Inhibitory neurogenic modulation of histamine-induced cutaneous plasma extravasation in the pigeon.

The neurohumoral modulation of the permeability increasing effect of histamine was studied in pigeon skin. Substances were administered through plasmapheresis capillaries inserted into the dorsal wing skin and the protein contents of the perfusates were determined by a quantitative method. The vascular labelling technique was also utilized to histologically identify leaky blood vessels. In the innervated skin histamine evoked a significant, dose-dependent plasma extravasation which was markedly augmented by the coadministration of a specific galanin receptor antagonist, galanin-1-16-bradykinin-2-9-amide (M35). Chronic cutaneous denervation per se resulted in a significant elevation of the permeability-enhancing effect of histamine. In the denervated skin this response was not affected by M35 but was significantly inhibited by galanin. It is concluded that in the normally innervated skin endogenous galanin may exert a neurogenic tonic inhibitory effect on histamine-induced plasma leakage. It is suggested that sensory nerves possess not only pro-inflammatory, but also anti-inflammatory (inhibitory) sensory-efferent functions.

Interleukin-1beta reduces temperature sensitivity but elevates thermal thresholds in different populations of warm-sensitive hypothalamic neurons in rat brain slices.

Extracellularly recorded firing rates of neurons in slices of the preoptic area and anterior hypothalamus (PO/AH) of the rat were determined during thermal stimulation. Human recombinant interleukin-1beta (20 ng/ml) did not influence temperature-insensitive neurons, but reduced the firing rate and thermosensitivity in linear warm-sensitive neurons, and shifted the thermal thresholds of activation in threshold warm- and cold-sensitive neurons by 1.1-2.3 degrees C to hyperthermic temperatures. The data support the suggestion that endogenous pyrogens may act on different populations of thermosensitive PO/AH neurons to induce fever. The shift of the thermal thresholds of activation of threshold warm- and cold-sensitive neurons in combination with the otherwise maintained temperature sensitivity of these neurons appears to play a major part for the controlled shift of body temperature and the maintenance of the elevated body temperature during cytokine-induced fever.

Effect of nociceptin and [Phe1psi (CH2-NH) Gly2]-nociceptin-(1-13)-NH2 on tonic activity of rat hypothalamic neurons.

The effect of nociceptin, an endogenous ligand for a unique member of the cloned opioid receptor family ORL1-receptor, on tonic activity of neurons in the preoptic area/anterior hypothalamus (PO/AH) has been examined in rat brain slices using extracellular recordings. Nociceptin (1, 10 and 100 nM) decreased dose-dependently tonic activity of PO/AH neurons. This effect was not significantly different from the effect of [Phe1psi (CH2-NH) Gly2]-nociceptin-(1-13)-NH2 (1, 10 and 100 nM), recently proposed as a selective antagonist of the nociceptin receptor. Thus, [Phe1psi (CH2-NH) Gly2]-nociceptin-(1-13)-NH2 appears to be an agonist rather than an antagonist of nociceptin (ORL1) receptor in rat PO/AH neurons. However, there was neither antagonism nor additive synergism when nociceptin and [Phe1psi (CH2-NH) Gly2]-nociceptin-(1-13)-NH2 were applied simultaneously at equimolar concentrations. The effect of nociceptin on tonic activity of rat PO/AH neurons was not blocked by selective mu-, kappa- and delta-opioid receptor antagonists (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), nor-binaltorphimine and naltrindol, respectively) at 10 times higher concentrations than nociceptin. These data suggest that the effect of nociceptin on tonic activity of PO/AH neurons is not due to an action on mu-, kappa-, or delta-opioid receptors but results from a specific effect on the ORL1-receptor.

Inhibitory modulation of cutaneous vascular responses by endogenous galanin in the pigeon.

The possible role of endogenous galanin in modulation of cutaneous vascular responses was studied in pigeons. Chemically induced plasma extravasation and regional skin blood flow changes were measured simultaneously with a capillary perfusion technique and a laser Doppler imager, respectively. Perfusion with both histamine and bradykinin increased plasma protein extravasation which was dose-dependently and significantly augmented by co-administration of M35, a specific galanin antagonist. This effect of M35 was abolished after chronic cutaneous denervation. In intact but not denervated skin, M35 increased the vasodilatatory effect of histamine, too. It is suggested that galanin-containing nerves may play an inhibitory efferent role in the modulation of cutaneous inflammatory responses.

Nociceptin/orphanin FQ: effects on thermoregulation in rats.

