuronal circuits of the spinal dorsal horn integrate sensory information from the periphery with inhibitory and facilitating input from higher central nervous system areas. Most previous work focused on projections descending from the hindbrain. Less is known about inputs descending from the cerebral cortex. Here, we identified cholecystokinin (CCK) positive layer 5 pyramidal neurons of the primary somatosensory cortex (CCK + S1-corticospinal tract [CST] neurons) as a major source of input to the spinal dorsal horn. We combined intersectional genetics and virus-mediated gene transfer to characterize CCK+ S1-CST neurons and to define their presynaptic input and postsynaptic target neurons. We found that S1-CST neurons constitute a heterogeneous population that can be subdivided into distinct molecular subgroups. Rabies-based retrograde tracing revealed monosynaptic input from layer 2/3 pyramidal neurons, from parvalbumin positive cortical interneurons, and from thalamic relay neurons in the ventral posterolateral nucleus. Wheat germ agglutinin-based anterograde tracing identified postsynaptic target neurons in dorsal horn laminae III and IV. About 60% of these neurons were inhibitory and about 60% of all spinal target neurons expressed the transcription factor c-Maf. The heterogeneous nature of both S1-CST neurons and their spinal targets suggest complex roles in the fine-tuning of sensory processing.

Inhibition of high voltage-activated calcium currents by L-glutamate receptor-mediated calcium influx.

The modulation of high voltage-activated (HVA) Ca2+ currents by L-glutamate and its agonists was investigated in cultured rat hypothalamic neurons. L-Glutamate and agonists selective for NMDA or non-NMDA receptors reversibly inhibited HVA Ca2+ currents. The putative presynaptic glutamate receptor agonist L-2-amino-4-phosphonobutyric acid and the selective metabotropic agonist trans-ACPD were ineffective. Inhibition was dependent on the presence of extracellular Ca2+ and blocked by internal perfusion of the cells with BAPTA. The calmodulin antagonists trifluoperazine and calmidazolium completely prevented the inhibition. Increases in the intracellular Ca2+ concentration due to Ca2+ influx through non-NMDA receptor channels were visualized using fura-2. These results indicate that not only NMDA but also non-NMDA receptor channels in these neurons are permeable for Ca2+ and that Ca2+ influx through these channels activates a calmodulin-dependent mechanism, which leads to HVA Ca2+ current inhibition.

Analgesic effect of clobazam in chronic low-back pain but not in experimentally induced pain.

BACKGROUND: Chronic pain is frequently associated with hypersensitivity of the nervous system, and drugs that increase central inhibition are therefore a potentially effective treatment. Benzodiazepines are potent modulators of GABAergic neurotransmission and are known to exert antihyperalgesic effects in rodents, but translation into patients are lacking. This study investigates the effect of the benzodiazepine clobazam in chronic low-back pain in humans. The aim of this study is to explore the effect of GABA modulation on chronic low-back pain and on quantitative sensory tests. METHODS: In this double-blind cross-over study, 49 patients with chronic low-back pain received a single oral dose of clobazam 20 mg or active placebo tolterodine 1 mg. Pain intensity on the 0-10 numeric rating scale and quantitative sensory tests were assessed during 2 h after drug intake. RESULTS: Pain intensity in the supine position was significantly reduced by clobazam compared to active placebo (60 min: 2.9 vs. 3.5, p = 0.008; 90 min: 2.7 vs. 3.3, p = 0.024; 120 min: 2.4 vs. 3.1, p = 0.005). Pain intensity in the sitting position was not significantly different between groups. No effects on quantitative sensory tests were observed. CONCLUSIONS: This study suggests that clobazam has an analgesic effect in patients with chronic low-back pain. Muscle relaxation or sedation may have contributed to the effect. Development of substances devoid of these side effects would offer the potential to further investigate the antihyperalgesic action of GABAergic compounds. SIGNIFICANCE: Modulation of GABAergic pain-inhibitory pathways may be a potential future therapeutic target.

Selective suppression of inhibitory synaptic transmission by nocistatin in the rat spinal cord dorsal horn.

Nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are two recently identified neuropeptides with opposing effects on several CNS functions, including spinal nociception. The cellular mechanisms that underlie this antagonism are not known. Here, we have investigated the effects of both peptides on synaptic transmission mediated by the three fast neurotransmitters l-glutamate, glycine, and GABA in the superficial layers of the rat spinal cord horn, which constitute the first important site of integration of nociceptive information in the pain pathway. NST selectively reduced transmitter release from inhibitory interneurons via a presynaptic Bordetella pertussis toxin-sensitive mechanism but left excitatory glutamatergic transmission unaffected. In contrast, N/OFQ only inhibited excitatory transmission. In the rat formalin test, an animal model of tonic pain in which N/OFQ exerts antinociceptive activity, NST induced profound hyperalgesia after intrathecal application. Similar to glycine and GABA(A) receptor antagonists, NST had no significant effects in the rat tail-flick test, a model of acute thermal pain. Our results provide a cellular basis for the antagonism of N/OFQ and NST and suggest the existence of a so far unidentified membrane receptor for NST. In addition, they support a role of NST as an endogenous inhibitor of glycinergic and GABAergic neurotransmission in the sensory part of the spinal cord and as a mediator of spinal hyperalgesia.

Capsaicin, protons and heat: new excitement about nociceptors.

The past few years have witnessed a remarkable progress in understanding the neurobiology of pain. Important advances have been made particularly in the field of peripheral signal transduction in nociceptors. Membrane receptors have been identified for capsaicin, a pungent ingredient of chilli peppers, protons (i.e. acidic solutions) and for heat, three stimuli that specifically excite nociceptors. Of particular interest appears to be the first cloned capsaicin receptor, VR1, which has been suggested to serve as an integrator of these three nociceptive stimuli. These findings not only give new insights into the molecular machinery of nociceptor activation and sensitization, but can also provide a rational basis for pharmacological research aiming for a new class of peripherally acting analgesics, which should selectively interfere with nociceptor activation.

Calcium-dependent inactivation of neuronal calcium channel currents is independent of calcineurin.

Dephosphorylation by the Ca2+/calmodulin-dependent phosphatase calcineurin has been suggested as an important mechanism of Ca2+-dependent inactivation of voltage-gated Ca2+ channels. We have tested whether calcineurin plays a role in the inactivation process of two types of high-voltage-activated Ca2+ channels (L and N type) widely expressed in the central nervous system, using the immunosuppressive drug FK506 (tacrolimus), which inhibits calcineurin after binding to intracellular FK506 binding proteins. Inactivation of L- and N-type Ca2+ channels was studied in a rat pituitary tumor cell line (GH3) and chicken dorsal root ganglion neurons, respectively. With the use of antisera directed against the calcineurin subunit B and the 12,000 mol. wt binding protein, we show that both proteins are present in the cytoplasm of GH3 cells and chicken dorsal root ganglion neurons. Ionic currents through voltage-gated Ca2+ channels were investigated in the perforated-patch and whole-cell configurations of the patch-clamp technique. The inactivation of L- as well as N-type Ca2+ currents could be well fitted with a bi-exponential function. Inactivation was largely reduced when Ba2+ substituted for extracellular Ca2+ or when the Ca2+ chelator EGTA was present intracellularly, indicating that both types of Ca2+ currents exhibited Ca2+-dependent inactivation. Extracellular (perforated-patch configuration) or intracellular (whole-cell configuration) application of FK506 to inactivate calcineurin had no effect on the amplitude and time-course of Ca2+ channel current inactivation of either L- or N-type Ca2+ channels. In addition, we found that recovery from inactivation and rundown of N-type Ca2+ channel currents were not affected by FK506. Our results provide direct evidence that the calcium-dependent enzyme calcineurin is not involved in the inactivation process of the two Ca2+ channel types which are important for neuronal functioning, such as gene expression and transmitter release.

Expression of cyclooxygenase-1 and -2 in normal and glaucomatous human eyes.

PURPOSE: Primary open-angle glaucoma (POAG) is the predominant form of chronic glaucoma, but the underlying pathologic mechanisms are largely unknown. Because prostaglandins (PGs) have been introduced into POAG treatment with remarkable success, this study was undertaken to investigate whether a change in the expression of the PG-synthesizing enzymes cyclooxygenase (COX)-1 and -2 might be involved in the pathogenesis of POAG. METHODS: Expression of COX-1 and -2 was assessed by confocal laser microscopy, immunohistochemistry, Western blot analysis, and real-time RT-PCR in human eyes with different forms of glaucoma (primary open-angle, angle-closure, congenital juvenile, and steroid-induced), as well as in age-matched control eyes. Additionally, PGE2 was measured in aqueous humor by means of an enzyme-linked immunoassay as a product of COX activity. RESULTS: In normal eyes, ocular COX-1 and -2 expression were largely confined to the nonpigmented secretory epithelium of the ciliary body. By immunohistochemistry and real-time RT-PCR, COX-2 expression was completely lost in the nonpigmented secretory epithelium of the ciliary body of eyes with end-stage POAG, whereas COX-1 expression was unchanged. By immunohistochemistry, in the ciliary bodies of eyes in five patients with diagnosis of early POAG, eyes in two had complete loss of COX-2 expression and in three showed only a few remaining scattered COX-2-expressing cells. COX-2 expression in the ciliary body was also lost in patients with steroid-induced glaucoma and was reduced in patients receiving topical steroid treatment. Eyes of patients with either congenital juvenile or angle-closure glaucoma showed COX-2 expression indistinguishable from control eyes. Aqueous humor of eyes with POAG contained significantly less PGE2 than control eyes. CONCLUSIONS: Both cyclooxygenase isoforms are constitutively expressed in the normal human eye. Specific loss of COX-2 expression in the nonpigmented secretory epithelium of the ciliary body appears to be linked to the occurrence of POAG and steroid-induced glaucoma.