Nociceptin/orphanin FQ is a 17 amino acid peptide which acts as a potent endogenous agonist of the opioid receptor-like 1 (ORL1) receptor. ORL1 receptor is a G protein-coupled unique member of the cloned opioid receptor family. We have investigated the effects of nociceptin (1, 10 and 100 nM) on the temperature sensitivity of neurons from the preoptic area of the anterior hypothalamus (PO/AH) in rat brain slices. The body temperature of male Wistar rats was measured after intrahypothalamic application of nociceptin (1 nM) via cannulas in the PO/AH. Low dose nociceptin (1 nM) significantly increased (p < 0.05) temperature sensitivity (TC) of warm-sensitive PO/AH neurons, while the high concentration (100 nM) decreased TC in both warm-sensitive and temperature-insensitive neurons. Similar agonistic activity was obtained after addition of [Phe1 psi (CH2-NH) Gly2]-nociceptin-(1-13)-NH2 (1, 10 and 100 nM), recently proposed to be a selective antagonist of the nociceptin receptor. Neither antagonism nor additive synergism were observed when nociceptin and [Phe1 psi (CH2-NH) Gly2]-nociceptin-(1-13)-NH2 were applied simultaneously in equimolar concentrations. The selective opioid OP3 receptor antagonist CTOP, the selective opioid OP2 receptor antagonist nor-binaltorphimine and selective opioid OP1 receptor antagonist naltrindol had no influence on the effects of nociceptin on temperature sensitivity in PO/AH neurons. In vivo experiments showed that nociceptin (1 nM; 1 microliter/rat) significantly decreased body temperature (p < 0.05) between 30 and 60 min after intrahypothalamic application. These data are in agreement with the hypothesis that the specific action of endogeous substances on body temperature appears to be closely related to a specific change in the temperature sensitivity of warm-sensitive PO/AH neurons.

The relationship between axonal spike shape and functional modality in cutaneous C-fibres in the pig and rat.

Axonal spike shape was examined in identified cutaneous C-fibres dissected from the saphenous nerves of anaesthetized pigs and rats, and was found to vary with functional class. In the pig, the action potential duration for heat nociceptor units (duration at half peak amplitude, 1.25 +/- 0.16 ms, mean +/- S.E.M., n=32) was significantly longer than the duration for polymodal nociceptive units (0.88 +/- 0.11 ms, n=32). Both classes of nociceptive C-fibre had action potentials of longer duration than the low-threshold mechanoreceptor units (0.49 +/- 0.04 ms, n=24) and the inexcitable C-fibres (0.56 +/- 0.06 ms, n=19). Undershoot durations were also longer in nociceptive than non-nociceptive C-fibres. In contrast, spike amplitudes were similar in all classes of C-afferent. In the rat, as in the pig, the polymodal nociceptor units had action potentials of longer duration (0.75 +/- 0.05 ms, n=73) than the mechanoreceptor units (0.60 +/- 0.01 ms, n=23). C-fibres identified as spontaneously active sympathetic efferent units had wider action potentials (main initial peak: 1.01 +/- 0.12 ms, n=22; undershoot: 4.1 +/- 1.23 ms, n=20) than the afferent C-fibres (main peak: 0.69 +/- 0.03 ms, n=130; undershoot: 1.4 +/- 0.09 ms, n=111). All rat C-fibre types had action potentials with main initial peaks of a similar height. However, cold thermoreceptor units had spikes with significantly smaller undershoots compared to nociceptive or inexcitable C-fibres. It is concluded that there are clear differences in axonal spike shape between the different functional classes of C-fibre and, in particular, that nociceptive C-afferents tend to have axonal action potentials of longer duration than non-nociceptive afferents. The ion channels responsible for the longer duration action potentials may provide a target for the development of highly selective analgesic drugs.

Galanin mediated inhibitory nervous modulation of cutaneous vascular reactions.

Noxious stimulation induces local inflammatory responses in a variety of mammals but these reactions are only faint in avian species. The possibility that endogenous galanin inhibits neurogenic vascular responses in avians was tested in the wing skin of anaesthetized pigeons. Intraarterial infusion of nanomolar concentrations of the specific galanin antagonist M35 dose dependently enhanced the small mustard oil induced increase of skin blood flow measured by means of a Laser Doppler Imager. Similarly, the small transient vasodilatation following electrical stimulation of a cutaneous nerve was also enhanced by M35. The effect of M35 was not observed after chronic denervation. Coperfusion of M35 dose dependently augmented the histamine and bradykinin induced plasma extravasation revealed by skin microdialyses, but this effect was abolished in the chronically denervated skin. However, chronic denervation per se enhanced the plasma extravasation induced by histamine but not by bradykinin and this effect was diminished by coperfusion of galanin. The results suggest an inhibitory modulation of cutaneous neurogenic inflammatory reactions by endogenous galanin in the pigeon.

Effects of kappa and delta opioid agonists on activity and thermosensitivity of rat hypothalamic neurons.

Extracellular recordings were made from 161 warm-sensitive, six cold-sensitive and 153 temperature-insensitive neurons in slices of the preoptic area/anterior hypothalamus (PO/AH) of rats, to investigate the effects of the kappa-receptor opioid agonist dynorphin A1-17 and the delta-receptor opioid agonist DPDPE on neuronal response characteristics. While 61% of the neurons exhibited kappa-receptors, delta-receptors were only present in 37% of the neurons. No co-localization was observed between kappa- and delta-receptors, whereas mu-receptors could be co-localized with kappa- as well as delta-receptors. Antagonistic effects on tonic activity were induced by different concentrations of the kappa-agonist dynorphin A1-17. At 0.5 nM, the excitatory effect was predominant, while 50% of the neurons were already inhibited at 5 nM and inhibition was the major effect at 100 nM. A significant increase in temperature sensitivity was observed in warm-sensitive neurons during administration of 0.5 nM dynorphin A1-17; in contrast, the temperature sensitivity was significantly decreased at the high dose of 100 nM. In most of the neurons responding to the delta-receptor agonist DPDPE (0.5-100 nM) the firing rate was decreased. The temperature sensitivity was only affected in warm-sensitive neurons, and was increased in the majority of neurons at 0.5 and 5 nM, but predominantly decreased at higher concentrations. The effects of low concentrations of dynorphin A1-17 and DPDPE were prevented by pre- and co-perfusion of the appropriate antagonists. The present results suggest that changes of the temperature sensitivity of warm-sensitive PO/AH neurons are an important mechanism for the effect of low doses of opioids on body temperature.

Evidence for an inhibition by endogenous galanin of neurogenic cutaneous vasodilatation in the pigeon.

The effect of high affinity galanin antagonist M35 on neurogenic cutaneous vasodilatation has been studied in the pigeon using a Laser Doppler Imager. Cutaneous application of mustard oil or antidromic electrical stimulation of a cutaneous nerve produced a small increase in skin blood flow. Close arterial injection of M35 prior to chemical or electrical stimulation resulted in a marked augmentation of the vasodilatory response. This effect was abolished by chronic denervation. The results suggest a nerve-mediated inhibitory effect of endogenous galanin on neurogenic cutaneous vasodilatation in the pigeon skin and provide the first experimental evidence for an inhibitory local regulatory function of cutaneous sensory nerves at least in the avian skin.

Efferent functions of C-fiber nociceptors.

C-fiber nociceptors not only serve afferent but also local efferent functions. The local efferent functions, such as vasodilatation, axon reflex flare reaction, plasma extravasation, and modulation of neuronal activity, are mediated via a local release of substance P, neurokinin A, and calcitonin gene-related peptide (CGRP) from the peripheral ending. CGRP is the main mediator of the capsaicin-induced flare reaction in the mammalian skin (including humans). In the pig skin the vasodilatation is due to activation of specific heat nociceptors. In the pigeon, antidromic vasodilatation is markedly inhibited by intrinsic galanin. Plasma extravasation in the pig skin blister base or using microdialysis can be evoked by histamine, but not by electrical stimulation or capsaicin. The neurogenic component of the histamine response (64%) appears to be mediated via NK2 receptors and can be modulated by CGRP. There is some evidence that the neuropeptides can also sensitize or stimulate nociceptors. Since in the fibromyalgia syndrome an increased sensitivity of the flare reaction has been observed, the hyperalgesia might be partly due to altered functions of C-fiber nociceptors.

Postnatal development of the autonomic and sensory innervation of the musculature in the rat urinary bladder.

The postnatal development of the innervation of the muscle layer in the rat urinary bladder was analysed in whole mount preparations using immunohistochemistry against protein gene-product 9.5 (PGP; general neuronal marker), growth-associated protein 43 (GAP), dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P (SP). Immunoreactive nerve fibres for all markers were already present at birth. The density of PGP- and GAP-positive nerve fibres was similar and remained constant throughout the postnatal development. The rank order of densities for the other markers relative to PGP was NPY (129-189%) > CGRP (20-63%) > SP (7-23%) > DBH (7-12%) > VIP (2-11%). While the density of presumably efferent VIP- and DBH-positive fibres did not change postnatally, NPY-positive fibres reached adult density at the fifth postnatal day. Sensory CGRP- and SP-positive nerve fibres approached adult levels at the end of the second week, shortly before the micturition reflex was completely developed. The data suggest that a sufficient relative density of sensory and certain efferent elements might be a prerequisite for the development of the mature micturition reflex.