Modulation of excitatory synaptic transmission by nociceptin in superficial dorsal horn neurones of the neonatal rat spinal cord.

1. The modulatory actions of nociceptin/orphanin FQ on excitatory synaptic transmission were studied in superficial dorsal horn neurones in transverse slices from 7 to 14 day old rats. 2. Glutamatergic excitatory postsynaptic currents (e.p.s.cs) were recorded from the somata of the neurones in the whole-cell patch-clamp configuration. E.p.s.cs were evoked by extracellular electrical stimulation (100 microns, 3-10 V) of the ipsilateral dorsal root entry zone by use of a glass electrode. E.p.s.cs with constant short latency (< 2.3 ms) and with no failures upon stimulation were assumed to be monosynaptic. These e.p.s.cs occurred with an average latency of 1.72 +/- 0.098 ms and exhibited a fast decay with a time constant, tau, of 4.8 +/- 0.53 ms (n = 30). 3. Nociceptin reversibly reduced the amplitudes of e.p.s.cs in a concentration-dependent manner in 25 out of 27 cells tested. Average maximum inhibition was 51.6 +/- 5.7% (mean +/- s.e.mean; n = 9), at concentrations > 3 microM. EC30 was 485 +/- 47 nM and the Hill coefficient was 1.29 +/- 0.09. 4. Inhibition of synaptic transmission by nociceptin (10 microM) was insensitive to the non-specific opioid receptor antagonist naloxone (10 microM) indicating that nociceptin did not act via classical opioid receptors. 5. In order to determine the site of action of nociceptin spontaneous miniature e.p.s.cs (m-e.p.s.cs) were recorded. Nociceptin reduced the frequency of m-e.p.s.cs in 6 out of 7 cells but had no effect on their amplitude distribution or on their time course. These findings suggest a pre- rather than a postsynaptic modulatory site of action. This is in line with the finding that current responses elicited by extracellular application of L-glutamate (10 microM) were not affected by nociceptin (10 microM; n = 7). 6. No positive correlation was found between the degree of inhibition by nociceptin (10 microM) and by the mixed delta- and mu-receptor agonist methionine-enkephalin (10 microM). This suggests that both neuropeptides acted on different but perhaps overlapping populations of synaptic connections. 7. Our results indicate that nociceptin inhibits excitatory synaptic transmission in the superficial layers of the rat dorsal horn by acting on presynaptic, presumably ORL1 receptors. This may be an important mechanism for spinal sensory information processing including nociception.

Selective growth inhibition of ductal pancreatic adenocarcinoma cells by the lysosomotropic agent chloroquine.

Pancreatic adenocarcinoma cells show characteristics of macrophages as phagocytosis and exocytosis. These phagocytic activities can be inhibited in macrophages by antirheumatic drugs, e.g. gold compounds and antimalarials. We have now tested the activity of auranofin and chloroquine to inhibit growth of a pancreatic tumor cell line (PaTu II) and of human foreskin fibroblasts (HFF) as a control. We found that auranofin (greater than or equal to 2 microM) inhibits both PaTu II cell and HFF growth. In contrast, chloroquine (4-18 microM) selectively interferes with the growth of PaTu II cells. This specific vulnerability of PaTu II cells was confirmed in cocultures of tumor cells and fibroblasts.

Differential analgesic effects of aspirin-like drugs.