Neurons in the chicken ureter are innervated by substance P- and calcitonin gene-related peptide-containing nerve fibres: immunohistochemical and electrophysiological evidence.

Numerous ganglia or single neurones immunoreactive to protein gene-product 9.5 (PGP) were demonstrated in the chicken ureter. Ganglia were observed in the main nerve trunks accompanying the ureter (400-2,000 cells), in the adventitia (1-45 cells; density; 79 +/- 12 ganglia/cm2; mean +/- S.E.M.), in the circular muscle (1-9 cells; 76 +/- 10 ganglia/cm2) and in the longitudinal muscle (1-8 cells; 232 +/- 41 ganglia/cm2). Most of the PGP-positive neurones in the nerve trunk ganglia (approximately 66%) and in the smooth muscle layers (85%) were encircled by a dense plexus of varicose nerve fibres containing both substance P (SP) and calcitonin gene-related peptide (CGRP). SP-positive somata were rarely observed. Immunogold electron microscopy revealed that SP- and CGRP-immunoreactivity were colocalised in the same dense core vesicles. A strong reduction of SP-positive nerve fibres was observed in organ cultures of the ureter, indicating their extrinsic origin. The fibres might originate from the dorsal root ganglia, where SP and CGRP were colocalised in 20-30% of the neurones. The sensitivity of ureteric neurones to SP and CGRP was investigated in recordings obtained from mechanosensitive nerve fibres with cell bodies located in or adjacent to the ureter (U-G units). The majority (71%) of the U-G units was excited by local application of SP in a dose-dependent manner. The SP-sensitive U-G neurones had higher mechanical thresholds (29 +/- 5 mmHg) as opposed to the SP-insensitive ones (10 +/- 3 mmHg). Repeated applications of high doses of SP to the U-G units resulted in desensitisation and reduced the response to mechanical stimuli. None of the U-G units responded to local application of CGRP, but all U-G units were excited by acetylcholine. The data support the hypothesis that SP-containing primary afferents are involved in the modulation of the activity of ureteric neurons in the chicken.

Neuronal basis for the hyperthermic effect of mu-opioid agonists in rats: decrease in temperature sensitivity of warm-sensitive hypothalamic neurons.

The effect of the selective mu-opioid receptor agonist Tyr-Pro-N-MePhe-D-NH2 (PL-017) on tonic activity and temperature sensitivity of neurons in the preoptic area/anterior hypothalamus (PO/AH) has been examined in rat brain slices using extracellular recordings. The tonic activity of both warm-sensitive and temperature-insensitive neurons was inhibited in a dose-dependent manner by superfusion with the mu-agonist (0.5-100 nM). The temperature sensitivity was selectively decreased in warm-sensitive neurons in concentrations up to 10 nM. Only in the high concentration of 100 nM did the mu-agonist reduce the temperature coefficient of both types of neurons. Pretreatment with equimolar concentrations of the mu-antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) prevented the effects of the mu-agonist (0.5-10 nM) on tonic activity as well as temperature sensitivity. The results suggest that a specific change of the temperature sensitivity of PO/AH neurons is involved in the hyperthermia induced by mu-agonists in rats.

The vasodilator component of neurogenic inflammation is caused by a special subclass of heat-sensitive nociceptors in the skin of the pig.

1. Skin blood flow has been imaged during stimulation of fine nerve filaments containing small numbers of identified C fibre units. Filaments were dissected from the saphenous nerve of anaesthetized pigs. 2. Stimulation of filaments containing C heat nociceptor units gave small areas of elevated blood flow (average increase 96%, n = 11) restricted to the afferent receptive field. The extent of the areas of raised blood flow was imaged completely for 8 units. The average extent of vasodilatation in the direction of greatest spread was 8 mm and the maximum spread in any unit was 13 mm. 3. Stimulation of C polymodal nociceptor units never caused increases in blood flow in or near their receptive fields. 4. Localized noxious stimuli (55 degrees C or intradermal injection of capsaicin) caused flare extending 7-15 mm in the same skin region. 5. In agreement with the axon reflex model, spread of flare was restricted to the zone innervated by the terminals of single C fibre units. 6. It is concluded that the C heat nociceptor units are the major class of afferent involved in the flare reaction in the skin of the pig. C polymodal nociceptor units do not appear to be involved in flare in this species. The probable situation in human skin, which is also innervated by heat nociceptors, is discussed.