Tissue damage, including that due to surgical manipulation, results in 2 distinct but connected changes in the pain perception pathway. Firstly, cells disintegrate at the site of tissue damage and release mediators, including prostaglandins. These mediators transform fine nerve endings, particularly high-threshold mechanoceptors, into nociceptors. In other words, fine nerve endings that are not normally activated by mechanical pressure or temperature changes become very sensitive and are depolarised after minor mechanical or thermal changes. Secondly, in the central nervous system (CNS) and, particularly, in the dorsal horn of the spinal cord, reflex activity is increased, metabolic activity of the neuronal cells is enhanced and, chronically, major rearrangements of mediator production and electrical activity of the dorsal horn cells may be observed. Both types of change contribute to the well known phenomenon of hyperalgesia, which is regularly observed in connection with tissue damage, including that produced by surgical manipulation. It has been shown that aspirin-like drugs reduce the enhanced nociceptor activity in damaged tissue, probably as a result of prostaglandin synthesis inhibition. Recently, there have been indications that these drugs may have an additional mechanism of action in the spinal cord or higher parts of the CNS. Using the pure enantiomers of flurbiprofen in pharmacodynamic experiments in the rat, we have observed that the R- and S-enantiomers may exert differential analgesic effects. The R-enantiomer, which does not inhibit cyclo-oxygenase in vitro, was almost as effective as the S-enantiomer, which does inhibit prostaglandin synthesis in different models of pain and nociception.(ABSTRACT TRUNCATED AT 250 WORDS)

Spinally delivered nociceptin/orphanin FQ reduces flinching behaviour in the rat formalin test.

The aims of this study were to investigate the dose-dependent effects of spinally delivered nociceptin (0.3, 1, 3.3 and 10 nmol) on flinching behaviour in the rat formalin test and whether these effects were influenced by the concomitant systemic administration of naloxone (3 mg/kg, i.p.). The effect of the highest nociceptin dose differed statistically from vehicle, 0.3 and 1 nmol nociceptin. Following the administration of 1, 3.3 or 10 nmol nociceptin mean total flinches decreased dose-dependently. The effects of 10 nmol nociceptin were not reversed by a high dose of naloxone. We observed a decrease in flinching behaviour with intrathecally to the lumbar enlargement delivered nociceptin and conclude that nociceptin has antinociceptive effects in the rat formalin test.

The role of the ORL1 receptor in the modulation of spinal neurotransmission by nociceptin/orphanin FQ and nocistatin.

Nociceptin/orphanin FQ and nocistatin are two neuropeptides with opposing effects on spinal neurotransmission and nociception. Nociceptin/orphanin FQ selectively suppresses excitatory glutamatergic neurotransmission, while nocistatin selectively interferes with glycinergic and gamma-aminobutyric acid (GABA)-ergic transmission. Here, we performed whole-cell patch-clamp recordings from superficial rat spinal cord dorsal horn neurons to investigate the role of the opioid receptor-like (ORL)1 receptor for modulatory actions of these peptides. The partial ORL1 receptor antagonist [phe1psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)NH(2) competitively reversed the effects of nociceptin/orphanin FQ on excitatory neurotransmission (estimated pA(2) 6.43), but left the suppression of inhibitory synaptic transmission by nocistatin unaffected. These results indicate that the inhibitory action of nociceptin/orphanin FQ on glutamatergic transmission is mediated via ORL1 receptors, while nocistatin acts via a different so far unidentified receptor.

Differential effects of ketamine enantiomers on NMDA receptor currents in cultured neurons.

The effects of R- and S-ketamine on N-methyl-D-aspartate receptor-activated cation currents (NMDA receptor currents) of voltage-clamped cultured rat hippocampal neurons were investigated using the whole-cell patch-clamp technique. Both enantiomers exhibited a voltage- and use-dependent blockade of NMDA receptor currents, with the S-enantiomer being about twice as potent as the R-enantiomer. Calculated relative forward and backward rates suggest that conformational differences influence the dissociation from the binding site more than the association with it.

Modulation of nitric oxide effects by flurbiprofen enantiomers and nefopam and its relation to antinociception.

We have examined the interactions of the analgesics, R- and S-flurbiprofen and nefopam, with nitric oxide (NO) in several experimental systems. Phenylephrine-precontracted rat aortic strips with intact endothelium were relaxed by R- and S-flurbiprofen and nefopam in a concentration-dependent manner. Removal of endothelium, inhibition of guanylate cyclase, inhibition of NO biosynthesis and inactivation of NO significantly reduced these relaxations. R- and S-flurbiprofen as well as nefopam enhanced the inhibition of platelet aggregation caused by rat peritoneal neutrophils or 3-morpholinosydnonimine. The antinociceptive effects of R- and S-flurbiprofen in the mouse writhing test as well as those of nefopam in the hot plate test were not significantly affected by administration of NO synthase inhibitors. We conclude that the increase in the biological activity of NO by R- and S-flurbiprofen and nefopam does not play a major role in the antinociceptive activity of the drugs, but might contribute to acute hypotension, a side-effect occasionally seen with flurbiprofen and nefopam.