Effects of GABA agonists and antagonists on temperature-sensitive neurones in the rat hypothalamus.

1. Extracellular recordings were obtained from 94 warm-sensitive, 6 cold-sensitive and 117 temperature-insensitive neurones in slices of the hypothalamic medial preoptic area of rats, to determine the effect of the GABAA agonist muscimol, the GABAA antagonist bicuculline, the GABAB agonist baclofen and the GABAB antagonist phaclofen on tonic activity and temperature sensitivity. 2. Muscimol and baclofen dose-dependently inhibited the tonic activity of 69% (36/52) and 97% (36/37) of the hypothalamic neurones, respectively, regardless of their type of thermosensitivity. In contrast, the GABAA antagonist bicuculline increased the tonic activity of the majority of neurones (58/83), while the GABAB antagonist phaclofen increased neuronal activity only in the high dose of 100 microM. 3. The temperature sensitivity of hypothalamic neurones was only changed by ligands of GABAB receptors, and this effect was restricted to warm-sensitive neurones. The temperature coefficient (TC) was significantly increased by the GABAB agonist baclofen (delta TC = 0.69 +/- 0.11 imp s-1 degree C-1, P < 0.01, n = 18). In contrast, the GABAB antagonist phaclofen (10 microM) decreased the temperature sensitivity (delta TC = -0.67 +/- 0.09 imp s-1 degree C-1, P < 0.01, n = 10) in doses which did not affect tonic activity. 4. The increase in temperature sensitivity due to the GABAB agonist baclofen was significantly enhanced by co-perfusion of the GABAA antagonist bicuculline, indicating an interaction of GABAA and GABAB receptor-mediated mechanisms with regard to neuronal thermosensitivity. 5. The results suggest that neurones in the medical preoptic area are subject to GABA-mediated tonic inhibition resulting in modulation of tonic activity and temperature sensitivity of warm-sensitive neurones possibly involved in the control of body temperature. The data support the hypothesis that the hypo- or hyperthermic action of an endogenous substance is related to its effect on the thermosensitivity rather than on tonic activity of hypothalamic neurones.

Substance P and calcitonin gene-related peptide in the chicken skin: distribution and cardiovascular effects.

A short vasodilatation but no plasma extravasation could be induced by antidromic stimulation of peripheral nerves in the chicken skin. Since in mammalian species the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) are involved in this mechanism, the distribution and the cardiovascular effect of these peptides were investigated in the chicken. In the skin, SP- and CGRP-immunoreactivity was found co-localized in the epidermis and dermis. On blood vessels, however, SP positive but CGRP negative nerve rfibres were observed. Systemic (i.v.) and local close arterial injection of SP produced dose-dependent cutaneous vasodilatation with threshold doses of 6.5 pmol/kg or 1 pmol, respectively. Neurokinin A and acetylcholine were about 20 to 50-fold less effective, when injected close arterially. Systemic injection of SP (5-1800 pmol/kg) dose-dependently evoked a short fall in blood pressure which was followed by a longer lasting pressor response. CGRP up to 800 pmol/kg did not change blood pressure but produced a pronounced tachycardia. Close arterial injection of CGRP resulted in variable bi- or triphasic vascular responses which consisted of vasodilatations and also vasoconstriction with thresholds between 0.25 and 65 pmol. The data also indicate that in the chicken, SP, and to a lesser extent CGRP, can be involved in antidromic vasodilatation.

Inhibition of the neurogenic inflammatory response by lidocaine in rat skin.

Axon reflex vasodilatation and neurogenic plasma extravasation are characteristic cutaneous vascular responses mediated by neuropeptides released from stimulated capsaicin-sensitive sensory nerve endings. Intracutaneous injections of local anaesthetics inhibit the axon-reflex flare elicited by chemical irritants in human skin. Results of earlier reports on the effects of local anaesthetics on neurogenic plasma extravasation are controversial. The aim of the present study, therefore, was to re-examine the effect of the local anaesthetic lidocaine on the neurogenic inflammatory response of rat skin. The effects of lidocaine on cutaneous inflammatory reactions were measured quantitatively by means of the Evans blue technique. Intracutaneous injection of lidocaine resulted in a dose-dependent inhibition of the neurogenic inflammation elicited by mustard oil and of the dye leakage response to compound 48/80 or histamine. It is suggested that the site of this inhibition is beyond the sensory nerve terminal, presumably at the level of the vascular endothelium.