Spinal inhibitory synaptic transmission in the glycine receptor mouse mutant spastic.

Inhibitory glycine receptor (GlyR) and GABA(A) receptor (GABA(A)R)-mediated synaptic transmission was examined in two strains of the GlyR mutant mouse spastic and the respective wild types. The mutants display a mild and a severe neurological phenotype. Electrically evoked postsynaptic whole-cell currents were recorded from alpha-motoneurons in lumbar spinal cord slices. Amplitudes of GlyR-mediated IPSCs were significantly reduced in the severe phenotype in comparison to the respective wild type and the mild phenotype mutants. Surprisingly, amplitudes of GABA(A)R-mediated IPSCs were also significantly reduced in both mutants. Fast time constants of the decay phase of IPSCs were slightly reduced for the GlyR-mediated IPSCs and significantly larger for the GABA(A)R-mediated IPSCs in both mutant strains.

The glycinergic control of spinal pain processing.

Alterations in synaptic transmission within the spinal cord dorsal horn play a key role in the development of pathological pain. While N-methyl-D-aspartate (NMDA) receptors and activity-dependent synaptic plasticity have been the focus of research for many years, recent evidence attributes very specific functions to inhibitory glycinergic and gamma-aminobutyric acid (GABA)-ergic neurotransmission in the generation of inflammatory and neuropathic pain. The central component of inflammatory pain originates from a disinhibition of dorsal horn neurons, which are relieved from glycinergic neurotransmission by the inflammatory mediator prostaglandin E2 (PGE2). PGE2 activates prostaglandin E receptors of the EP2 subtype and leads to a protein kinase A-dependent phosphorylation and inhibition of glycine receptors containing the alpha3 subunit (GlyRalpha3). This GlyRalpha3 is distinctly expressed in the superficial dorsal horn, where nociceptive afferents terminate. Other but probably very similar disinhibitory mechanisms may well contribute to abnormal pain occurring after peripheral nerve injury.

Role of interleukin-8 in neutrophil signaling.

Interleukin-8 was originally discovered as one of the first chemokines activating neutrophil granulocytes (neutrophils) after secretion by lipopolysaccharide-stimulated monocytes. A wealth of information has been gathered concerning the intracellular events mediated by interleukin-8 and the role of interleukin-8 in numerous physiologic and pathophysiologic processes. We discuss recent advances in the understanding of the initial intracellular signals elicited by interleukin-8. Detailed investigation of these events has led to the identification of subtle but significant differences in the signal transduction processes evoked by interleukin-8 receptors. In particular, much has been learned concerning differences in the cellular mechanisms leading to desensitization, internalization, and recycling of interleukin-8 receptors, and functional consequences of interleukin-8 receptor diversity are now being unraveled.

Synaptic connectivity in cultured hypothalamic neuronal networks.

We have developed a novel approach to analyze the synaptic connectivity of spontaneously active networks of hypothalamic neurons in culture. Synaptic connections were identified by recording simultaneously from pairs of neurons using the whole cell configuration of the patch-clamp technique and testing for evoked postsynaptic current responses to electrical stimulation of one of the neurons. Excitatory and inhibitory responses were distinguished on the basis of their voltage and time dependence. The distribution of latencies between presynaptic stimulation and postsynaptic response showed multiple peaks at regular intervals, suggesting that responses via both monosynaptic and polysynaptic paths were recorded. The probability that an excitatory event is transmitted to another excitatory neuron and results in an above-threshold stimulation was found to be only one in three to four. This low value indicates that in addition to evoked synaptic responses other sources of excitatory drive must contribute to the spontaneous activity observed in these networks. The various types of synaptic connections (excitatory and inhibitory, monosynaptic, and polysynaptic) were counted, and the observations analyzed using a probabilistic model of the network structure. This analysis provides estimates for the ratio of inhibitory to excitatory neurons in the network (1:1.5) and for the ratio of postsynaptic cells receiving input from a single GABAergic or glutamatergic neuron (3:1). The total number of inhibitory synaptic connections was twice that of excitatory connections. Cell pairs mutually connected by an excitatory and an inhibitory synapse occurred significantly more often than predicted by a random process. These results suggests that the formation of neuronal networks in vitro is controlled by cellular mechanisms that favor inhibitory connections in general and specifically enhance the formation of reciprocal connections between pairs of excitatory and inhibitory neurons. These mechanisms may contribute to network formation and function in vivo.